Gamete and Embryo Manipulation

Laboratory Environment

Conditions in the IVF laboratory must be tightly regulated to replicate physiologic parameters.
The laboratory macroenvironment should be free of contaminants and the air quality rigorously controlled.
Establishment of a suitable microenvironment for laboratory procedures involves quality control to maintain consistent pH and temperature.

In vivo, sperm and egg unite, and preimplantation embryos develop within specific, narrowly defined, physiologic parameters under sterile conditions. Part of the laboratory’s task is to recapitulate these conditions as closely as possible. Because of this, the presence of pollutants can have significant adverse effects on the reproductive system. ^, These contaminants can be microbial, inorganic (i.e., carbon monoxide, nitrous oxide) or volatile organic chemicals (VOC). They can come from the exterior environment (general pollution, construction, and industrial hazards) or can originate internally (released from paint, cleaning fluids, flooring, cabinets, and equipment).

Macroenvironment

In order to minimize exposures to contaminants, IVF laboratories must rigorously control air quality. To date, there is no standard practice for optimization of IVF outcomes. ^, However, IVF laboratories generally have their own central air handling unit (AHU) and/or mobile filter units where the outside (plus recirculating) air is forced through high-efficiency particle air (HEPA) filters, along with activated charcoal, potassium permanganate filters, and/or activated carbon filters. ^, The HEPA filters remove particulate matter (i.e., >0.3 μm in size) and absorb mold spores and bacteria. The carbon or charcoal filters remove VOCs. In addition, many laboratories have filters, housed within the HVAC ducts, that use chemical/absorbent filtration and/or UV light irradiation to further clean the air and remove microbes and particulates (>0.01 μm in size), bacteria, and viruses. To maintain a clean air environment, the laboratory should have a high tightness factor (walls and ceilings should have no penetrations), multiple air exchanges per hour (i.e., 15–25) and be under positive pressure to prevent contaminants from adjacent rooms entering the laboratory.

To minimize VOC emissions within the laboratory, all furniture should be stainless steel. The laboratory should have smooth, nonporous walls and lighting should be within sealed lighting units in order to reduce airborne particles. Inline HEPA and activated carbon filters, which supply the equipment within the laboratory (i.e., incubators), should be present to remove potential contaminants) such as benzene, isopropanol, and pentane from compressed gas (N ₂ and CO ₂ ).

A major task for the laboratory is to ensure that a uniform and optimal environment is maintained. It is therefore necessary to use laboratory equipment and media that provide ideal conditions for embryo development: stable temperatures (37°C) and a pH range of 7.2–7.4. Fluctuations in temperature have been shown to be detrimental to the oocyte microtubular system, causing reductions in spindle size, disorganization of microtubules within the spindle, and, in some cases, complete absence of microtubules. ^, Interference with spindle organization can prevent chromosome disassociation, resulting in aneuploidy. Changes to pH could perturb metabolic homeostasis, stimulate or prevent induction of specific molecular pathways, alter gene expression profiles, and influence embryo development.

IVF Laboratories have many pieces of equipment, including incubators, microscopes, lasers, heating stages, water baths, and storage dewars. There are a variety of options for each piece of equipment and the choice may depend on cost, specific procedures, flow and size of the laboratory. Incubators may be typical box or benchtop incubators. The incubators may be humidified or dry and/or capable of running at low oxygen tension. Stereoscopes may be used for stripping the oocytes, while inverted microscopes with micromanipulators are used to perform ICSI or an embryo biopsy. Regardless of selection, appropriate equipment quality control measures should be performed (an example schedule is shown in Table 36.1 ).

Table 36.1

Sample Quality Control (QC) Schedule

Location	Instrument	QC Performed	Frequency
Embryology lab	Incubator	Temperature and gas level recording pH ^∗ Water level Viasensor ^† of CO ₂ , O ₂	Daily ^∗ 3x weekly Monthly
	Isolette	Viasensor ^† of CO ₂ , O ₂ Temperature and gas level recording	Daily
	Heated stage surface temperatures	Recording	Daily
Gas room	CO ₂ /nitrogen and mixed gas supply	Pressure recording	Daily
Andrology	Heat blocks	Temperature recording	Daily
	Water filter	Level check	Daily
	Computer-assisted sperm analysis	Validation by latex beads with different concentrations	Daily
Alarm system		Alarm monitoring	Daily
Cryo lab	Nitrogen dewars	Refill	Biweekly

† Viasensor: verification of CO ₂ incubator levels; ∗recommended frequency of pH measurements.

