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. This document is intentionally written as an educational template that looks and reads like a true clinical SOP manual. It is intended for laboratories to adapt into their own controlling manual by inserting validated local setpoints, workflow details, device- or media-specific instructions, form identifiers, escalation pathways and approval signatures. Once locally completed, reviewed, approved and issued under document control, the adopted version becomes the laboratory's operational source of truth.

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, including requirements set forth by applicable accreditation bodies (e.g., CAP, CLIA) and federal regulations (e.g., FDA 21 CFR Part 1271) as well as any applicable state licensure requirements.
• 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. In the locally adopted version, each critical operational parameter shall be classified and controlled as one of the following: a published reference standard, a locally validated operational setpoint, or a manufacturer-specific instruction for use. Exact local values, tolerances, timing windows, alarm limits, grading thresholds and workflow-specific decision points shall be inserted into the adopted version and linked to the corresponding worksheet, log, validation record, appendix or controlled form so that staff are never required to infer which value governs practice.

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 vacuum or level loss.

1.6 Regulatory and Accreditation Framework

This laboratory operates in compliance with applicable federal, state and accreditation requirements. These include, but are not limited to:

• Clinical Laboratory Improvement Amendments (CLIA): The laboratory shall maintain CLIA certification at the applicable complexity level and comply with all CLIA requirements for personnel, quality control, proficiency testing and recordkeeping.
• College of American Pathologists (CAP): Where applicable, the laboratory shall comply with CAP Laboratory Accreditation Program requirements, including participation in CAP proficiency surveys and adherence to CAP checklists for reproductive laboratory standards.
• FDA 21 CFR Part 1271: The laboratory shall comply with FDA regulations governing human cells, tissues, and cellular and tissue-based products (HCT/Ps), including donor eligibility determination, current good tissue practice (cGTP), and adverse event reporting requirements as applicable.
• State Licensure: The laboratory shall comply with all applicable state regulations governing IVF laboratory operations. Some states, including New York, California and others, impose requirements beyond federal minimums. Staff shall be aware of and adhere to state-specific requirements.
• ASRM and ESHRE Guidelines: Professional society guidelines from ASRM and ESHRE shall inform all procedures, grading criteria and quality standards, as detailed throughout this manual.

This manual is intended to support accreditation compliance but does not substitute for direct review of current accreditation checklists, regulations or institutional policies. The laboratory director is responsible for ensuring that this manual is updated when regulatory or accreditation requirements change.

1.7 Template Adoption and Control Status

This version is a template procedure manual intended to help laboratories build a true controlling laboratory procedure manual. It can be used as a drafting, harmonization and review tool. It intentionally omits certain site-specific details, such as exact incubator setpoints, alarm limits, witness language, form numbers, equipment identifiers, staffing assignments and escalation contacts, so those details can be inserted by the adopting laboratory. A laboratory shall not rely on this template as its controlling manual until those local details are completed, validated where required, approved and issued under local document control.

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.
• Sperm cryopreservation.
• Cryostorage inventory management.
• Specimen transport and offsite storage coordination.
• Donor gamete handling where applicable.
• 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. In the adopted local version, this manual is the governing procedural reference for the activities within its scope, and supporting materials shall be aligned to it.

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.

4.1 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:

• Perform hand hygiene and enter the laboratory according to laboratory dress and access requirements.
• Confirm that the laboratory is clean, orderly and suitable for work.
• Verify function of required equipment including microscopes, incubators, hot blocks, warming stages and any equipment needed for the scheduled cases.
• Record temperatures of all monitored devices required by the daily QC schedule.
• Verify incubator gas values using the approved digital gas analyzer. Incubators used for embryo culture should maintain a physiologic environment within range of manufactured recommendations.
• Confirm that gas supplies, alarms and back-up systems are in normal status when applicable.
• 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.
• Confirm that sharps and biohazard containers are present and not overfilled.
• Review the daily clinical schedule and identify all procedures planned for the day.
• 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?
• 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.

5.1 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, oocyte retrieval, 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 (semen collection cups, etc.).
• Sperm preparation tubes and devices.
• Oocyte dishes, insemination dishes, stripping dishes, injection dishes and culture dishes.
• Biopsy dishes and tubes.
• Cryopreservation devices (straws, cryodevices, etc.) 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 and after oocyte retrieval.
• 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

Operational note: 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.

6.1 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 (scrubs, 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:

• Remove unnecessary items from the work surface.
• Disinfect the surface with approved laboratory disinfectant and allow to dry.
• Arrange sterile and non-sterile items so that sterile handling can proceed without cross-contamination.
• 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

Operational note: 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.

7.1 Purpose

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

7.2 Procedure

• Assemble all ingredients and verify identity and expiration.
• Prepare the solution using the approved formula.
• Mix until homogeneous.
• Filter through an approved sterile filter of appropriate pore size into a sterile labeled container.
• Label the container with solution name, date prepared, expiration date, preparer initials and any required storage condition.
• 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.

8.1 Purpose

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

8.2 Procedure

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

8.3 Rejection Criteria

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

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

Operational note: 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.

10.1 Purpose

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

10.2 Receipt Procedure

• Confirm that the specimen container is labeled with approved identifiers and matches the requisition and patient record.
• Record date and time of collection if available and date and time of receipt.
• Record specimen source (e.g., fresh ejaculation, aspirate) and intended use.
• Assign or confirm the laboratory case record.
• 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

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

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 sample 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

• Verify patient identifiers on the specimen container, requisition and laboratory worksheet.
• Confirm specimen type and intended use.
• 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.
• Mix gently before aliquoting so the sample is representative.
• Perform the required pre-processing semen assessment, typically including concentration and motility and any additional parameters used by the laboratory.
• Select the preparation method based on specimen quality, expected sperm recovery needs and the planned fertilization method.

11.4 Density Gradient Preparation

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

11.5 SwimCount® Harvester Preparation

• Verify patient identifiers on the specimen container, device and worksheet.
• Confirm that the device is within expiration date, intact and suitable for clinical use.
• Allow the semen specimen to liquefy fully unless otherwise directed.
• Gently mix the liquefied specimen to ensure a representative sample without creating excessive bubbles.
• Label all receiving tubes or dishes before beginning.
• Prepare the device exactly as required by the manufacturer instructions and the laboratory workflow.
• Load the semen specimen into the device using the volume designated by the current laboratory validation or manufacturer instructions.
• Place the loaded device under the environmental conditions specified by the manufacturer (typically 37°C).
• 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.
• At the end of the migration interval, harvest the motile sperm-enriched fraction from the designated collection chamber using the supplied sterile syringe.
• Examine the harvested fraction for concentration and motility.
• Hold the final preparation under controlled conditions at room temperature 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

Operational note: 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.

12.1 Purpose

To prepare previously cryopreserved sperm for IVF after thawing.

12.2 Procedure

• Verify the specimen designation against the patient record, thaw plan and storage inventory before removal from storage.
• Confirm witness according to laboratory policy before thawing begins.
• Retrieve the correct vial, straw or other storage unit from cryostorage and minimize warming during handling. Use proper PPE to protect against cryogenic exposure.
• 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.
• Once thawed, transfer the sample to an appropriately labeled sterile tube.
• Assess post-thaw appearance, motility and concentration as applicable. Cryopreserved sperm often show reduced motility compared with fresh sperm.
• If cryoprotectant removal is required, dilute or wash the sample gradually to reduce osmotic stress.
• Centrifuge using the laboratory's standard thaw-preparation recovery setting when concentration or wash recovery is needed.
• Remove the supernatant carefully and resuspend the recovered sperm fraction in approved insemination medium.
• 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.
• Thawed sperm can also be processed using density gradient or the SwimCount® Harvester using documented laboratory protocols.

12.3 Documentation

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

12.4 Teaching Point

Operational note: 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.

13.1 Purpose

To process testicular or epididymal sperm specimens for ICSI.

13.2 Procedure

• Verify specimen identity, specimen source and intended use before opening or processing the sample.
• If the specimen is a fresh tissue sample, transfer small portions to a sterile processing dish containing handling medium.
• Using sterile needles or fine sterile forceps, gently tease the tissue to unravel the tubules and flatten to expel the contents. The contents can then be gently disrupted to allow the cells to disperse.
• If the specimen is an aspirate, mix gently and examine a small aliquot microscopically.
• Examine the processed sample under the inverted microscope for sperm presence, approximate abundance and motility when visible. Record whether sperm are present and motile.
• 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.
• Remove excess debris when possible without risking loss of rare sperm.
• Prepare the final suspension in a small volume of handling medium suitable for ICSI or other intended use.
• If no motile sperm or very few are seen initially, continue a careful microscopic search using the laboratory's standard search method and consider reassessment after brief incubation. Any sperm that are seen can be removed from the sample with an ICSI pipette or hatching pipette and placed in a separate dish ready for ICSI. Communicate with the physician about potential use of immotile sperm or testicular sperm extraction.
• 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 very low sperm numbers and substantial cellular debris. 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

Operational note: 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.

14.1 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 must remove seminal plasma and debris thoroughly and include as many good quality sperm as possible from the initial sample.

