Chapter 9 – Laboratory Procedures for Assisted Reproduction




Abstract




Successful assisted reproduction treatment is critically dependent on consistent laboratory performance. Each laboratory process, from collection of oocytes and preparation of sperm for use in fertilisation in vitro, through embryo culture, assessment, selection for transfer, biopsy for genetic testing, to the storage (cryopreservation) of gametes and embryos for use in later treatment, carries an inherent risk of damage, whether mechanical or through exposure to suboptimal conditions outside the body, with consequences for the chance of successful clinical outcome. Training, competency in specific, unique technical skills and consistent performance of all laboratory practitioners are vital in the ART laboratory. Procedures must be carried out meticulously, adhering to standard operating procedures, with precise attention to detail. Strict adherence to guidelines issued by regulatory and professional bodies is necessary and essential in order to minimise risk and maximise performance. The implementation of a Quality Management System ensures consistent, optimised performance, and facilitates risk assessment and root cause analyses.





Chapter 9 Laboratory Procedures for Assisted Reproduction



Virginia N. Bolton



9.1 Background


From the time of ovulation and fertilisation until the developing embryo reaches the uterus and implants some days later, the mammalian embryo is a free-floating entity within the female genital tract. It is this unique feature that has made the development of assisted reproduction therapies possible. The earliest studies, undertaken in the rabbit and mouse, led developmental biologists to develop culture conditions that allowed the successful fertilisation of eggs in vitro (literally ‘in glass’), the culture of developing embryos and finally embryo transfer, culminating in the first live birth following IVF and embryo transfer, in the mouse, in 1958.


The transposition of skills and techniques developed in the animal research laboratory to clinical application in the treatment of infertility has entailed decades of refinement of the early techniques for achieving successful fertilisation and embryo culture in vitro. The introduction of legislation and associated regulations in the United Kingdom (the Human Fertilisation & Embryology Act 1990; amended in 2008) and Europe (the EU Tissues and Cells Directive), guidelines from professional bodies [1, 2] and the drive to improve success rates have led to the development of relatively sophisticated laboratories and procedures that serve this sector today.



9.2 The Laboratory Environment


The impact of variables such as patient demographics, ovarian stimulation regimens and clinical oocyte recovery on gamete quality are outside the control of the laboratory practitioners. However, once the patient’s gametes have been passed to the laboratory, the primary objective is to maintain constant and consistent conditions, within prescribed acceptable ranges, in order to minimise any damage that may compromise their viability. A consistent, optimised environment in the laboratory, within individual workstations and critical items of equipment; the consistent performance of individual practitioners; and the selection of appropriate culture media, reagents, equipment and consumables will all have a critical bearing on treatment outcomes (Table 9.1).




Table 9.1 Factors affecting outcome in the assisted reproduction laboratory
































































Building/construction Non-toxic materials, glues, paints
Filtered lighting to block UV irradiation
Design Proximity to clinical areas
Ergonomically designed to maximise efficiency and minimise risk
Restricted access for designated staff
Storage for consumables
Disposal of waste
Adjacent cryostore



  • Air quality



  • (minimum Grade D; optimally Grade C or higher [1])

Positive pressure
HEPA filtered
Activated carbon filters to reduce volatile organic carbons (VOCs)
Critical equipment Laminar flow hoods (vertical laminar flow; Class II)
Refrigerators
Incubators
Warmed worksurfaces/test tube holders
Culture media Core components: Water
Salts (maintain osmolality)
Energy substrate
Buffer (pH 7.2–7.4)
Protein supplement
Gas phase and buffers in air: HEPES or MOPS buffer
In incubators: sodium bicarbonate buffer and 5%–6% CO2
Low O2 (5%) in incubators is recommended
Staff competency Training and assessment
Adherence to standard operating procedures (SOPs)
Attention to detail

Minimising variation in every element within the assisted reproduction laboratory is ensured through the implementation of a quality management system (QMS [3]). Hazards that must be controlled include exposure to infectious organisms and environmental toxins, to fluctuations in temperature and pH and to physical damage sustained during manipulation and micromanipulation.


Monitoring and regulation of environmental variables are achieved using an accurate, objective monitoring system with associated alarms, ensuring that critical equipment functions within prescribed ranges at all times. This includes the monitoring of temperature in all working areas (heated stages in laminar flow hoods; microscope stages), incubators, fridges and freezers and tanks of liquid nitrogen (Dewars) in which cryopreserved material is stored. In addition, mechanisms should be in place to monitor the levels of individual gases within incubators.