Additionally, quality control testing must be performed on all consumables (e.g., media and plastic ware) that are either newly introduced into the laboratory or are a recurring supply (i.e., a new lot). The purpose of the testing is to ensure there are no embryotoxic substances being introduced into the system. This testing is performed using a bioassay (e.g., mouse embryo development, human sperm survival assay), which compares the results of new material to control material that has been previously used successfully in the laboratory.

Risk management is a critical aspect of the IVF laboratory. Good laboratory practice (GLP) and total quality management (TQM) are critical to ensure adherence to standard operating protocols (SOP) and minimize laboratory errors. New technologies, such as electronic witnessing systems using radio frequency identification (RFID) or barcodes, are being implemented in laboratories to track patient samples throughout the entire IVF process to minimize the potential for human error.

Microenvironment

The media system is a vital component in the handling of gametes and embryos. The goal is to emulate the in vivo environment as closely as possible. However, the in vivo milieu is dynamic while the in vitro environment is static. Therefore, a single ideal culture medium is elusive. ^, Today, the media used in IVF laboratories consider these and other physiological issues and provide the most appropriate components at various stages of the IVF process. ^,

Media can be classified based on nutrients (simple or complex) or culture process (one-step, sequential). ^, Numerous studies on embryo metabolism as well as examination of tubal and uterine fluid composition have led to advances in media. There are several complex formulations, each composed of various amounts of substrates including ions, carbohydrates, amino acids, protein, antioxidants, antibiotics, buffers, and chelators. The media system the laboratory utilizes is dependent on the overall culture system and clinical outcomes. ^,

Media can also be classified based on the buffering system in use. As mentioned previously, the pH of the media is critical to embryo development. The pH of the surrounding environment influences sperm function, embryo metabolism, and organelle localization. Safety concerns and outcomes with respect to human embryo development have narrowed the buffering systems in use within the IVF laboratory. The buffering systems most utilized depend on the particular purpose and equipment available ( Table 36.2 ). Typically, sodium bicarbonate buffered-based medium is used for culture purposes due to detailed studies showing superior embryo development in the presence of CO ₂ over room atmosphere. The pH is maintained by way of gas equilibrium and requires expensive equipment with carbon dioxide (CO ₂ ) capabilities. The dissolved CO ₂ is a weak carbonic acid, and the equipment must be adjusted to maintain a desired pH unique to the media. Media that is sodium bicarbonate controlled and remains in room atmosphere (outside of a CO ₂ -controlled environment) even for a short time is susceptible to significant pH changes. Therefore, for procedures that are performed in room atmosphere, the inclusion of a zwitterionic buffer such as MOPS (pH = 6.5–7.9, pK _a of 7.0–7.3) or HEPES (pH = 6.8–8.2, pK _a of 7.2–7.5) is required. Both are effective and have been shown to work well clinically. ^, This nonbicarbonate medium is mainly used for procedures such as oocyte collection, oocyte stripping, ICSI, and biopsy. Alternatively, laboratories may have isolettes where CO ₂ can be supplied and controlled, and a bicarbonate buffer system can be utilized.

Table 36.2

Common Buffering System Media Utilization Within an IVF Laboratory

Noncarbonated-Based Media	Procedure
Noncarbonated-Based Media	Sperm Washing	OR	ICSI	Embryo Transfer	Cryopreservation	Embryo Culture	D3 Biopsy	D5 Biopsy
HEPES	Yes	Yes	Yes	Yes	Yes	No	Yes ∗	Yes
MOPS	Yes	Yes	Yes	Yes	Yes	No	Yes ∗	Yes
HEPES/MOPS mixture	Yes	Unknown	Yes	Unknown	Unknown	No	Yes ∗	Yes
Bicarbonate/CO ₂	Unknown	Yes ∗∗	Yes ∗∗	Yes ∗∗	Unknown	Yes	No	No

∗ HEPES or MOPS without Ca ²⁺ /Mg ²⁺ is used for D3 embryo biopsy which allows for easier blastomere removal.

