A Workbook on Human Spermatozoa and Assisted Conception Sonia Malik, Ashok Agarwal
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Equipment and Culture Media1

RK Sharma
A well-planned laboratory is the foundation of a successful in vitro fertilization (IVF) practice. There are several factors that should be taken into consideration when designing the laboratory. The equipment you choose is as important as the laboratory space itself. The success of an IVF lab is dependent on the conditions in the laboratory. The media you use should be conducive to the environment and the working culture of the laboratory.
Workstations need to be positioned sensibly and logically to make working more practical and accident free. Efficiency and safety should be considered when positioning incubators, centrifuges, flow cabinets and cryopreservation equipment.
In addition, local health and safety guidelines must be complied with. A sterile environment should be maintained in the laboratory with rigorous daily cleaning of floors and surfaces. This is of particular importance in areas where tissue culture techniques are applied, such as media making, to prevent any microbial contamination. If the andrology and embryology areas are within a single setting, it is advisable to allocate separate incubators for each to prevent temperature and pH fluctuations every time the incubator door is opened.
The IVF laboratory should be located in a low traffic area and entry restrictions should apply. Careful attention has to be paid to the type of paint used as the use of solvent-based paints is lethal to embryos. Low fume or water-based paint should be used on the walls and surrounding area. It is recommended that the paintwork should be completed at least two weeks before starting laboratory work involving oocytes and embryos. The use of strong adhesives also should be avoided for the same reasons.
It is best to have vinyl or single-layered flooring which is antistatic. Wooden or tiled floors should be avoided as their crevices and ridges can trap dirt and they are more difficult to clean. The benches should be of an adequate height to enable comfortable working. Adjustable height chairs are quite useful. These should be PVC or leather-coated to avoid the accumulation of dust in fabric covers.
The laboratory should ideally be located next to the theater where oocyte recovery and embryo transfers are carried out. The size of the laboratory depends on several factors, such as the number of embryologists, the equipment, the number of cycles performed and whether specialized treatments are performed (such as preimplantation genetic diagnosis on sperm 2washing for HIV positive patients). The laboratory should have an adequate space to ensure aseptic and optimal handling of the gametes and the embryos at all times.
  • Incubators
  • Isolates
  • IVF workstation
  • Stereo zoom microscope
  • Aspiration pump
  • Laminar flow
  • Ambient filter system
  • Test tube warmers
  • Heater plates
  • Slow freezing devices
  • Makler counting chambers
  • Phase contrast microscope
  • Centrifuge
  • Refrigerators
  • Micromanipulators with inverted microscope
  • Laser system
  • Automatic sperm analyzers
  • Sperm class analyzer
  • Antivibration platform and table.
These are virtual fallopian tubes and high performing incubators are critical for the success of an IVF lab. Reliability and consistency are absolutely necessary.
The incubator can be water jacketed or air jacketed. Water jacketed incubators have a large thermal mass, so do not cool down as much when opening the door and during power failures. Air-jacketed technology is lightweighted and is of low maintenance. Therefore, such technology provides additional protection of high temperature contamination control. Air-jacketed incubators are preferred for better decontamination function.
The inner chambers of incubators may have a steel, copper or copper enriched steel inner sheet. CO2 incubators with copper in their cabinet are designed to deter contamination. As the copper breaks down, it releases copper oxide, which destroys microbes present in the chamber. The microbiocidal action is only when copper comes in contact with water, however, they are difficult to maintain and require frequent cleaning. Unless new particles are exposed by some sort of metal removal process like sanding down the inner chamber, the antimicrobial benefits would slowly disappear. For low maintenance and easy cleaning, steel lined cabinets are preferred.
Incubation of live cells requires precise control of environmental parameters such as temperature, humidity and pH. Carbon dioxide lowers the pH in the chamber of an incubator 3to levels similar to that of the natural mammalian body environment and is a critical element in incubation. The sensors for controlling CO2 are based on Thermal conductivity (TC) or Infrared (IR) technology. TC sensors provide accurate CO2 control in applications where temperature and humidity values are consistent. IR sensors are recommended in uses where temperature and humidity are changed frequently. Further the IR sensors can be single or dual, of them the latter is better. These sensors could have remote alarm contacts by SIM message or connection to inhouse alarm system.
The door of incubators is equally important for maintaining humidity, temperature and pH. Instead of single door, multiple door screen is beneficial for culturing oocytes and embryos as this will minimize the rate of CO2 loss and rapid drops in temperature when the door is opened. The six door model is preferred over others for better temperature and CO2 control.