9.2.1 The Cryostore


The hazards associated with the use of liquid nitrogen mean that as well as compulsory safety training and equipment to protect staff, the tanks of liquid nitrogen (Dewars) used for storing cryopreserved embryos and gametes must be located separately from, but in close proximity to the assisted reproduction laboratories. Oxygen monitors, and associated audible and visible alarms, must be installed in the cryostore to alert staff both inside and outside the laboratory suite to potentially fatal low atmospheric oxygen. Appropriate flooring material, that can withstand cryodamage caused by spillages of liquid nitrogen, must be installed.



9.2.2 Witnessing


To prevent any mismatches of gametes or embryos, it must be possible to identify unambiguously every sample of gametes or embryos at all stages of the laboratory and treatment processes. Meticulous systems must be in place, so that each step involving the movement of gametes or embryos between containers (test tubes, petri dishes and cryopreservation holding devices) is witnessed. To minimise the risk of involuntary automaticity during witnessing, it is recommended that an objective, electronic witnessing system, such as bar coding or radio frequency identification (RFID), should be used for every step where it is physically possible.



9.3 Use of Sterile Working Areas Within the Laboratory


Laminar flow hoods must be used in the laboratory wherever a sterile working area is required. The type of hood used for different workstations in the laboratory depends upon the level of protection against microbial contamination required (Figure 9.1).





Figure 9.1 Diagrams illustrating air flow in vertical or horizontal laminar flow and Class II hoods. Laminar flow hoods provide protection for the product but not the operator. (a) Horizontal laminar flow. (b) Vertical laminar flow. Room air is drawn into the high-efficiency particulate air (HEPA) filter; 99.99% particle-free air is forced across the work surface. (c) Class II hoods provide protection for the product, the operator and the environment.


Class II cabinets provide protection for the operator and the environment as well as for the product. These hoods must be used for procedures when the operator is handling body fluids (follicular fluid, blood, semen) including




  • Oocyte retrieval



  • Surgical sperm collection



  • Semen analysis



  • Semen preparation



  • Semen cryopreservation and warming



  • Thawing cryopreserved semen/surgically collected sperm


Vertical or horizontal laminar flow hoods provide protection for the product and equipment, but not for the operator or the environment. These hoods are used for all procedures where containers (petri dishes/flasks/vials/test tubes) of culture medium and reagents that contain, or will be used to contain gametes and/or embryos are open to the atmosphere, including




  • Preparation of culture tubes and dishes



  • Decanting media and reagents



  • IVF insemination



  • All procedures when oocytes/embryos are moved between dishes



  • Denudation of oocytes



  • Intracytoplasmic sperm injection (ICSI)



  • Embryo biopsy



  • Cryopreservation of oocytes and embryos



  • Thawing/warming of oocytes and embryos



9.4 Use of Buffers in Culture Media


Dishes and test tubes of culture medium for use in gamete preparation and embryo culture are prepared using sterile techniques in laminar flow hoods and must be warmed to 37°C before use. Medium in culture dishes may be overlaid with pharmaceutical grade light mineral oil, to minimise temperature, osmolality and pH fluctuations in the medium, especially when dishes are exposed to the atmosphere during procedures outside the incubator.



9.4.1 Working in Air


When carrying out procedures outside the incubator, such as during oocyte retrieval and ICSI, if the procedure entails working at the microscope for any length of time, it is common to use media that are buffered with HEPES or MOPS, which maintain physiological pH in atmospheric levels of CO2.



9.4.2 Culture in Incubators


Incubators are maintained with an atmosphere of 5%–6% CO2 and culture media used for incubation are buffered with sodium bicarbonate. With bicarbonate-buffered culture medium, CO2 dissolves into the medium and reacts with water to form carbonic acid according to the Henderson–Hasselbalch equation (Figure 9.2). The concentration of sodium bicarbonate in the culture medium must be matched with the level of CO2 in the atmosphere in the incubator to achieve the appropriate, physiological pH.





Figure 9.2 The Henderson–Hasselbalch equation: pH of buffers.