14.2 Procedure

• Verify specimen identity and intended use for IUI.
• Allow the specimen to liquefy fully unless pre-liquefied on receipt.
• Perform the initial assessment required by laboratory policy, including volume and motility-related observations.
• Select the preparation method used by the laboratory for IUI, most commonly density gradient preparation, SwimCount® Harvester or sperm wash.
• 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.
• If using SwimCount® Harvester, prepare and load the device according to the current laboratory protocol and manufacturer instructions, allow the migration interval to proceed undisturbed, then harvest the motile sperm-enriched fraction and assess whether the recovered sample is suitable for intrauterine insemination or requires further concentration.
• 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.
• Assess the final preparation for volume, concentration, motility and total motile sperm when applicable.
• Label the final preparation clearly and keep it under appropriate conditions until released to the clinical team.
• Transfer the prepared specimen to the clinical team using the laboratory's chain-of-custody process.

14.3 Teaching Point

Operational note: 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.

15.1 Purpose

To define the procedure for freezing and cryopreserving ejaculated, epididymal or testicular sperm specimens for future clinical use, with sufficient detail for trainees to understand the sequence of steps, the rationale for cryoprotectant use and the requirements for identity control and storage accuracy.

15.2 Scope

This procedure applies to all sperm cryopreservation cases performed in the laboratory, including ejaculated samples from patients undergoing IVF, patients banking sperm for future fertility preservation and surgical sperm samples that are not used entirely on the day of retrieval.

15.3 Consent and Documentation

Written informed consent shall be obtained before cryopreservation. Consent documents shall specify the intended use of the sample, storage duration, disposition in the event of patient death, incapacity or failure to maintain contact, and any restrictions on donor eligibility. Consent shall be reviewed and confirmed present before processing begins.

15.4 Initial Assessment

• Verify patient identifiers on the specimen container, requisition and laboratory worksheet.
• Perform the required pre-freeze semen assessment, typically including volume, concentration, motility (total and progressive) and any additional parameters used by the laboratory.
• Assess liquefaction and viscosity. Allow the specimen to liquefy fully unless otherwise directed.
• Document pre-freeze findings to allow comparison with post-thaw results.

15.5 Cryoprotectant Preparation

Prepare the cryoprotectant medium according to the manufacturer instructions and the laboratory's validated protocol. Common cryoprotectant agents include glycerol-based formulations. The cryoprotectant medium shall be within its validated expiration and prepared at room temperature or as specified. Confirm identity and lot number and record on the worksheet.

15.6 Procedure

• Label all cryodevices (straws, vials, etc) with at least two approved patient identifiers before loading. Labels must be durable at cryogenic temperatures.
• Confirm witness verification of patient identity before loading begins.
• Mix the semen specimen gently so the sample is uniform. Add the cryoprotectant medium to the specimen in a dropwise or gradual manner using the laboratory's validated ratio to minimize osmotic shock.
• Mix the specimen and cryoprotectant medium gently and allow equilibration for the time defined by the laboratory protocol (commonly 5–10 minutes at room temperature).
• Load the cryoprotected suspension into labeled cryostraws or vials using the standard filling technique. Seal each straw or cap each vial immediately.
• Place loaded straws or vials into the appropriate rack, cane or goblet for programmed or vapor freezing.
• If using a controlled-rate freezer, load the specimen holder into the unit and initiate the validated freeze program. If using vapor-phase cooling, suspend straws in the nitrogen vapor for the time defined by the laboratory protocol before plunging.
• Plunge the straws or vials into liquid nitrogen at the appropriate step and transfer to the assigned long-term storage cane, goblet or rack.
• Record the storage location (tank, cane, goblet, slot) immediately in the cryostorage inventory log and electronic system.

15.7 Post-Freeze Quality Check

A post-thaw check of a test straw (or a separate aliquot prepared for this purpose) should be performed according to the laboratory's validation policy. Post-thaw total motile sperm count (TMSC) and progressive motility should be recorded. Findings should be documented and communicated to the clinical team and patient.

15.8 Documentation

Document the following:

• Patient identifiers.
• Date of cryopreservation.
• Pre-freeze semen analysis parameters.
• Cryoprotectant medium lot number and preparation details.
• Number of straws or vials frozen.
• Freeze method used.
• Storage tank, cane and slot location for each unit.
• Operator identity and witness identity.
• Post-thaw check findings when performed.
• Any deviation or unusual finding.

15.9 Teaching Point

Operational note: Sperm cryopreservation is a chain of small steps where each one matters. Proper cryoprotectant medium addition, adequate equilibration, controlled cooling and accurate storage location recording are all essential. Post-thaw assessment closes the loop and provides the data needed for clinical planning.

16.1 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.

16.2 Scope

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

16.3 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.

16.4 Preparation Before Retrieval

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

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

16.5 Receipt of Follicular Aspirates

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

16.6 Identification of Oocytes

Gently pour or transfer the aspirate into the scanning dish or approved retrieval vessel. Examine the aspirate systematically under the stereomicroscope at the magnification normally used by the laboratory for retrieval scanning. Scan the entire dish in a consistent pattern so that no portion of the aspirate is missed. 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. Carefully inspect dense cellular material or tissue fragments when an oocyte may be embedded. Before discarding the original aspirate tube, inspect the tube wall and bottom for any COC that may remain adherent.

16.7 Washing and Transfer to Culture

• Pick up each identified COC using an approved handling pipette.
• Transfer the complex through the laboratory's designated wash droplets or dishes to remove excess blood and debris.
• Trim cumulus cells if required by laboratory workflow.
• Place washed oocytes into the labeled pre-equilibrated culture dish containing the approved culture medium.
• Group oocytes in the dish according to laboratory workflow while preserving patient identity and traceability.
• Return the culture dish to controlled incubation conditions promptly after transfer.

16.8 Retrieval Count and Communication

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

16.9 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).

16.10 Teaching Point

Operational note: A trainee should understand that retrieval scanning is a systematic search process. Missed oocytes usually result from inconsistent scanning technique. Recording unusual COC characteristics supports clinical decision-making and may inform fertilization strategy.

17.1 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.

17.2 Scope

This procedure applies to all oocytes designated for conventional insemination.

17.3 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.

17.4 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 hours after insemination (Day 1), consistent with ESHRE/Alpha guidelines.

17.5 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.

17.6 Preparation

Before insemination:

• Confirm patient identity on the oocyte dish, sperm preparation tube and worksheet.
• Confirm the required witness step according to laboratory policy.
• Confirm the number of oocytes designated for conventional insemination.
• Confirm that the insemination dish has been prepared with equilibrated medium under appropriate culture conditions.
• 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.
• 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.
• Review whether the case includes any specific instruction regarding low sperm count, low motility, split insemination plan or fallback plan for ICSI.

17.7 Procedure

• Remove the oocyte dish from the incubator only when ready to inseminate.
• Review the oocyte identity again before sperm is added.
• Place or confirm placement of the oocytes into the designated insemination droplet or well.
• Inspect the dish to confirm that the number of oocytes present matches the expected count.
• Mix the prepared sperm suspension gently so the suspension is uniform.
• Sperm can be added to a separate droplet or dish before adding oocytes, which allows for checking and adjusting the concentration before adding oocytes, or added directly to the dish or drops containing the oocytes, depending on the laboratory protocol. Add the appropriate volume of sperm suspension to the insemination droplet or well to achieve the target insemination concentration.
• 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.
• Confirm that the final insemination dish is properly labeled and contains the intended oocytes.
• Return the dish promptly to the incubator. Minimize time outside the incubator to preserve pH and temperature.
• Record the exact or approximate time of insemination according to laboratory practice.

17.8 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.

17.9 Post-Insemination Handling

Maintain the dish under stable incubator conditions until the planned fertilization check (approximately 16–18 hours after insemination). Avoid unnecessary disturbance of the insemination dish. Use a denudation pipette to remove cumulus cells prior to fertilization check. If any issue occurs during insemination, including dish contamination, labeling discrepancy or concern about sperm adequacy, notify laboratory leadership immediately and document the event.

17.10 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).

18.1 Purpose

To define the procedure for removal of cumulus and corona cells from oocytes for maturity assessment and preparation for ICSI.

18.2 Scope

This procedure applies to oocytes designated for denudation (typically those intended for ICSI). Denudation is not performed on oocytes designated for conventional insemination.

18.3 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.

18.4 Preparation

Before stripping:

• Prepare and label the stripping dish, wash droplets and post-stripping culture dish.
• 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 and exposure time should be minimized to reduce potential toxicity.
• Confirm patient identity on all dishes and the worksheet.
• Confirm the timing of the denudation procedure according to laboratory workflow.
• Arrange denuding pipettes from larger bore to smaller bore so that progressive removal of cells can be done efficiently. Be cautious using smaller diameters as this can increase the risk of damage to the oocytes.
• Limit the number of oocytes handled at one time to the number that can be denuded promptly without prolonged exposure outside controlled conditions.

18.5 Procedure

• Remove the oocytes designated for stripping from incubation when ready to perform the procedure. Keep them in a warmed environment until denuding begins.
• Transfer the cumulus-oocyte complexes into the hyaluronidase-containing droplet or dish. Expose the oocytes only briefly (usually ≤30 seconds), just long enough to loosen the cumulus matrix so mechanical denudation can proceed.
• Transfer the oocytes into wash droplets and begin mechanical denudation using denuding pipettes of appropriate bore size.
• Move the oocytes through progressively smaller denuding pipettes as needed, using gentle aspiration and expulsion to remove residual cells.
• 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.
• Transfer the oocytes into the appropriately labeled post-stripping culture dish and return promptly to the incubator.