∗∗ Only under the CO ₂ equipment environment.

In addition to the environment itself, laboratories must be staffed adequately with properly trained personnel. The work in the laboratory is extremely manpower intensive. Laboratory personnel must have discipline, be detail oriented, and function as team players. The number of staff depends on the number of procedures and type of procedures that are performed in the laboratory. In an IVF lab that is performing all procedures, a minimum of 2 embryologists is required, with an additional embryologist for every 100 to 150 cycles. Each laboratory procedure is standardized. Embryologists follow standard operating procedures (SOP) created for every procedure performed in the laboratory. The adherence to the SOP ensures consistency and reduces systematic errors. As part of the quality assurance/control system, analytics are periodically performed and each embryologist participates in internal and/or external quality assurance programs for each procedure.

Procedures

The procedures performed in the laboratory are specific to the developmental stage of gametes and embryos ( Fig. 36.1 ). Additionally, embryologists must coordinate efforts, so the timing of events is carried out precisely. The following sections will discuss the underlying concepts and provide a detailed description of common procedures.

Gamete Handling

Oocytes are isolated from follicular fluid at retrieval and assessed for morphologic appearance. An expanded cumulus-oocyte complex (COC) is suggestive of oocyte maturity.
Cumulus cells surrounding the oocyte are denuded prior to oocyte cryopreservation and intracytoplasmic sperm injection (ICSI), allowing for assessment of the zona pellucida, perivitelline space, and oocyte cytoplasm. In addition, this allows for evaluation of nuclear maturation by detection of the presence (or absence) of a germinal vesicle (GV) and a polar body (MII).
Ejaculated sperm is processed by a simple wash, migration (swim-up), or density-gradient technique.
Surgically retrieved sperm are processed according to the method in which the sample was obtained: epididymal samples by density gradient, and testicular samples by mechanical separation and dissection.

Oocyte Collection

The oocyte harvest entails aspirating follicular contents from antral follicles. The follicular fluid is transferred to the laboratory for evaluation. Approximately 75% to 80% of the follicular aspirates will have an oocyte. The size of the follicle dictates the likelihood of oocyte recovery and the likelihood that it is mature ( Fig. 36.2 ). Nuclear and cytoplasmic maturation of the oocyte are critical for normal fertilization and embryo development.

Fig. 36.2, Odds of aspiration of a nuclear mature oocyte (Metaphase II, MII) relative to lead group >18 mm

In order to prepare for an oocyte collection, each individual case must be prepared for in advance. The day before retrieval, culture dishes are made and equilibrated under controlled conditions with a constant pH and stable temperature at 37°C (see above).

Once the follicular fluid is collected, it is passed to the IVF lab, where oocytes are identified using a stereomicroscope and heated stage, usually at 8 to 60 times magnification. The embryologist pours the contents of each tube into a petri dish, forming a thin layer of fluid that can be quickly scanned for the presence of an oocyte. The oocytes are then washed in bicarbonate-based culture medium, transferred into preequilibrated media, and stored in a CO ₂ environment (i.e., a CO ₂ incubator) for incubation until further processing.

Assessment of the Oocyte

Techniques that assess the nuclear and cytoplasmic maturation of the oocyte fall into two major categories: invasive and noninvasive. Common practice in embryology laboratories includes a noninvasive morphologic assessment using light microscopy. Each oocyte is assessed for nuclear maturation and morphologic characteristics that include evaluating each of the anatomical components of the oocyte (cumulus, zona pellucida, perivitelline space, polar body, and cytoplasm) ( Fig. 36.3 ). The morphologic features of the oocyte have been studied as a factor that may be associated with pregnancy outcomes. However, a consensus on whether each morphologic characteristic is associated with outcomes has been difficult because many investigators have used different terminology to define similar observations. Although no consensus exists, it is common practice to record these observations. An oocyte scoring system has been developed that considers all morphologic characteristics.

Fig. 36.3, The cumulus-oocyte-complex (COC).