Dishes and tubes of bicarbonate-buffered medium must be allowed to equilibrate in the incubator before use, usually overnight but at least for several hours, depending on the volumes of medium used, in an atmosphere of between 5% and 6% CO2 according to the concentration of sodium bicarbonate in the medium.



9.5 Oocyte Retrieval


Oocyte retrieval must be carried out in a Class II laminar flow hood, which provides protection not only to the environment and the product, but also for the operator working with body fluids. Aspirated follicular fluid is collected into test tubes held in a warmed, monitored heated block maintained at 37°C. Tubes of follicular fluid are passed from the operating theatre to the (ideally) adjacent laboratory, where the bloodstained fluid is tipped into a shallow petri dish on a surface warmed to 37°C and examined for the presence of oocyte–cumulus complexes (OCCs) (Figure 9.3).





Figure 9.3 Oocyte retrieval: visualisation of the oocyte–cumulus complex. (a) Oocyte retrieval: using a sterile Pasteur pipette, the follicular fluid is expelled into a petri dish on the warm stage of a stereomicroscope in a Class II laminar flow hood; the oocyte–cumulus complex can be seen with the naked eye in the blood-stained fluid. (b) The oocyte within the oocyte cumulus complex, as visualised using the stereomicroscope.


Working at a stereomicroscope with a warmed stage within the Class II laminar flow hood, using sterile technique, each OCC is aspirated gently using a polished Pasteur pipette and rinsed through petri dishes containing fresh, warmed culture medium before being placed into labelled dishes. Each dish is witnessed and uniquely identified as that of the patient undergoing the procedure. Dishes containing OCCS are placed into an incubator positioned adjacent to the workstation.


The pH of the culture medium used, of which numerous products are commercially available, is maintained using the appropriate buffer to maintain physiological pH in the working environment (Table 9.1).


Appropriate decontamination procedures must be carried out between each patient’s oocyte retrieval procedure.



9.6 Semen Analysis and Sperm Preparation for Insemination In Vitro


The sperm for insemination may be prepared from fresh or frozen (cryopreserved) semen, or from fresh or frozen (cryopreserved) surgically collected sperm samples. Samples must be processed in a Class II laminar flow hood, as for oocyte retrieval, to provide protection for the operator as well as for the environment and the sample [4].



9.6.1 Semen Analysis


Before preparation, samples are assessed for sperm concentration, motility and morphology [5]. Routine semen analysis should be carried out using phase contrast microscopy, and motility analysis using warmed microscope stages at 37°C. Ideally, bright field microscopy should be available for the examination of sperm morphology. Neubauer haemocytometers should be used for assessing sperm concentration (Figure 9.4). Alternative counting chambers are available (e.g. Makler) but are less accurate.





Figure 9.4 Use of the Neubauer counting chamber for sperm concentration assessment. (a) The Neubauer counting chamber. Note the Newton’s rings between the coverslip and the chamber; the appearance of the Newton’s rings confirms that the coverslip is attached appropriately, the chamber depth is fixed correctly, and the sperm concentration assessment will be accurate. (b) Diagrammatic representation of the side view of the counting chamber. (c) Diagrammatic representation of the counting grid of the chamber as visualised using the microscope. (d) Diagrammatic representation of a higher magnification of the counting grid with sperm suspension ready for concentration assessment.


Computer-assisted semen analysis (CASA) may be used to reduce inter-operator variation, but where this is not available, the subjective nature of most aspects of semen assessment mean that it is important to carry out internal quality assurance for all semen analysis practitioners within each laboratory, in conjunction with regular external quality assurance exercises, such as the UK National External Quality Assessment Service (NEQAS), both to assess performance and to minimise variation between and within laboratories.



9.6.2 Sperm Preparation


Sperm must be prepared before use in insemination in vitro, to remove the seminal fluid, non-sperm cells and contaminating micro-organisms from fresh and cryopreserved semen samples. Preparation will yield a concentrated suspension of sperm, in culture medium, with improved motility, and may enhance the concentration of those with normal morphology.


All procedures are carried out using sterile technique in a Class II laminar flow hood, and care is taken throughout preparation to protect sperm samples from extreme fluctuations in temperature and pH. Appropriate decontamination procedures must be carried out between processing samples from different patients. To avoid the risk of cross-contamination and mismatches between patients, laboratory staff should process one sample at a time, and at each step during preparation where the sample is transferred between containers, unique identifying labels on each matching container must be witnessed.