18.6 Precautions

• Avoid prolonged exposure to hyaluronidase.
• 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 and can be removed from the oocyte selection at this time.

18.7 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.

18.8 Teaching Point

Operational note: 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. If oocytes appear to be fracturing during this process, discontinue immediately and seek advice from senior laboratory personnel.

19.1 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.

19.2 Scope

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

19.3 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.

19.4 Preparation of Equipment

Before beginning ICSI:

• 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.
• Confirm that the injection dish, sperm handling dish and post-injection culture dish are properly labeled.
• Confirm that the required microtools are correctly mounted and functioning.
• Prepare all media, PVP and oil according to laboratory policy. Confirm that the work area is organized so that identity control is maintained throughout the case.

19.5 Preparation of Oocytes and Sperm

• Confirm patient identity on all dishes, sperm materials and records.
• 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.
• Confirm the required witness step before injection according to laboratory policy.
• 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.

19.6 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.

19.7 Injection Procedure

• Place a manageable number of mature oocytes into the injection dish. Keep remaining oocytes in the incubator, or similar conditions, until ready.
• 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.
• Immobilize the selected sperm by the ICSI method (e.g., by crushing the tail against the dish). Aspirate the sperm into the injection pipette.
• Penetrate the zona pellucida and oolemma with the injection pipette using the laboratory's standard micromanipulation technique. Make sure the membrane has been breached, then deposit the sperm into the oocyte cytoplasm.
• Withdraw the injection pipette carefully. Verify that the oolemma has resealed and that no cytoplasmic material is aspirated.
• Inspect the oocyte briefly for survival and obvious lysis. If lysis occurs, document and continue with caution. Oocytes that appear to be lysed at this stage can be placed in a separate drop after ICSI to avoid any negative factors of degeneration affecting other oocytes.
• Transfer each injected oocyte to the labeled post-injection culture dish. Keep the injection dish clean and ready for the next oocyte.
• Continue until all designated mature oocytes have been injected or the case plan has been completed.
• Return the dish promptly to the incubator.

19.8 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.
• Sperm that are difficult to immobilize or poorly suited for injection.
• 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.

19.9 Post-Procedure Review

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

19.10 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.

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 hours 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

• Prepare labeled assessment dishes if embryo movement is required by laboratory workflow.
• Confirm patient identity on the dish and record. Witness if required.
• Minimize the time the dish is outside the incubator. Set up the stereomicroscope or inverted microscope for pronuclear assessment.

20.4 Procedure

• Remove the dish from the incubator at the scheduled time.
• Examine each oocyte or zygote using the microscope appropriate for pronuclear assessment.
• 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.
• Separate into clearly labeled media drops under oil. 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.
• 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.
• 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.

21.1 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. Assessments on day 6 and 7 can also be performed to determine if any additional later developing blastocysts are suitable for biopsy/cryopreservation.

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

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

• What stage is the embryo at right now? (e.g., 4-cell, 8-cell, morula, blastocyst)
• Does the morphology fit what is expected for that stage and day of development? (e.g., number of cells, compaction, blastocyst expansion)
• Are there findings that suggest lower developmental potential or degeneration? (e.g., severe fragmentation, multinucleation, poor compaction)
• 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 4 assessment may not be performed in all laboratories based on lab-specific policies.
• Day 5/6/7 (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.

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 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.

22.1 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

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

22.5 Procedure

• Move the embryo into the designated treatment dish.
• Position the embryo so the zona can be treated at a site away from blastomeres or key cellular structures.
• Focus the treatment area clearly.
• 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.
• Reassess the embryo immediately after treatment for evidence of unintended damage.
• 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

Operational note: The goal is controlled zona manipulation, not excessive treatment. Less is often better when the indication is valid. Over-exposure to laser can damage embryos.

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 both cleavage-stage and trophectoderm (TE) biopsy procedures performed by the laboratory. These two biopsy approaches differ in timing, developmental criteria, cell number sampled and post-biopsy handling; both are described below.

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:

• Review the biopsy plan and confirm embryo identity.
• 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.
• Prepare the biopsy dish, post-biopsy culture dish and all required records.
• Confirm the required witness step before the biopsy begins and again at any required loading or tube confirmation step.
• Confirm function of the microscope, micromanipulators, biopsy pipette, holding pipette and laser or other associated equipment.

23.5 Cleavage-Stage Biopsy

Cleavage-stage biopsy is performed on Day 3 embryos (63–65 hours post-insemination) and involves the removal of one or two blastomeres from an 8-cell or greater embryo. This approach is less common than TE biopsy and is performed primarily for specific clinical indications or where blastocyst culture is not appropriate. Pre-biopsy confirmation:

• The embryo should ideally have at least 6 cells and show minimal fragmentation. Highly fragmented embryos carry greater risk from blastomere removal.
• Confirm that the embryo has a zona pellucida suitable for opening.
• Confirm that the patient's testing plan is compatible with single-cell or dual-cell analysis.

Procedure:

• Move the Day 3 embryo into the biopsy dish. Maintain temperature and pH.
• Stabilize the embryo using the holding pipette.
• Create an opening in the zona using laser-assisted drilling at a location away from blastomeres.
• Insert the biopsy pipette through the zona opening and gently aspirate one or, if specified, two blastomeres. Aim for blastomeres with a visible nucleus; anucleate fragments should not be biopsied.
• Separate the blastomere(s) from the embryo using laser-assisted or mechanical separation.
• Confirm that the embryo remains intact and transfer promptly to the labeled post-biopsy culture dish.
• Load the blastomere(s) into the corresponding labeled biopsy tube as described in section 23.8.

23.6 Trophectoderm (TE) Biopsy

TE biopsy is performed on Day 5, 6 or 7 blastocysts and involves removal of 5–8 trophectoderm cells. It is the current preferred biopsy approach for preimplantation genetic testing (PGT) because it provides more cellular material for analysis while leaving the inner cell mass (ICM) intact. Pre-biopsy confirmation:

• The blastocyst should have an expanded or fully expanded cavity (Gardner grade 3–6) with a visible, accessible trophectoderm.
• Confirm that the embryos designated for biopsy meet developmental criteria. Blastocysts that are collapsed, poorly expanded or degenerating should be reassessed before proceeding.
• Laser-assisted hatching should be performed on Day 3 or 4 to facilitate trophectoderm herniation and improve accessibility for Day 5/6 biopsy. Confirm this was completed if planned.

Procedure:

• Move the expanded blastocyst into the biopsy dish. Maintain temperature and pH.
• Stabilize the embryo using the holding pipette.
• Orient the embryo so that the trophectoderm cells selected for biopsy are accessible and the ICM is positioned away from the biopsy site (typically at 6 or 12 o'clock).
• If a herniated TE strand is present, gently draw it into the biopsy pipette. If no herniation is present, create a small zona opening and coax TE cells into the pipette.
• Draw 5–8 TE cells into the biopsy pipette without aspirating the ICM.
• Separate the TE cells using laser-assisted cutting at the base of the TE strand, applying the minimum number of laser pulses required to achieve clean separation.
• Confirm that the embryo remains intact with the ICM protected. Transfer promptly to the labeled post-biopsy culture dish.
• Load the biopsy sample as described in section 23.8.

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, 1–2 blastomeres for Day 3).
• 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

• Prepare the corresponding labeled biopsy tube with proper buffer.
• 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 external DNA or other embryos.
• Confirm that the embryo number, tube number and worksheet match exactly.
• Confirm witness at the required checkpoint.
• 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

• Return biopsied embryos promptly to the incubator or proceed to cryopreservation according to the clinical plan.
• 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).
• 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 type (cleavage-stage or TE).
• 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).

24.1 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:

• 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.
• Confirm witness according to laboratory policy.
• Prepare and label all cryodishes, cryodevices (e.g., Cryolocks, straws, etc), storage records and associated worksheets before embryos are moved. Labels must be legible, water-resistant and durable at cryogenic temperatures.
• 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.
• 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.
• Arrange the workspace so embryos can be processed one at a time or in small controlled groups without confusion. Ensure proper PPE (cryogloves, safety glasses, etc) and that the workstation is free of clutter.

24.5 Embryo Selection

• Confirm which embryos are designated for cryopreservation based on the laboratory and clinical plan.
• Verify that embryo identity and numbering in the dish match the worksheet or disposition record. Use witness verification.
• Record the number, stage (e.g., blastocyst grade) and quality of embryos selected for cryopreservation. Consult the Gardner grading system for blastocyst quality.
• 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, due to potential toxicity.
• Loading the cryodevice efficiently with minimal fluid volume.
• Recording the exact storage location immediately.

24.7 Cryopreservation Procedure

• Move the embryo or embryos into the vitrification solutions as detailed and defined by the media manufacturer.
• 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.
• Complete the vitrification step by placing the loaded cryodevice into liquid nitrogen.
• Secure the cryodevice in the assigned protective hardware if applicable (e.g., straw sleeve, goblet holder).
• 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.
• 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 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

• Confirm that the number of embryos cryopreserved matches the number documented on the worksheet and in the cryostorage inventory.
• Confirm that all cryodevice identifiers match the worksheet and storage record. Reconcile any discrepancy immediately.
• Confirm that the storage location entry is complete and accurate. Include tank number, cane/goblet position and slot number.
• 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).