Oocytes collected at retrieval are difficult to visualize because they are surrounded by a cumulus mass composed of follicular cells in a polymerized matrix of hyaluronic acid ( Fig. 36.3 B–C). Therefore, in the case of conventional insemination (see below) the morphologic assessment is limited to the cumulus-oocyte complex (COC). The volume, density, and expansion of the cumulus cells (CC) can suggest the stage of oocyte nuclear maturity. The grading of cumulus expansion is a visual inspection of whether the cells are compact or expanded. Compact cumulus cells take on a darker appearance due to the dense association of these cells around the oocyte ( Fig. 36.3 B). They are usually associated with immature oocytes, while expanding or fully expanded cumulus cells are usually associated with mature oocytes ( Fig. 36.3 C).

Unlike conventional insemination, in which intact mature COC are inseminated, fertilization with micromanipulation (ICSI) requires denudation of oocytes (i.e., removal of the surrounding cumulus and corona cells). The denudation allows for an additional detailed evaluation of each component of the oocyte ( Fig. 36.4 ). Similar to planned fertilization with ICSI, oocytes that will undergo cryopreservation for future use also require denudation after retrieval. When oocyte cryopreservation occurs within two hours of retrieval, embryo quality and clinical outcomes are improved. Since zona hardening has been reported prior to insemination of thawed oocytes, ICSI is typically required for future fertilization.

Starting from the outermost layer, the oocyte is encased in a thick glycoprotein shell called the zona pellucida (ZP). The ZP normally averages 15 μm to 20 μm in width and is intact and translucent in a mature oocyte ( Fig. 36.3 A). Studies have evaluated whether the darkness and/or thickness of the ZP are associated with clinical outcomes. ^, While darkness has shown little correlation with clinical outcomes, the thinner ZP has been associated with higher fertilization rates. ^, Other reported alterations of ZP morphology, including fractured or broken ZP, are found to be unassociated with clinical outcomes. The etiology presumably results from postmaturity of the oocyte or excess pressure during oocyte aspiration.

The space between the ZP and the oolemma is called the perivitelline space. Although several studies have suggested that an enlarged perivitelline space ( Fig. 36.5 F ) is associated with poorer fertilization and embryo development, there is not a clear consensus. ^, ^, ^, An enlarged perivitelline space has been hypothesized to relate to oocyte postmaturity. Sometimes the perivitelline space may contain granules that do not appear to influence oocyte or embryo development. The first polar body appears in the perivitelline space. Unusually large polar bodies may indicate disturbances in the position of the meiotic spindle, which may occur during oocyte aging. Accordingly, a large polar body has been associated with poor embryo quality and low pregnancy rates. Fragmentation of the first polar body has not been consistently identified as an abnormality.

The cytoplasm of a normal oocyte is typically uniform, translucent, and free of inclusions. A relatively common abnormality that can be visualized by light microscopy is central granularity ( Fig. 36.5 B). This granular area likely represents a polarized distribution of mitochondria and may have implications for cytoplasmic maturity. Studies evaluating granularity, either generalized or central, have failed to demonstrate a consistent association with clinical outcomes. ^, While some studies have shown that cytoplasmic darkness is associated with poorer fertilization rates or embryo development, others have not. ^, It has also been hypothesized that localized dark and granular cytoplasm may be related to atresia.

The presence of vacuoles, an aggregation of smooth endoplasmic reticulum (SER) within the cytoplasm, has been associated with decreased fertilization, embryo quality, and ongoing pregnancy rates ( Fig. 36.5 C). Vacuoles may be associated with oocyte aging and degenerative processes in oocytes. Case reports have suggested that the presence of vacuoles is associated with an increased risk of Beckwith–Wiedemann syndrome, diaphragmatic hernia, multiple malformations, and ventricular septal defect. A recent study reported healthy live births from embryos derived from such oocytes and recommended a careful follow-up of the children born. As a result, it is recommended that this cytoplasmic abnormality be noted. Inclusions, including refractile bodies, are inconsistently associated with a compromise in embryo quality and pregnancy outcomes. While their cellular basis is unknown, the presence of inclusions within the oocyte may be associated with atresia.