Sperm is most commonly prepared using either a buoyant density gradient centrifugation or ‘swim-up’ technique (Figure 9.5), although for severely oligozoospermic or cryptozoospermic samples (Table 9.2) where few motile sperm are present, other techniques may be used.




Table 9.2 WHO reference values for normal semen [5]












































Parameter WHO criteria Diagnosis if criteria not met
Volume ≥1.5 mL Aspermia (where there is no ejaculate); possible retrograde ejaculation
Appearance Grey and opaque
pH ≥7.2
Concentration (× 106/mL) ≥15 Oligozoospermia Cryptozoospermia (sperm only seen after centrifugation); azoospermia (no sperm present)
Motility (% progressive) ≥32 Asthenozoospermia Necrozoospermia (only dead sperm present)
Normal morphology (%) ≥4 Teratozoospermia Globozoospermia (round heads; no acrosome)
Leucocytes (× 106/mL) ≤1 Infection


9.6.2.1 Buoyant Density (Isopycnic) Centrifugation

This separation technique relies on the different buoyant densities of cells and debris in the sperm sample (semen; surgically collected testicular sperm; post-ejaculatory urine in cases of retrograde ejaculation). Separation according to buoyant density is achieved by centrifugation of the sample for 20 minutes at 300 g (1,500–1,600 rpm) on a discontinuous, two-layer gradient of colloidal silane-coated silica particles (usually 80%–90% and 45–55% suspensions in HEPES- or MOPS-buffered medium). Motile, morphologically normal spermatozoa become concentrated at the bottom of the gradient, and can be collected for use in insemination, leaving immotile, abnormal forms, cells and other contaminants elsewhere in the column.



9.6.2.2 Swim-Up

Up to 1 mL of warmed, equilibrated bicarbonate-buffered culture medium is carefully layered over the semen sample in a test tube. Alternatively, the semen sample may be first diluted 1:2 with warmed, equilibrated medium, and the suspension centrifuged for 10 minutes at 1,500 rpm. After discarding the supernatant, the pellet (20–50 µL) is gently resuspended and overlaid with 1 mL of fresh warmed, equilibrated culture medium. With both approaches, the test tube of sperm overlaid with culture medium is placed, inclined at an angle of 45°, in an incubator at 37°C in an atmosphere of 5% or 6% CO2 as appropriate for the culture medium used. After incubation for approximately 1 hour, the upper layer of culture medium is collected and transferred into a fresh tube.



9.6.2.3 Wash and Spin

In cases of severe oligozoospermia (<0.1 × 106/mL) and cryptozoospermia (Table 9.2) it may be appropriate simply to centrifuge the entire sample, diluted at least 1:2 with warmed, equilibrated culture medium, in order to concentrate the small number of sperm present into a 20- to 50-µL pellet.



9.6.3 Sperm Vitality Tests


Where sperm are present in the sample, but few or no motile forms are seen, it is possible to assess whether or not any of the immotile forms are viable using sperm vitality tests. Such tests determine the proportion of live, membrane-intact spermatozoa, either by dye exclusion (e.g. eosin–nigrosin staining) or by osmoregulatory capacity demonstrated by swelling of the sperm tail under hypo-osmotic conditions (the hypo-osmotic swelling [HOS] test). Sperm staining techniques destroy living cells and can only be used diagnostically, but the HOS test does not affect sperm viability and may be used on samples intended for use in treatment.



9.6.4 Chemical Motility Enhancers


Where so few or no motile sperm are seen after preparation of a sample, such that there will be difficulty finding sufficient motile forms to carry out ICSI, the motility of immotile, viable spermatozoa may be stimulated using preparations containing a chemical motility enhancer (pentoxifylline or theophylline). These members of the xanthine family inhibit phosphodiesterase activity and increase levels of intracellular cyclic adenosine monophosphate (cAMP), which plays a role in sperm motility. Clinical grade preparations of chemical motility enhancers are available commercially, specifically for use in assisted reproduction procedures. Their addition to immotile sperm in samples such as surgically retrieved epididymal or testicular sperm samples may induce motility, significantly reducing the time taken to identify and select motile sperm for use in ICSI. Fertilisation, pregnancy and live birth can be achieved with treated sperm, but use of chemical motility enhancers should be restricted to cases where it is considered essential by the experienced ICSI practitioner; where live births have been reported following pentoxifylline or theophylline treatment, there is no evidence of anomalies in the offspring, but larger follow-up studies are needed to confirm their safety.