25.1 Purpose

To define the procedure for vitrification of mature oocytes for fertility preservation or donor oocyte programs, with sufficient detail for trainees to understand the technical steps, the biological sensitivity of the mature oocyte to cryoprotectant exposure, and the identity control requirements specific to oocyte storage.

25.2 Scope

This procedure applies to all cases of mature oocyte vitrification, including elective fertility preservation (social or medical), egg banking for donor programs and cases where oocytes are vitrified due to the absence of sperm on the day of retrieval. Immature oocytes (germinal vesicle or metaphase I) are generally not vitrified for clinical use unless specifically indicated and validated by the laboratory.

25.3 Background and Biological Considerations

The mature metaphase II oocyte is particularly sensitive to cryopreservation because of its large size, high water content, temperature-sensitive meiotic spindle and cortical granule arrangement. Key considerations include:

• The meiotic spindle is susceptible to cold shock and depolymerization. Warming the oocyte through the vitrification solutions at controlled temperature minimizes spindle disruption.
• Zona hardening can occur after vitrification, which may reduce fertilization rate with conventional insemination. ICSI is therefore the standard fertilization method for vitrified-warmed oocytes.
• Exposure to cryoprotectants must be carefully timed to achieve dehydration without toxicity. Vitrification solutions typically consist of an equilibration solution (lower cryoprotectant concentration) followed by a vitrification solution (higher concentration). Timing in the final vitrification solution is strictly limited.

25.4 Consent

Written informed consent shall be obtained before oocyte vitrification. Consent shall address disposition options including future use, donation, research and discard, and shall specify instructions in the event of patient death, incapacity or prolonged loss of contact. For medical fertility preservation patients, the clinical indication should be documented.

25.5 Preparation

Before vitrification:

• Confirm patient identity on all dishes, cryodevices and worksheets.
• Confirm witness according to laboratory policy.
• Confirm that the oocytes designated for vitrification are mature (MII with first polar body). Document the maturity status of each oocyte.
• Prepare the equilibration and vitrification solutions according to the manufacturer's instructions and the laboratory's validated protocol. Confirm that solutions are within expiration and at the required temperature.
• Label all cryodevices with at least two approved patient identifiers. Labels must be durable at cryogenic temperatures.
• Assign a storage location before beginning. Confirm that the receiving tank, cane and goblet are ready.

25.6 Procedure

• Remove oocyte culture dish from incubator when ready to begin.
• Transfer oocytes into the equilibration solution. Allow the oocytes to equilibrate for the time defined by the laboratory's validated protocol (typically 5–15 minutes). Oocytes will initially shrink due to osmotic efflux of water, then re-expand as cryoprotectant enters the cell. Full re-expansion signals adequate equilibration.
• Transfer the equilibrated oocytes into the vitrification solution. Exposure time in the vitrification solution is strictly limited (typically less than 60 seconds) because of the high cryoprotectant concentration.
• During the final exposure interval, load each oocyte individually onto the labeled cryodevice in the minimum fluid volume required. Work efficiently; time starts from first contact with the vitrification solution.
• Plunge the loaded cryodevice into liquid nitrogen immediately upon completing the loading step.
• Secure the cryodevice in the assigned protective hardware and transfer to the assigned storage location without delay.
• Record the storage location (tank, cane, goblet, slot and device identifier) immediately.
• Repeat for remaining oocytes. Do not allow oocytes to wait in the vitrification solution beyond the validated exposure time. Process oocytes individually or in small groups of two or three.

25.7 Post-Procedure Reconciliation

• Confirm that the number of oocytes vitrified matches the number on the worksheet and in the cryostorage inventory.
• Confirm that all cryodevice identifiers and storage location entries are complete and accurate.
• Document the final disposition of the case (number vitrified, number not vitrified due to immaturity or quality).

25.8 Documentation

Document the following:

• Patient identifiers.
• Date and time of vitrification.
• Number of oocytes vitrified and maturity status (MII) of each.
• Cryoprotectant solutions used, lot numbers and expiration dates.
• Cryodevice identifier for each oocyte or group.
• Storage tank, cane, goblet and slot location.
• Operator identity and witness identity.
• Any deviation or unusual observation (e.g., oocyte degeneration during cryoprotectant exposure, equipment failure).

25.9 Teaching Point

Operational note: Timing in the vitrification solution is non-negotiable. Prepare everything before oocytes touch the vitrification solution, and work with confident speed.

26.1 Purpose

To define the procedure for warming vitrified embryos and oocytes prior to clinical use, including preparation steps, the warming sequence, post-warm assessment and communication with the clinical team.

26.2 Scope

This procedure applies to all vitrified embryo warming (for fresh or frozen-thaw embryo transfer), vitrified oocyte warming (for insemination after fertility preservation or from a donor oocyte program) and any other warming of cryopreserved reproductive specimens stored by vitrification.

26.3 Responsibility

Only trained and authorized personnel may perform warming procedures. The embryologist performing the warm is responsible for confirming identity, retrieving the correct cryodevice, executing the warming sequence, assessing post-warm survival and viability, and documenting the result.

26.4 Preparation

Before warming:

• Review the clinical plan and confirm which cryodevice(s) are designated for warming. Identify the patient, cycle type and planned procedure (e.g., fresh cycle blastocyst transfer, donor oocyte insemination).
• Confirm that the cryostorage inventory identifies the correct cryodevice(s). Verify tank, cane, goblet and slot.
• Confirm witness according to laboratory policy before retrieval from storage.
• Prepare all warming solutions according to the manufacturer's instructions and the laboratory's validated warming protocol. Warming solutions typically consist of a thawing solution (high sucrose concentration to drive osmotic re-expansion), a dilution solution and a washing solution. Confirm solutions are within expiration and at the appropriate temperature.
• Prepare and label the post-warming culture dish or insemination dish with approved patient identifiers.
• Confirm that the incubator, warming stage or holding block is within the acceptable temperature range.

26.5 Retrieval of Cryodevice

• Wear appropriate PPE (insulated cryogloves, safety glasses, etc.) before approaching the liquid nitrogen tank.
• Retrieve the assigned cane or goblet from the storage tank, working quickly to minimize ambient warming.
• Identify the correct cryodevice by its label. Confirm identity with the witness before removing from the goblet or cane.
• Keep the cryodevice submerged in liquid nitrogen or in the vapor phase until ready to begin the warming step. Do not allow the device to warm prematurely.

26.6 Embryo Warming Procedure

The warming procedure must be performed exactly according to the manufacturer's validated protocol for the vitrification system in use. The sequence below represents the general approach for most commercial vitrification systems; the laboratory's specific solution names, volumes, temperatures and timing shall govern:

• Plunge the cryodevice directly into the pre-warmed thawing solution (typically at 37°C) for the time specified by the protocol (commonly 1 minute).
• Transfer the embryo(s) into the dilution solution for the next timed interval. The cryoprotectant concentration continues to fall.
• Transfer through one or two wash solutions of decreasing sucrose concentration to complete cryoprotectant removal and osmotic re-equilibration.
• Transfer the embryo(s) to the labeled post-warming culture dish containing equilibrated culture medium.
• Place the culture dish promptly into the incubator. Allow the embryo(s) to recover for the time defined by laboratory policy before performing the post-warm survival assessment.

26.7 Oocyte Warming Procedure

Oocyte warming follows the same general solution sequence as embryo warming using the validated oocyte vitrification system:

• Plunge the cryodevice into the thawing solution at 37°C. Gently pipette to assist oocyte release from the device if required.
• Transfer through the dilution and wash solutions according to the validated protocol.
• Transfer the oocyte(s) to the labeled post-warming dish and place in the incubator.
• Allow a recovery period before ICSI. A minimum recovery interval of 1–2 hours post-warming is commonly used before insemination, though the exact interval shall be determined by the laboratory's validation and the clinical plan.
• Assess maturity and survival before proceeding to ICSI. Confirm the presence of the first polar body and intact zona. Degenerated or lysed oocytes shall not be inseminated.

26.8 Post-Warm Assessment

For embryos:

• Assess each embryo for survival after the recovery period. Survival criteria are stage-dependent: a blastocyst that re-expands or shows trophectoderm activity is considered viable; a collapsed blastocyst with no re-expansion after an adequate recovery period is considered degenerated.
• For Day 3 warmed embryos, assess cell number, fragmentation and evidence of continued development.
• Document the number of embryos warmed and the number surviving.

For oocytes:

• Assess for intact zona, clear cytoplasm and absence of degeneration signs (dark cytoplasm, fragmentation, lysis).
• Document the number of oocytes warmed and the number surviving.
• If survival is unexpectedly low (e.g., <70%), notify laboratory leadership and consider reviewing the cryopreservation and warming records for the case.

26.9 Communication

Communicate the post-warm survival result to the clinical team promptly. If the number of viable embryos or oocytes is fewer than expected, the physician should be notified so that the clinical plan can be adjusted as needed (e.g., warming an additional cryodevice, adjusting the transfer plan).

26.10 Documentation

Document the following:

• Patient identifiers.
• Date and time of warming.
• Cryodevice identifier(s) warmed.
• Number and stage of embryos or number of oocytes warmed.
• Post-warm survival (number surviving and any degenerated).
• Operator identity and witness identity.
• Any deviation or unusual observation (e.g., cryodevice damage, premature warming, failed re-expansion after recovery period).