Various shapes and sizes of the oocyte have been observed. The normal size of a human oocyte is roughly 120 μm in diameter. Oocytes with a diameter of 200 μm or more are commonly called “giant” oocytes ( Fig. 36.5 G). These oocytes are thought to be giant due to errors in mitosis during proliferation of the oogonia. As a result, these oocytes are likely diploid and the resultant embryos are at increased risk for digynic triploidy, mosaicism, or polyploidy. The presence of a giant egg in a cohort of oocytes is not associated with overall decreased pregnancy outcomes, but some data has shown that embryos derived from the cohort may show an increased incidence of abnormalities in cleavage rate.

Microscopic observation of denuded oocytes allows for assessment of the presence or absence of a germinal vesicle (GV). Fig. 36.6 shows three stages of oocyte maturation. On average, 3.9% of the intact oocytes are in the metaphase I (MI) stage, having undergone breakdown of the germinal vesicle but not extrusion of the first polar body. Approximately 10.3% of the intact oocytes are at the germinal vesicle stage, and approximately 85.8% are in the metaphase II stage, defined by the presence of the first polar body. ICSI is carried out on metaphase II oocytes because only such oocytes have reached the haploid state and can thus be fertilized normally. It has been reported that 74% of the MI oocytes completed meiosis within 20 hours of retrieval. Another study reported that 27% of MI oocytes extruded their polar bodies within 4 hours of the egg retrieval. Oocytes from this study were injected on the day of the egg retrieval in parallel to the oocytes retrieved at MII. This study demonstrated that MI oocytes that completed their maturation in vitro had a lower fertilization rate compared to those aspirated at MII. However, there were no differences observed in embryo quality between the two groups of oocytes. Additional studies support these observations, showing that in vitro matured oocytes can be normally fertilized; however, embryos derived from these oocytes rarely result in pregnancies. ^, These results suggest that rescue of MI oocytes from patients with few MII oocytes may increase the number of embryos for transfer, but the chance to improve pregnancy rates by this procedure is minimal. Germinal vesicle-stage oocytes require overnight incubation (24 to 48 hours) in culture media supplemented with gonadotropins in order to reach the MII stage. ^, Few pregnancies have been reported from oocytes that were retrieved at the GV stage, although standard IVF treatment with controlled ovarian hyperstimulation was performed. ^, Because of the poor results, GV oocytes are usually discarded. Denuded and rinsed oocytes are incubated until the time of microinjection.

Fig. 36.6, Stages of oocyte maturation .

Polscope imaging provides valuable information on the structure and architecture of the meiotic spindle. The integrity of the meiotic spindle in MII oocytes is crucial for normal fertilization and subsequent development. Overall, meiotic spindles can be detected in up to 91% of human metaphase II oocytes at the time of ICSI. Studies have shown the presence of a birefringent spindle in human oocytes can predict not only a higher fertilization rate but also greater embryo developmental competence. High degrees of misalignment of the meiotic spindle and the first polar body have also been documented and are thought to occur as a result of polar body displacement during cumulus corona removal. ^, The relative position of the spindle within the oocyte, however, has little influence on the developmental potential of the resulting embryos. ^,

Because most oocytes possess spindles, the mere presence of a spindle is likely of limited value. Whether quantitative spindle analysis (estimating the density of tubules within the spindle) offers added value remains to be seen. So far, visualization of the spindle and the appearance of the first polar body are accurate indicators of oocyte maturity and may help to determine the timing of ICSI (see ICSI).

The developmental competence of the oocyte has also been assessed through a biomarker approach. Several strategies have been applied, including a comparison of transcriptomes, proteins (proteomics) or metabolites (metabolomics) of granulosa cells (GC) and/or CCs or follicular fluid (FF). Evidence suggests that CC from follicles of embryos that result in a pregnancy may have differential expression. Others have shown that gene expression profiling in either GC or CC has no association with fertilization outcomes but may be associated with advanced maternal age. ^, Estes et al. found that the FF proteome in women ≤ 32 years of age predicted ovarian response and live birth. Another study reported that oocytes resulting in pregnancy presented high amounts of proteins, and oocytes resulting in no pregnancy presented high amounts of ubiquitinated peptides. Several investigators have demonstrated associations between the FF metabolome and the developmental competence of the oocytes. Although intriguing, the available testing is not ready for routine clinical use. However, given that they represent cellular regulatory processes, biomarkers may be more informative in the future than morphology.