9.7 Insemination In Vitro



9.7.1 Conventional Insemination (IVF)


For normal semen samples [5] (Table 9.2), it is usual practice to inseminate OCCs at approximately 4–6 hours after oocyte retrieval using conventional IVF. Usually, up to five OCCs will be incubated in 1 mL of culture medium. With gametes for only one set of patients in a workstation at any time, and with appropriate witnessing, insemination is carried out at a stereomicroscope with a warmed stage at 37°C in a laminar flow hood, using a graduated pipette with a sterile, disposable tip that must be discarded between patients. An appropriate volume of the prepared, concentrated sperm suspension (usually 5–20 µL) is expelled into each drop of culture medium containing OCCs, to give an insemination concentration of 50–100 × 103/mL motile spermatozoa. The mixed OCCs and spermatozoa are incubated overnight, and the oocytes checked for evidence of fertilisation 18–20 hours post insemination.



9.7.2 Intracytoplasmic Sperm Injection


First described in 1992, ICSI is now an established technique used routinely in the assisted reproduction laboratory. ICSI involves the selection and injection of a single motile spermatozoon into each mature oocyte in a cohort in order to maximise the chance of fertilisation where




  • Initial semen analysis shows the sample is suboptimal in one or more parameters (Table 9.2).



  • Sperm have been collected surgically (epididymal or testicular) [6].



  • Frozen semen samples have shown poor survival on thawing.



  • The final sperm preparation intended for use in IVF insemination does not meet the laboratory’s criteria for conventional IVF.



  • A clinically significant titre of antisperm antibodies is present.



  • Previous IVF treatment cycle(s) have resulted unexpectedly in complete fertilisation failure.



  • Cryopreserved oocytes are used.


No definitive guidelines have been developed for the routine use of ICSI to achieve fertilisation in vitro outside these criteria. Although its efficacy when used for other indications remains disputed, ICSI may be used routinely in some assisted reproduction centres for




  • Unexplained infertility



  • Women of advanced maternal age (usually ≥40 years)



  • Where oocyte quality is deemed to be poor according to the laboratory’s own criteria



  • Patients whose embryos showed poor development in previous treatment cycle(s)



9.7.2.1 Preparation of Oocytes for ICSI

Prior to ICSI, the cumulus and corona radiata cells surrounding each oocyte must be removed in a procedure known as denudation, usually carried out 3–4 hours after oocyte retrieval. Using sterile technique and appropriate witnessing, working in a laminar flow hood at a stereomicroscope with a warmed stage at 37°C, the first stage of denudation is enzymatic digestion of the matrix of hyaluronan oligosaccharide chains cross-linked by hyaluronan binding proteins and proteoglycans between the cumulus granulosa cells and oocyte. The OCCs are incubated in hyaluronidase for 30–45 seconds, after which any remaining cumulus and corona radiata cells are removed mechanically, using fine (0.01 mm internal diameter) pipettes. Clinical grade human recombinant hyaluronidase is available commercially, specifically for use in oocyte denudation for assisted reproduction, and is usually added to HEPES- or MOPS-buffered culture medium, since dishes containing oocytes may be exposed to air for several minutes during the procedure.


Once denuded, each oocyte can be assessed for maturity using a stereomicroscope, and those that are mature (metaphase II stage of meiosis; MII), as indicated by the extrusion of the first polar body, are selected for injection (Figure 9.5). Oocytes that are still at the germinal vesicle (GV) stage will usually degenerate in culture without undergoing maturation in vitro and are discarded. Any MI oocytes may be returned to culture in the incubator and examined at intervals throughout the day of oocyte retrieval. Those that mature to the MII stage, extruding the first polar body after further incubation, may be added to the patient’s cohort to be used for ICSI. MII and MI oocytes are returned to fresh dishes of warmed, pre-equilibrated bicarbonate-buffered medium before they are returned to the incubator for incubation until ICSI is performed, at around 4–6 hours following oocyte retrieval.


Feb 26, 2021 | Posted by in GYNECOLOGY | Comments Off on Chapter 9 – Laboratory Procedures for Assisted Reproduction
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