26.11 Teaching Point

Operational note: The warming sequence is as important as the vitrification sequence. Moving too slowly through the thawing solution, skipping wash steps or failing to keep solutions at the correct temperature can cause ice recrystallization or osmotic injury. Post-warm survival should be communicated quickly so the clinical team can respond.

27.1 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.

27.2 Scope

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

27.3 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.

27.4 Preparation

Before the transfer:

• Review the transfer paperwork and embryo inventory.
• Confirm the transfer plan, including the number and stage of embryos to be transferred and whether genetic results or other selection criteria apply.
• Verify the patient name and date of birth with the patient and clinical team.
• Retrieve the correct embryo transfer dish from the incubator. Confirm dish identity against all transfer documents with a witness according to policy. Keep the embryo transfer workstation at conditions similar to the incubator environment.
• Prepare the catheter, syringe and required sterile materials. Prime the catheter with culture medium and remove air bubbles according to the laboratory method.
• Confirm the physician is ready before loading the catheter.
• Keep embryo exposure outside the incubator as short as practical during the loading process.
• Capture an image of the embryo or embryos if this is the laboratory standard.

27.5 Embryo Selection

• Identify the embryo or embryos designated for transfer.
• Confirm that the selected embryos match the transfer plan and the record. Use witness verification.
• Review embryo stage, quality and day of development before loading. High-quality embryos are preferred for single embryo transfer.
• Move the selected embryo or embryos into the transfer droplet using approved handling technique.

27.6 Catheter Loading

• Prepare the catheter using transfer medium and remove air bubbles according to the laboratory method.
• 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/bubbles to help visualize the transfer column.
• Load the embryo or embryos into the catheter using the loading volume in current use. Ensure the embryo is positioned in the correct part of the column. Sometimes an air bubble is created at the tip of the catheter to create an echogenic marker; alternatively, echogenic embryo transfer catheters can be used.
• Inspect the loaded catheter before handing it to the physician. Confirm identity and that the catheter is not kinked or leaking.
• Hand the catheter to the physician using sterile technique and without unnecessary delay.

27.7 What to Watch During the Transfer

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.

27.8 Post-Transfer Check

• Receive the catheter immediately after transfer.
• Examine the catheter under the microscope according to laboratory policy.
• 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.

27.9 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.

28.1 Purpose

To define the procedures for receipt, verification, handling and tracking of donor sperm and donor oocytes, including the additional identity control, consent and regulatory requirements that apply to the use of third-party gametes.

28.2 Scope

This section applies to all cases involving the use of donor sperm (from a licensed sperm bank or directed donor) and donor oocytes (from a recruited egg donor, egg bank or shared-donor program). It supplements the relevant sections on sperm preparation, oocyte handling and cryopreservation, which continue to apply.

28.3 Regulatory Requirements

Donor gametes are regulated as human cells, tissues and cellular and tissue-based products (HCT/Ps) under FDA 21 CFR Part 1271. The laboratory shall ensure that:

• Anonymous donor sperm and oocytes used in the laboratory have been sourced from FDA-registered tissue establishments that have performed donor eligibility determination, including required infectious disease testing, in accordance with current good tissue practice (cGTP).
• Documentation of donor eligibility (or documented ineligibility with a physician-authorized exception) shall be on file before donor gametes are used clinically.
• Any adverse reaction associated with a donor gamete shall be reported to the supplying tissue establishment and to FDA as required.
• Directed donation cases (known donors) shall follow the laboratory's directed donor protocol, which may involve different testing timelines as permitted by regulation and physician order.

28.4 Consent Verification

Before any donor gamete procedure is performed:

• Confirm that written informed consent has been obtained from the recipient(s) for use of donor gametes.
• For egg donation cycles, confirm that the egg donor's consent for oocyte retrieval and donation is on file.
• Confirm that the identity of donor gametes and recipient patient(s) are correctly matched in the laboratory record and that the chain of consent is documented.

28.5 Donor Sperm Receipt and Verification

• Confirm that the donor sperm shipment is accompanied by the required documentation, including donor identification code, lot number, vial count, infectious disease testing results or certification of donor eligibility, and storage and handling instructions.
• Inspect the shipping container for integrity and temperature compliance. Record the condition of the shipment on receipt.
• Confirm the number of vials received against the packing list. Document any discrepancy.
• Assign the donor sperm to the correct patient record using the clinic's matching system. Never use donor identifiers that could reveal donor identity to patients unless an identity-release arrangement is in place and documented.
• Store the donor sperm vials in the designated cryostorage location and record the storage location in the inventory.
• Confirm that the vials are labeled with a coded donor identifier that can be traced to the tissue establishment's records, but that does not identify the donor to the patient without authorization.

28.6 Donor Oocyte Receipt and Verification (from Egg Bank)

• Confirm that the donor oocyte shipment is accompanied by required documentation: donor identification code, oocyte count, infectious disease testing results or certification of donor eligibility, lot or cohort information, and shipping vessel validation data.
• Inspect the dry shipper for temperature compliance. A shipping vessel that has lost cryogenic temperature shall not be opened until the tissue establishment has been notified and an assessment is made.
• Confirm the number of oocytes received against the packing list. Document any discrepancy immediately.
• Assign oocytes to the correct recipient patient record. Confirm that donor identification is traceable but not revealing of donor identity unless authorized.
• If oocytes are to be stored temporarily before warming, transfer to the designated cryostorage location and record the storage assignment.

28.7 Fresh Egg Donation Cycle Coordination

For fresh or shared egg donation cycles involving an in-house or recruited donor:

• Confirm that the donor's retrieval plan has been coordinated with recipient cycle timing.
• Verify that donor infectious disease testing and eligibility documentation are complete before the donor undergoes retrieval.
• Prepare recipient-labeled dishes and worksheets before retrieval. The donor identity and recipient identity must be clearly separated in all records. Oocytes from one donor may not be mixed with oocytes from another donor at any step.
• If oocytes from a single retrieval are being allocated to multiple recipients, define the allocation plan before retrieval begins and confirm the allocation with the clinical team and laboratory leadership. Each recipient's allocated oocytes must be handled and tracked independently.
• Confirm witness verification at each step where donor oocytes are associated with recipient labels.

28.8 Donor Sperm Preparation

Donor sperm preparation follows the same procedure as described in Sections 11 and 12 for ejaculated or frozen sperm, with the following additional requirements:

• Confirm the donor vial code and the recipient patient record match the clinical order before removing the vial from storage.
• Confirm witness before opening or thawing the donor vial.
• Document the donor identification code on the laboratory worksheet and all relevant records. The donor code shall be traceable to the tissue establishment's records but is not the donor's personal identity.
• Any unused portion of a donor vial shall be handled according to laboratory policy and the tissue establishment's instructions. Refreezing may not be appropriate for all products; confirm before doing so.

28.9 Adverse Event Reporting

If a recipient or offspring experiences an adverse event that may be related to a donor gamete (e.g., infectious disease transmission, genetic disease attributed to the donor), the laboratory shall notify the supplying tissue establishment and comply with applicable FDA adverse event reporting requirements. All documentation related to the donor lot and recipient outcome shall be preserved.

28.10 Documentation

Document the following for all donor gamete cases:

• Recipient patient identifiers.
• Donor identification code (coded, not personal identity) and tissue establishment name.
• Donor eligibility documentation on file (lot number or certification reference).
• Number of donor sperm vials or donor oocytes received, used and stored.
• Witness identity at key steps.
• Any deviation, discrepancy or adverse finding.

28.11 Teaching Point

Operational note: Donor gamete cases add a second layer of identity that must be tracked simultaneously: the donor identity (coded) and the recipient identity. A mix-up at any step could result in a recipient using an unintended donor's gametes, which has profound legal, ethical and medical consequences. The witness process is especially critical in donor cases.

29.1 Purpose

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

29.2 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.

29.3 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.

29.4 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.

29.5 Daily Quality Control Principles

Before patient-use procedures begin each day:

• Confirm that each required piece of equipment is powered on or in normal operating state.
• Check displays, alarm indicators and environmental readings where applicable.
• Record required temperature, gas or status values in the QC log.
• Review results for acceptability before using the equipment for patient specimens.
• If a result is abnormal, investigate before proceeding. Do not place specimens into equipment with unknown or out-of-range conditions.

29.6 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 a minimum of three times per week or continuously monitored with level probes. Thermographic monitoring can be used as an additional highly sensitive method to observe for vacuum failure.

29.7 Out-of-Range Results or Equipment Failure

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

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

29.8 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.

29.9 Teaching Point

Operational note: 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.

30.1 Purpose

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

30.2 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.

30.3 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.

30.4 Procedure

• Use the designated thermometer, display, monitoring system or validated measurement device for each unit.
• Read and record the temperature carefully.
• Compare the observed value with the acceptable range assigned to that equipment.
• Initial or sign the temperature log.
• If the temperature is outside range, repeat the check and confirm whether the result is real.
• If the result remains abnormal, follow the out-of-range procedure.

30.5 Out-of-Range Response

If temperature is outside acceptable range:

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

31.1 Purpose

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

31.2 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.

31.3 Daily Procedure

• Review the incubator display for temperature and gas values.
• Record the displayed values on the incubator QC log.
• Verify gas concentration using the approved digital gas analyzer according to the established schedule. When an independent temperature verification device is used, record that value as well.
• Compare all readings with the acceptable range assigned to that incubator.
• Confirm that alarms are active and not in fault status when applicable.