Sperm Collection

Sperm collection is either obtained through ejaculation (i.e., masturbation, electroejaculation) or by surgical retrieval. Evaluation and assessment of semen are very important for the diagnosis of infertility and treatment decisions. The collection method for IVF is dependent on the clinical scenario ( Chapter 35 ). The collection, processing, and assessment of the sperm is dependent on the method of collection and is discussed below. The choice of sperm preparation technique depends on the nature of the semen sample, particularly with respect to motile count, the ratio between motile and immotile count, volume and presence of antibodies, agglutination, pus cells, or debris.

The majority of sperm collections are obtained through masturbation into a collection cup. The container to collect the sample has to be wide-necked plastic or sterile glass, and it must be cytotoxically tested. It must be labeled with the complete patient name, identification number, date of birth, date and time of collection, and number of days of abstinence. Semen samples are collected after 2 to 10 days of abstinence. Shorter periods of abstinence may be associated with lower volume but higher motility, whereas longer abstinence can lead to reduced sperm motility. ^, In cases of male infertility, shorter durations of abstinence (1 day) may be associated with higher motility and better morphology. The use of lubricant (other than mineral oil or selected lubricants) is discouraged because they may be spermicidal. Other collection methods include the use of silastic condoms during intercourse.

Most of the samples should be collected on site in a private room set aside for this purpose. The room can be adjacent to the lab and have a pass-through for drop off, or alternatively, it can be given to a staff member with proper chain of custody (for specimen verification) protocol. If the patient cannot produce on site for personal reasons, the patient can do so at home or a place nearby that will allow sample delivery to the laboratory within an hour. Samples should be kept at room temperature during transport (20°C–37°C) and can stay at room temperature in the laboratory for up to one hour in order for liquefaction of the semen to occur.

Assessment of Ejaculated Sperm

A semen assessment is performed prior to processing the sperm for IVF and entails a sperm count and motility evaluation ( Table 36.3 ). The assessment dictates how the sperm is processed and the type of procedure used for insemination. The sperm count and motility assessment are obtained through standard procedures. The goal of preparation of sperm is to concentrate the motile spermatozoa into a fraction that is free of seminal plasma and debris. The sperm preparation methods attempt to mimic the natural process in which viable sperm are separated from the seminal plasma and other constituents as they migrate through the cervix. The seminal plasma contains substances that inhibit capacitation and prevent fertilization. Sperm processing should be performed within 30 minutes if liquefaction is completed or no later than one hour after production. If delayed, the uncontrolled production of reactive oxygen species (ROS) that exceeds the antioxidant activity of seminal plasma will lead to oxidative stress and result in decreased sperm capacity. ROS are produced by leukocytes and other debris found in seminal plasma. ^, After sperm processing, the insemination process ideally should be performed within 4 to 6 hours. After 4 to 6 hours, sperm DNA fragmentation may be increased. There are several sperm preparation techniques described below.

Table 36.3

Semen Parameter Reference Value According to World Health Organization (WHO) Manuals

Semen parameters	WHO, 2010
Volume	1.5 mL
Concentration	15 x 10 ⁶ /mL
Total sperm concentration	39 x 10 ⁶
% Motile	40%
Progressive motility ^†	32% (a+b)
Vitality ^‡	58%
Morphology (Kruger strict)	4%
Leukocyte count	<1 x 10 ⁶ /mL
Values obtained from lower fifth centile value		Strict morphology analysis: observed morphologies in the sample include (a) large acrosome, (d) tail defect, (l) leukocyte, (m) macrocephalic head, (n) normal morphology, (r) round head, (t) tapered head

† a—rapid progression (>25 μm/seconds), b—sluggish (5–25 μm/seconds).

‡ % alive.

Processing Techniques

Simple Wash

A simple washing procedure provides the highest yield of sperm and is adequate if semen samples are of good quality. The procedure entails adding culture medium that is a balanced salt solution supplemented with protein and an appropriate buffer. The semen sample is mixed with the buffered medium and centrifuged. The pellet is resuspended in the medium and centrifuged again. The centrifugation speed (i.e., 300 g; 1000 rpm) and number of washes should be minimized to avoid the production of ROS within the pellet. Generation of reactive oxygen species may reduce the function and motility of sperm, cause peroxidation of sperm plasma membranes, decrease oolemma binding and fusion, and cause fertilization to fail. The number of washes to remove seminal plasma can be reduced by increasing the volume of medium in each tube. Although quite successful, a simple wash alone for IVF is rare due to persistent debris (i.e., leukocytes, epithelial cells) and nonviable sperm that are present in the pellet, which may inhibit capacitation and the ability of the viable sperm to fertilize the oocyte.