31.4 If the Incubator Is Out of Range

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

32.1 Purpose

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

32.2 Scope

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

32.3 Preparation

Before use:

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

32.4 Procedure

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

32.5 Abnormal Results

If the gas reading is outside acceptable range:

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

33.1 Purpose

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

33.2 Scope

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

33.3 Procedure

• Identify the thermometer or probe to be verified.
• Compare it against the laboratory's traceable reference standard using the approved calibration setup.
• Record the observed value of the device and the reference value.
• 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.
• Document the date, result and initials of the person performing the verification.

33.4 Frequency

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

34.1 Purpose

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

34.2 Preparation

Before use:

• Confirm that the meter is clean and functioning.
• Confirm that the probe is properly stored and not damaged.
• Assemble the required calibration buffers as recommended.
• Allow buffers and samples to reach the appropriate temperature if required by the laboratory method.

34.3 Calibration Procedure

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

34.4 Sample Measurement

• Rinse the probe after calibration.
• Place the probe into the sample to be measured.
• Allow the reading to stabilize.
• Record the pH value. Rinse the probe after the measurement and return it to storage solution as required.

34.5 If Calibration Fails

If the pH meter does not calibrate properly:

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

35.1 Purpose

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

35.2 Receipt of Materials

When supplies are received:

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

35.3 Storage and Rotation

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

35.4 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.

36.1 Purpose

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

36.2 General Procedure

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

36.3 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.

37.1 Purpose

To define the core chemical safety requirements for the laboratory.

37.2 General Requirements

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

37.3 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.

38.1 Purpose

To define requirements for monitoring and maintaining air quality in the IVF laboratory, with particular attention to volatile organic compounds (VOCs) and other airborne contaminants that can adversely affect gamete and embryo viability.

38.2 Background

The IVF laboratory environment must be protected from airborne contaminants including:

• Volatile organic compounds (VOCs) – emitted from cleaning agents, plastics, adhesives, floor coatings, furniture, construction materials and solvents. VOC exposure has been associated with reduced fertilization rates, impaired embryo development and reduced implantation rates.
• Particulate matter – dust, skin cells, aerosols and other airborne particles that may contaminate open culture systems.
• Microbial contamination – bacteria, fungi and endotoxins that may be present in poorly maintained HVAC systems or introduced by traffic.
• CO and CO₂ – combustion byproducts that can alter pH in open systems.

ESHRE and ASRM guidance emphasizes that the laboratory should maintain clean air conditions appropriate for an embryology laboratory, and that HEPA filtration combined with activated carbon filtration is the current standard for VOC removal in IVF laboratory HVAC systems.

38.3 Facility and HVAC Requirements

• The IVF laboratory HVAC system shall provide HEPA-filtered, positively pressurized supply air to maintain a clean environment relative to adjacent corridors and support areas.
• The HVAC system shall incorporate activated carbon or equivalent filtration to reduce VOC levels in the laboratory.
• The laboratory shall be under positive air pressure relative to non-laboratory areas to minimize inflow of contaminants from outside the laboratory.
• Temperature and humidity shall be maintained within the ranges defined by the laboratory and appropriate for embryo culture conditions.
• HVAC filters (HEPA and activated carbon) shall be replaced at the intervals defined by the facility management plan and manufacturer recommendations. Replacement dates shall be documented.

38.4 VOC Monitoring

The laboratory shall monitor total VOC (TVOC) levels at defined intervals. Monitoring may be performed by:

• Continuous electronic VOC sensors placed within the laboratory, particularly near laminar flow hoods and incubator areas.
• Periodic sampling using calibrated analytical methods (e.g., photoionization detector, gas chromatography) to measure TVOC concentrations.
• Post-construction or post-renovation air quality assessment before patient specimens are handled in the affected area.

Acceptable TVOC levels shall be defined by the laboratory in accordance with current guidance. As a general reference, many IVF laboratories target TVOC levels below 0.5 mg/m³, though the laboratory's validated acceptable range shall govern.

38.5 Sources of Contamination to Minimize

Staff and laboratory management shall take the following precautions:

• Avoid bringing scented products (perfumes, scented lotions, scented hand sanitizers) into the laboratory.
• Use only cleaning and disinfection agents validated for use in the IVF laboratory. Avoid solvent-based cleaning products, bleach-containing compounds (unless specifically validated) and highly volatile agents in areas where specimens are handled.
• Allow adequate off-gassing time for new materials (equipment, plastics, gloves, culture materials) before introducing them into the laboratory. Follow manufacturer guidance and the laboratory's validation policy.
• Avoid construction, renovation or installation work adjacent to the laboratory during active patient cycles whenever possible.
• Restrict traffic in the laboratory to authorized personnel.

38.6 Laminar Flow Hood Filter Maintenance

HEPA filters within laminar flow hoods shall be certified at least annually by a qualified certifier in accordance with NSF/ANSI 49 standards. Certification records shall be maintained. Any laminar flow hood that fails certification shall be removed from patient use until repairs and re-certification are complete.

38.7 Post-Construction and Post-Renovation Procedures

Following any construction, renovation or major equipment installation adjacent to or within the laboratory:

• The laboratory shall not be used for patient specimen handling until air quality has been assessed and VOC levels confirmed within acceptable range.
• Off-gassing time shall be provided for new materials and surfaces, typically a minimum of 24–72 hours of continuous HVAC operation with fresh air exchange, or as determined by the air quality assessment result.
• A written record of the air quality assessment and clearance for use shall be maintained.

38.8 Documentation

Document the following:

• TVOC monitoring results, including date, location and measured value.
• HEPA and activated carbon filter replacement dates and records.
• Laminar flow hood certification dates and results.
• Any deviation, corrective action or investigation related to air quality.
• Post-construction or post-renovation air quality clearance records.

38.9 Teaching Point

Operational note: Air quality is invisible but its effects on embryo culture are measurable. A laboratory that maintains low VOC levels, positive pressure, regular filter maintenance and rigorous staff compliance with fragrance-free and chemical-use policies creates a measurably better environment for embryo development.

39.1 Purpose

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

39.2 Start-of-Day Check

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

39.3 Use During the Day

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

39.4 Cleaning and Maintenance

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

40.1 Purpose

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

40.2 Start-Up Procedure

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

40.3 Use During Procedures

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

40.4 Shutdown and Maintenance

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

41.1 Purpose

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

41.2 Routine Monitoring

• Check tank liquid nitrogen 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.
• Record the value or status in the tank log.
• Confirm alarm status if the tank is alarmed. Alarm systems should be tested regularly.
• Inspect the tank exterior for frost pattern changes, physical damage or other abnormal conditions.
• 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.

41.3 Filling Procedure

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

41.4 Abnormal Tank Conditions

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

• Notify laboratory leadership immediately.
• Assess whether specimens need transfer to an alternate tank. Prepare an emergency transfer plan.
• Always have an empty dewar or tank available to move cryopreserved materials to in the case of vacuum failure.
• Document the event and action taken. Consider contacting the tank manufacturer or facility maintenance for inspection.

42.1 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.

42.2 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 to detect dangerous O₂ depletion.

42.3 Internal Movement of Specimens

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

42.4 Transport to or from Offsite Storage

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

42.5 Tank Monitoring

All cryostorage tanks shall be monitored using the laboratory’s approved manual and/or electronic monitoring systems. The purpose of monitoring is to detect level loss, vacuum failure, abnormal temperature change, or other signs of tank malfunction before specimen safety is compromised.

Monitoring shall include, as applicable:

• Scheduled manual level or weight checks
• Continuous electronic monitoring and alarm notification
• Inspection for unusual frost patterns, external cooling, or physical damage
• Confirmation that remote alerts and call pathways are functioning properly

Any abnormal result or alarm condition shall be treated as time-sensitive. Laboratory personnel shall immediately assess the tank, determine whether specimens are at risk, initiate transfer to a validated alternate tank if required, and fully document the event and corrective action.

42.6 Teaching Point

Cryostorage safety depends on disciplined monitoring, not assumption. Most serious storage failures are not caused by one dramatic event, but by missed warning signs, delayed response, or incomplete documentation. Staff should understand that level checks, alarm review, visual inspection, and immediate escalation are all part of specimen protection.

43.1 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.

43.2 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.

43.3 Critical Witness Points

Witnessing shall occur, as applicable, before or during:

• Sperm preparation release for use.
• Oocyte retrieval.
• 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.
• Donor gamete association with recipient record.

43.4 Witness Procedure

• The operator presents the labeled materials and record.
• 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.
• The witness confirms the match verbally or through the approved system.
• The witness documents the check according to laboratory policy (initials, date and time or electronic signature).
• If there is any discrepancy, the procedure stops until the issue is resolved.
• The operator does not proceed until the witness step is complete.

43.5 Teaching Point

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

44.1 Purpose

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

44.2 Initial Training

Before independent work, personnel shall complete:

• Laboratory safety orientation.
• Biohazard training.
• Chemical hygiene training.
• Occupational safety training required by the institution.
• Review of the procedure manual.
• Supervised observation of assigned procedures.
• Supervised hands-on practice for assigned procedures.
• 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, cryostorage management and air quality requirements.

44.3 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.

44.4 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.

44.5 Teaching Point

Operational note: 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.

45.1 Purpose

To define the controlled forms required for clinical documentation, specimen tracking and regulatory compliance in the IVF laboratory.

45.2 Required Forms

The laboratory shall maintain controlled forms for each of the following areas. All forms shall be current, legible and completed in full. Electronic forms shall maintain audit trails, version control and support secure electronic signatures.

45.2.1 Patient and Cycle Documentation

• Patient demographic and cycle information form.
• Laboratory cycle checklist (confirming consents, orders and preparation steps before each case).
• Cryopreservation consent form – specifying disposition options for embryos, oocytes and sperm (must address future use, donation, research or discard, and disposition in the event of death, separation or abandonment).
• Informed consent acknowledgment for each procedure (retrieval, ICSI, biopsy, transfer).

45.2.2 Andrology Records

• Semen receipt log.
• Semen analysis worksheet.
• Sperm preparation worksheet (IVF and IUI).
• Sperm cryopreservation and post-thaw assessment record.

45.2.3 Embryology Records

• Oocyte retrieval worksheet (oocyte count, COC morphology observations).
• Insemination/ICSI record (method, timing, sperm dose, oocyte count, witness).
• Fertilization assessment record (PN status for each oocyte).
• Embryo culture and development record (daily assessments, grading).
• Assisted hatching record.
• Embryo biopsy record (embryo designation, tube number, cell count, genetic lab destination, witness).
• Embryo transfer record (embryo selection, catheter loading, post-transfer check, witness).

45.2.4 Cryostorage Records

• Embryo vitrification record (device label, storage location, witness).
• Oocyte vitrification record (device label, maturity status, storage location, witness).
• Embryo/oocyte warming record (cryodevice identifier, post-warm survival, witness).
• Sperm cryopreservation record (vials frozen, storage location, witness).
• Cryostorage inventory (all patients, all specimens, tank-cane-goblet-slot location, current status).
• Specimen transfer or disposition record.

45.2.5 Donor Gamete Records

• Donor sperm receipt form (donor identification code, tissue establishment, eligibility documentation reference, vial count).
• Donor oocyte receipt form (donor identification code, tissue establishment, eligibility documentation reference, oocyte count).
• Donor-to-recipient allocation record (confirming match of donor gametes to recipient cycle).

45.2.6 Regulatory and HCT/P Records

• Donor eligibility determination record (or certificate of eligibility from supplying tissue establishment) for all donor gamete cases.
• Adverse event report form (for reportable adverse reactions related to HCT/Ps).

45.3 Document Management

Controlled forms shall be version-controlled. Superseded form versions shall be removed from active use when a new version is approved. The date of form approval and version number shall be visible on the form. Electronic forms shall support version tracking and prevent use of outdated versions.

46.1 Purpose

To define the controlled QC logs and monitoring records required for daily laboratory operations, equipment management and regulatory compliance.

46.2 Required QC Logs

The laboratory shall maintain active, controlled logs for the following:

46.2.1 Daily Environmental Monitoring

• Incubator temperature log (each incubator, each day, including display value and independent thermometer reading when applicable).
• Incubator CO₂ and O₂ gas log (each incubator, verified by digital gas analyzer at defined frequency).
• Laminar flow hood or clean bench temperature log (when heated surfaces are used for embryo handling).
• Room temperature and humidity log (when applicable to the laboratory environment standard).

46.2.2 Calibration and Verification Logs

• Thermometer calibration log (date, device identifier, reference standard value, device reading, pass/fail, initials).
• pH meter calibration log (date, buffer values used, calibration result, initials).
• Digital gas analyzer calibration and maintenance log.

46.2.3 Equipment Maintenance Logs

• Incubator cleaning and maintenance log (date, tasks performed, technician identity).
• Laminar flow hood certification log (date of certification, certifier name, HEPA test result, pass/fail).
• Laminar flow hood daily use and surface disinfection log.
• Microscope and micromanipulator maintenance log.
• Autoclave or dry heat sterilizer log (cycle date, cycle parameters, load contents, result).

46.2.4 Cryostorage Monitoring Logs

• LN₂ tank level log (each tank, at least three times per week or continuous monitoring record).
• LN₂ fill log (date, tank identifier, level before and after fill, operator initials).
• Tank alarm test log (alarm type tested, result, initials).
• Cryostorage inventory log (see Section 45 for inventory form requirements).

46.2.5 Reagent and Media Traceability Logs

• Media and reagent receipt log (item name, manufacturer, lot number, expiration date, receipt date, storage location).
• Media in-use log (lot number, date opened, dish preparation records linking to case records).
• Recall log (records of manufacturer alerts, lot-specific actions taken).

46.2.6 Air Quality Log (see Section 38)

• TVOC monitoring log (date, location, measured value, acceptable range, initials).
• Filter replacement log (HVAC HEPA and activated carbon, date replaced, initials).

46.3 Log Review

QC logs shall be reviewed by the laboratory director or designee at defined intervals (typically monthly at a minimum) to identify trends, deviations and corrective actions. Evidence of review shall be documented.

46.4 Completion Standard

Entries shall be made at the time the activity is performed or immediately thereafter. Blank spaces shall not appear in required fields; if no entry is applicable, the field shall be marked per laboratory policy. Logs shall not be altered retroactively without a documented correction.

47.1 Exposure Control

The laboratory shall maintain a written exposure control policy addressing bloodborne pathogens, personal protective equipment (PPE), post-exposure response, spill response and biohazardous waste handling. The policy shall comply with OSHA Bloodborne Pathogen Standard (29 CFR 1910.1030). Staff shall receive annual bloodborne pathogen training. Staff shall know how to access and use spill kits and eyewash stations. Post-exposure response procedures (including chain of notification, medical evaluation and incident reporting) shall be documented and reviewed regularly.

47.2 OSHA and Safety Compliance

The laboratory shall comply with applicable occupational safety requirements including staff training, hazard communication (HazCom/GHS), accident prevention and incident reporting. A written Chemical Hygiene Plan shall be maintained as required by the OSHA Laboratory Standard (29 CFR 1910.1450) and shall be reviewed annually.

47.3 Latex Allergy

The laboratory shall maintain practices to reduce latex exposure for patients and staff with known or suspected sensitivity. Latex-free gloves and equipment shall be used when sensitivity has been identified or when laboratory policy requires latex-free practice. Staff shall be aware of the signs of latex reaction and the procedures for responding to an allergic event.

47.4 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. Upon receipt of a recall or alert:

• The laboratory director or designee shall review the recall and determine whether affected materials or equipment are in use.
• Affected materials shall be quarantined and, if clinically feasible, removed from patient use.
• Patients whose care may have been affected shall be identified and the clinical team notified.
• The recall shall be documented, including the action taken and outcome.
• FDA MedWatch reports shall be filed when applicable.

47.5 Adverse Events

The laboratory shall maintain documented procedures for reporting, investigating and managing adverse laboratory events and adverse patient reactions. This includes:

• Internal adverse event reporting: any laboratory event that causes patient harm, near-miss events, specimen misidentification events, equipment failures affecting specimens and medication or reagent errors shall be reported through the laboratory's internal incident reporting system.
• External adverse event reporting: reportable events under applicable regulations (e.g., FDA MedWatch for HCT/P-related reactions, state laboratory reporting requirements) shall be reported within required timeframes.
• Root cause analysis: significant adverse events shall undergo root cause analysis to identify contributing factors and implement corrective actions.
• Corrective action and follow-up: corrective actions shall be documented, implemented and reviewed for effectiveness.

47.6 Deviation and Corrective Action Policy

Any deviation from a controlled procedure shall be documented at the time it occurs. A deviation report shall include a description of what occurred, the potential impact on specimens or patient care, immediate corrective action taken, root cause assessment and preventive measures. Deviations shall be reviewed by the laboratory director and, where required, by the quality committee.

47.7 Cryostorage Security and Access Policy

Access to cryostorage areas shall be restricted to authorized personnel. Cryostorage tanks shall be in a monitored, alarmed, well-ventilated and secured area. No specimens shall be removed from cryostorage without a written order, confirmed identity check and witness documentation. Unauthorized access to cryostorage is a serious violation and shall be reported to the laboratory director immediately.

47.8 Patient Confidentiality and Privacy

All patient information, specimen identifiers and laboratory records shall be treated as confidential medical information. Access to patient records shall be limited to authorized personnel. Electronic records shall be protected by role-based access controls. Paper records shall be stored securely. Staff shall not discuss patient information in public areas or share it with unauthorized individuals. Compliance with HIPAA and applicable state privacy laws is required of all laboratory staff.

47.9 Informed Consent Policy

Informed consent shall be obtained by the clinical team before the laboratory performs any invasive or irreversible procedure. The laboratory shall not proceed with sperm retrieval, ICSI, biopsy, cryopreservation, disposal or use of donor gametes without confirmed documentation of appropriate consent. If consent documentation is missing or incomplete at the time of the scheduled procedure, the procedure shall be held and the clinical team notified. The laboratory shall maintain a record confirming that consent was reviewed before each procedure.

A.1 Values Linked to Published Guidance

Reference Standard 1. Embryo-culture incubation environment

ASRM states that a tri-gas mix of CO₂ 5%–7%, O₂ 5%, and N₂ 88%–90% best mimics physiologic conditions for growing preimplantation embryos. The same document notes that CO₂ adjustment is the key variable used to maintain pH, typically around 7.25–7.35, although no single optimal pH has been defined.