Migration: Swim-Up

Motile sperm can be further selected by their ability to swim out of seminal plasma and into the culture medium. The swim-up is a very common sperm processing technique for IVF. There are multiple ways to perform a swim-up procedure. The technique involves first washing the semen sample and then placing semen underneath a small volume of culture medium (between 0.5 and 1 mL of media). After one hour of incubation (swim-up time), the supernatant is removed; this will contain highly motile sperm (more than 90% motile spermatozoa). This is then centrifuged and resuspended in a small volume of culture medium and may be used directly. This technique is simple and cheap and does not require sophisticated equipment or highly specialized skills. It is most often used for “normal” samples (average or good sperm concentration and motility) given it has a low yield.

Density Gradient

Density gradient separates sperm based on their density. Although the initial preparation was removed from the market due to possible contamination of endotoxins, there are now several types of density media based on silane-coated silica particles that have been proven to have very low toxicity. Density gradients can either be continuous or discontinuous. The type of gradient is based on whether there is a continuous gradient from the top to the bottom of the tube or if there are a number of layers of decreasing densities that are placed on top of each other. Numerous commercial products are available. The most common gradient used for sperm processing in IVF is discontinuous, where a volume of a low suspension (40%–45%) is layered over a high suspension (80%–90%) of silane-coated silica particles. The semen sample is placed on top of the suspension and centrifuged. The principle of this procedure is based on morphologically normal sperm, abnormal sperm, and debris having different densities. As a result of the differences in density, normal motile sperm cells penetrate the higher densities in the direction of the centrifugation force, while immotile or abnormal morphological sperm are retained at the boundaries of interphases. This technique isolates the subpopulation of sperm with the best motility, morphology, and nuclear and mitochondrial DNA quality. ^, Density gradient processing of sperm has been associated with the selection of high quality sperm and higher assisted conception rates than swim-up. ^, The density gradient is the most widely used method in IVF laboratories for sperm processing, after masturbation and electroejaculation.

Glass Wool Filtration

The glass wool filtration separates motile sperm from immotile sperm, debris, and leukocytes prior to centrifugation. The process involves removing the debris from the ejaculate through mechanical retention and adhesion to glass fibers. Since the whole ejaculate is filtrated, ejaculate from patients with oligozoospermia can be processed. This technique eliminates up to 90% of leukocytes contaminating the semen and therefore significantly reduces ROS. After the filtration process, the semen is centrifuged to remove the seminal plasma. Glass wool filtration is an easy technique that can be used to recover sperm with good motility.

Magnetic Activated Cell Sorting (MACS)

MACS separates apoptotic from nonapoptotic sperm on a molecular level. Apoptotic sperm externalize phosphatidyl serine residues, which bind to annexin V. ^, The process entails mixing a semen sample after double density gradient centrifugation with superparamagnetic beads that are conjugated with specific antibodies to annexin V for 15 minutes. The mixture is loaded onto a separation column, which is placed in a magnetic field. The nonapoptotic sperm (annexin V-negative) do not bind to the beads and pass through the column. The fraction of the sample that does not bind has better morphology and higher fertilization potential than sperm separated by density gradient alone. ^, ^, While some clinical studies have shown improvement in cryosurvival rates and pregnancy rates with the addition of MACS for sperm selection, others have not. More studies are needed before MACS can be routinely used for sperm selection in clinical laboratories.