Source: https://www.asrm.org/practice-guidance/practice-committee-documents/comprehensive-guidance-for-human-embryology-andrology-and-endocrinology-laboratories-management-and-operations-a-committee-opinion-2022/

Reference Standard 2. Buffered handling media

ESHRE states that buffered media such as HEPES or MOPS should be kept in atmospheric air, whereas bicarbonate-buffered media should be kept in 5%–7% CO₂.

Source: https://www.eshre.eu/en/Guidelines-and-Legal/Guidelines/Oocyte-and-embryo-morphology-assessment

Reference Standard 3. Oocyte retrieval and early handling

ESHRE states that oocyte retrieval is particularly sensitive to temperature and pH and that appropriate equipment should maintain oocytes close to 37°C. Flushing media, collection tubes, and dishes should be pre-warmed.

Source: https://www.eshre.eu/Guidelines-and-Legal/Guidelines/Revised-guidelines-for-good-practice-in-IVF-laboratories-(2015).aspx

Reference Standard 4. Semen sample transport and early handling

ESHRE states that, after collection, semen samples should be delivered to the laboratory as soon as possible while avoiding extreme temperatures below 20°C and above 37°C, and that sperm analysis and preparation should start within 1 hour of collection.

Source: https://www.eshre.eu/Guidelines-and-Legal/Guidelines/Revised-guidelines-for-good-practice-in-IVF-laboratories-(2015).aspx

Reference Standard 5. Morphology assessment

The current ESHRE/ALPHA Istanbul Consensus update should be the laboratory's reference framework for oocyte, zygote, embryo, and blastocyst morphology terminology and embryo-ranking language.

Source: https://www.eshre.eu/en/Guidelines-and-Legal/Guidelines/Oocyte-and-embryo-morphology-assessment

A.2 Values That Should Usually Be Defined Locally in the Controlled Version

• Exact incubator temperature setpoint(s), acceptable tolerance, and alarm limits.
• Exact incubator CO₂ and, where applicable, O₂ setpoints, acceptable tolerance, and independent verification frequency.
• Laboratory-specific validated culture pH range for each media system in use.
• pH meter calibration acceptance criteria, calibration frequency, and corrective-action pathway.
• Heated-stage, warming-block, hood warming-surface, and transport-device setpoints and acceptable tolerances.
• Room temperature and humidity acceptable ranges for monitored areas.
• Refrigerator, freezer, and any -20°C or -80°C storage ranges and alarm thresholds.
• Cryostorage monitoring thresholds, fill schedules, response times, and alarm-escalation chain.
• Media equilibration times, handling times outside the incubator, and any local witness-language requirements.
• TVOC acceptable range for the laboratory environment.
• Sperm cryoprotectant medium formulation and validated freeze/thaw parameters.
• Post-thaw survival thresholds for embryos and oocytes that trigger clinical team notification.

C.1 Abbreviations

The following abbreviations are used throughout this manual:

2PN – Two pronuclei (normal fertilization pattern)

ART – Assisted reproductive technology

ASRM – American Society for Reproductive Medicine

CAP – College of American Pathologists

cGTP – Current good tissue practice

CLIA – Clinical Laboratory Improvement Amendments

CO₂ – Carbon dioxide

COC – Cumulus-oocyte complex

ESHRE – European Society of Human Reproduction and Embryology

ET – Embryo transfer

FDA – U.S. Food and Drug Administration

FET – Frozen embryo transfer

GV – Germinal vesicle (immature oocyte stage)

HCT/P – Human cells, tissues, and cellular and tissue-based products

HEPA – High-efficiency particulate air (filtration)

HIPAA – Health Insurance Portability and Accountability Act

HVAC – Heating, ventilation, and air conditioning

ICM – Inner cell mass

ICSI – Intracytoplasmic sperm injection

IUI – Intrauterine insemination

IVF – In vitro fertilization

LN₂ – Liquid nitrogen

MII – Metaphase II (mature oocyte stage)

MI – Metaphase I (immature oocyte stage)

MOPS – 3-(N-morpholino)propanesulfonic acid (a zwitterionic buffer)

NPB – Nucleolar precursor body

O₂ – Oxygen

OSHA – Occupational Safety and Health Administration

PB – Polar body

PGT – Preimplantation genetic testing

PN – Pronucleus / pronuclei

PPE – Personal protective equipment

PVP – Polyvinylpyrrolidone (used in ICSI for sperm immobilization)

QA – Quality assurance

QC – Quality control

SDS – Safety data sheet

TE – Trophectoderm

TMSC – Total motile sperm count

TVOC – Total volatile organic compounds

VOC – Volatile organic compound

C.2 Glossary of Terms

Blastocyst: A stage of embryo development (Day 5–7) characterized by an expanding fluid-filled cavity (blastocoele), an inner cell mass (ICM) and a surrounding layer of trophectoderm cells.

Blastomere: An individual cell within a cleavage-stage embryo.

Chain of custody: The documented, unbroken sequence of possession and handling of a specimen from receipt through final disposition.

Cleavage stage: Embryo development on Days 2–3 post-insemination, characterized by cell division without increase in overall size.

Compaction: The process by which blastomeres maximize cell-to-cell contact on Day 4, forming a morula in preparation for blastocyst development.

Controlled-rate freezer: A device that cools specimens at a defined programmed rate, used for slow-freeze cryopreservation of sperm and some embryos.

Cryodevice: Any device used to carry and store vitrified specimens in liquid nitrogen, including cryoloops, cryotops, Cryolock devices and sealed vitrification straws.

Cryoprotectant: A chemical agent that reduces ice crystal formation during freezing. Common cryoprotectants in IVF include DMSO, glycerol, ethylene glycol and propylene glycol.

Cumulus-oocyte complex (COC): The oocyte surrounded by its associated cumulus granulosa cells, as it appears at retrieval.

Denudation: The mechanical and enzymatic removal of cumulus and corona cells from an oocyte, typically performed before ICSI.

Donor eligibility determination: The regulatory process of evaluating a donor’s suitability based on screening and testing for communicable diseases, as required by FDA 21 CFR Part 1271.

Dry shipper: A cryogenic transport vessel that holds liquid nitrogen absorbed in a porous matrix, allowing safe transport of cryopreserved specimens without free liquid nitrogen.

Fragmentation: The presence of anucleate cytoplasmic fragments within a cleavage-stage embryo, associated with reduced developmental potential when severe.

Gardner grading: A widely used blastocyst grading system describing blastocyst expansion (grades 1–6) and separately grading the ICM and TE (grades A, B or C).

Germinal vesicle (GV): The nucleus of an immature oocyte at the prophase I stage, visible as a large vesicular structure. GV oocytes are not yet competent for fertilization.

Goblet: A container within a cryostorage cane that holds cryodevices for organized storage in a liquid nitrogen tank.

Hatching: The process by which a blastocyst escapes from the zona pellucida, required for implantation. Assisted hatching artificially creates an opening or thinning in the zona to facilitate this process.

Hyaluronidase: An enzyme used to digest the hyaluronic acid matrix of the cumulus complex, loosening it to facilitate mechanical denudation.

Inner cell mass (ICM): The cluster of cells within the blastocyst that will form the fetus and associated structures. Protected during TE biopsy.

Intracytoplasmic sperm injection (ICSI): A fertilization technique in which a single sperm is injected directly into the oocyte cytoplasm using micromanipulation.

Liquefaction: The process by which semen transitions from a gel to a liquid state after ejaculation, typically within 15–60 minutes.

Metaphase II (MII): The mature stage of an oocyte, arrested at the second meiotic division. The first polar body has been extruded and the oocyte is competent for fertilization.

Morula: An embryo stage on Day 4 post-insemination, characterized by compaction of blastomeres into a solid cell mass.

Multinucleation: The presence of two or more nuclei within a single blastomere, associated with chromosomal abnormalities and poor developmental potential.

Nucleolar precursor body (NPB): Spherical structures visible in the pronuclei of a zygote; their number, size and symmetry can be used in zygote scoring.

Oolemma: The plasma membrane of the oocyte.

Perivitelline space: The space between the oocyte plasma membrane (oolemma) and the inner surface of the zona pellucida.

Polar body: A small cell extruded from the oocyte during meiosis. The first polar body is extruded at ovulation; the second is extruded at fertilization.

Polyvinylpyrrolidone (PVP): A viscous medium used during ICSI to slow sperm movement and assist in sperm immobilization.

Pronucleus (PN): A membrane-bound structure containing the genetic material from one gamete, present in the zygote shortly after fertilization. Normal fertilization is indicated by two pronuclei (2PN).

Trophectoderm (TE): The outer layer of cells in a blastocyst that will form the placenta and extraembryonic membranes. The cells sampled during TE biopsy.

Vitrification: An ultra-rapid cryopreservation method in which high concentrations of cryoprotectants and extremely rapid cooling rates produce a glass-like solid state, avoiding ice crystal formation.

Witness: A qualified second staff member, or an approved electronic system, who independently verifies specimen identity at defined critical checkpoints.

Zona pellucida: The glycoprotein shell surrounding the oocyte and early embryo, which is penetrated during fertilization (ICSI) and must be escaped during implantation.

Zygote: A fertilized oocyte from the time of fertilization until the first cleavage division; typically assessed for pronuclear status on Day 1.