Sperm Stimulation—Pentoxifylline

The goal in any ICSI procedure is to use spermatozoa that are viable. Sperm motility is the best indicator that both the functional (protective) plasma membrane and the metabolic processes are in place. Spermatozoa retrieved from the testis are in a different physiological state than sperm that have been transported through the epididymis. These sperm (fresh or frozen and thawed) often have extremely low motility or are immotile, making it difficult to identify viable sperm for injection. Sperm motility can be stimulated by a variety of chemicals. One of the most commonly utilized chemicals for stimulating sperm motility is pentoxifylline. ^, It can be administered orally for 3 to 6 months, or directly applied in solution to processed sperm and incubated 1 hour prior to use. Pentoxifylline is a nonspecific inhibitor of phosphodiesterase that has stimulatory effects on sperm motility. The stimulatory effect is attributed to elevated intracellular levels of cyclic adenosine monophosphate (cAMP) via inhibition of its breakdown by cAMP phosphodiesterase. Pentoxifylline is also reported to enhance the acrosome reaction due to the increased levels of cAMP. ^, Overstimulation of sperm with pentoxifylline can induce premature acrosome reaction. Therefore, use of this chemical should be limited to situations in which no motile sperm can be readily identified.

Novel Techniques

Some novel techniques are now being assessed to aid in sperm preparation by reducing the presence of ROS. ^, The introduction of antioxidants in the sperm preparation media or removal of the centrifugation step may limit ROS levels. Electrophoretic sperm isolation and microfluidic sperm processing isolate sperm from the seminal plasma without centrifugation and are being investigated as methods of preparation that minimize ROS. It is unknown whether these techniques will improve clinical outcomes. ^,

Surgical Sperm Retrieval

Several sperm retrieval methods have been developed to retrieve sperm from the epididymis and/or testes in men with azoospermia. The technique employed depends on the clinical scenario (discussed in Chapter 35 ). The evaluation and processing of the sample is dependent on the surgical technique and site of collection. Sperm retrieval should be carried out either on the day of oocyte retrieval or the day before, depending on the laboratory workload. Additionally, the surgically retrieved specimen may be incubated for up to several days if the sample is poor and requires further maturation. All surgical collections require ICSI for optimal fertilization (see below).

Collection, Processing, and Assessment of Surgical Sperm

Epididymal Sample

The procedure to collect sperm from the epididymis is either a MESA (microsurgical epididymal sperm aspiration) or a PESA (percutaneous epididymal sperm aspiration). PESA is a blind procedure. The surgical approach depends on physician preference and clinical scenario. A fine needle is used to puncture the epididymis, and the epididymal fluid is collected by fine-gauge needle aspiration. MESA is an open surgical sperm retrieval procedure that uses an operating microscopy to locate the tubules of the epididymis. Once they are located, the tubules are opened and the spermatic fluid that flows out is aspirated. Regardless of the approach, the epididymal fluid is placed on a petri dish and examined under an inverted microscope (at 400 times magnification) to confirm the presence of motile sperm. In most cases, the amount of debris is less than ejaculated samples and contains a high concentration of motile sperm (1×10 sperm/μL). The preparation technique to isolate the motile sperm is based on the sperm density and motility and the amount of cellular debris. A density gradient is typically used unless the sperm density is low, in which case a simple wash is employed.

Testicular Sample

The procedure to collect sperm from the testes may either be a TESA (testicular sperm aspiration) or a TESE (testicular sperm extraction). TESA samples are obtained with a wide-bore needle injected percutaneously into the testis. TESE is an open technique that removes several segments of testicular tissue. TESA samples are evaluated for motile or immotile spermatozoa with a stereomicroscope. Using fine needle dissection, the sperm is identified and separated from the seminiferous tubules, and surrounding tissue. The majority of the sperm found are immature, although some sperm are motile, or “twitching.” TESE samples contain a large amount of cellular debris. Finding sperm in the testicular tissue can be a laborious task and can take several hours to process depending on the degree of sperm production and the etiology of testicular failure. There are multiple processing methods to identify the sperm. Typically, the testicular tissue is evaluated under a stereomicroscope, to identify seminiferous tubules and to remove blood clots. Following identification, the testicular sample is processed by dispersion of the tubules by mechanical mincing and/or enzymatic digestion. Once homogenized, the sample is evaluated using an inverted microscope (400 times magnification) to identify the presence of sperm. The sperm is freed from the seminiferous tubules and other debris by dissection.

You're Reading a Preview

Become a Clinical Tree membership for Full access and enjoy Unlimited articles

Become membership

If you are a member. Log in here