The incubators are needed to maintain humidity by incorporating water trays. Nowadays unique panless humidity system is available which generates relatively high humidity protecting cultures and prevents them from drying out. Humidity recovery times are up to five times faster than those of standard water tray. Humidity poses the risk of fungal and bacterial contamination. Some incubators come with a built-in ‘disinfection routine’ module which is useful for cleaning (the walls and plates heat up to 90°C) aka CONTRACAN. These cycles could be moist heat cycle of 90°C or moist plus dry heat. The Galaxy incubators from RS Biotech have a high-heat decon feature, which the Hera-cell line also has. The decon feature is nice, as it heats up the incubator to 200 degrees to kill any germs.
Incubators may have internal fan to circulate the gas and heat. Incubator that does not have a fan or other moving parts reduces contamination and particle production. But these incubators have some problems in maintaining pH.
Another vital consideration is to have a CO2 or TRI Gas incubators. For blastocyst culture, low oxygen is required. Current literature suggests the use of 5% O2 for extended culture However, studies with mouse ova have shown spindle damage and unaligned chromosomes in low oxygen tensions (Hu et al. 2001). Some incubators have got HEPA filter for inner circulating air. These filters need to be changed every six months.
Incubators should be cleaned at least once a month with 70% ethanol (analar grade) and flushed out with one liter of sterile water (tissue culture grade non-pyrogenic). They should be left running overnight after cleaning before culturing gametes or embryos.
Benchtop Incubators
These are the new generation incubators and are compact with a very small chamber volume. They can easily fit over the workstation. They reduce transit time between the incubator and the workstation, besides low gas consumption.
Their main advantage is the rapid recovery of CO2 and temperature within three minutes of opening the chamber lid. It does this by purging the gas at a rate of 25 mL/min in the chamber. It uses a tri gas mixture of 5% O2, 6% CO2 and 89% nitrogen, which is quite expensive compared to pure clinical grade CO2. They can be used for conventional IVF.
Temperature recovery after a 5-s door opening/closing procedure is approximately 5 minutes for the mini-incubator and 30 minutes for the conventional incubator. The oxygen 4concentration return is significantly improved in the mini-incubator (3.0 +/–0 min) compared with the conventional incubator (7.8 +/–0.9 min). Both the early-stage good embryo formation rate and the good blastocyst formation rate are significantly higher in the mini-incubator (39.5% and 15.1%) than the conventional incubator (28.4% and 7.8%). The microenvironment maintenance ability of incubators appears to significantly influence the formation of good embryos (Fujiwara et al. 2007).
The benchtop incubator is much easier to clean than the conventional one. It uses 150 mL of water as opposed to 5 l with the conventional one. However, they can only contain a maximum of eight dishes which means that bigger laboratories may need three to four incubators.
Benchtop incubators have to be installed with autodiallers that will dial out in cases of incubator emergencies. In case of a power cut or CO2 depletion, the chamber will maintain its pH and temperature for about an hour if unopened which gives the embryologist some time to rectify the problem. Incubators should also have two tanks of CO2/mixed gas attached to a changeover unit, in case one runs out so that the other is activated immediately. All fittings and silcone tubing should be checked for leaks. Incubators should be fully serviced twice yearly.
MINC or BT37 both are capable of supporting embryo growth with reduced oxygen levels and both have broadly similar features. BT37 takes care of issues of cranked and water clogged humidity tubing by employing tube guides and heaters. It also comes with built-in battery power which lasts for two hours. There is also a visual indication for active humidification. The heating plate(s) are calibrated at six different points as against other incubators.
Laminar Flow
The laminar flow hood provides an aseptic work area while allowing the containment of infectious splashes or aerosols generated by many microbiological procedures. Three kinds of laminar flow hoods, designated as Classes I, II and III, have been developed to meet varying research and clinical needs.
Class I laminar flow hoods offer significant levels of protection to laboratory personnel and to the environment when used with various microbiological techniques but they do not provide cultures protection from contamination. They are similar in design and air flow characteristics to chemical fume hoods.
Class II laminar flow hoods are designed for work involving BSL-1, 2 and 3 materials, and they also provide an aseptic environment necessary for cell culture experiments. A Class II biosafety cabinet should be used for handling potentially hazardous materials (e.g. primate-derived cultures, virally infected cultures, radioisotopes, carcinogenic or toxic reagents).
Class III biosafety cabinets are gas-tight and they provide the highest attainable level of protection to personnel and the environment. A Class III biosafety cabinet is required for work involving known human pathogens and other BSL-4 material.
Laminar flow hoods protect the working enviroment from dust and other airborn contaminants by maintaining a constant, unidirectional flow of HEPA-filtered air over the work 5area. The flow can be horizontal, blowing parallel to the work surface, or it can be vertical, blowing from the top of the cabinet onto the work surface.
Depending on its design, a horizontal flow hood provides protection to the culture (if the air flowing towards the user) or to the user (if the air is drawn in through the front of the cabinet by negative air pressure inside). Vertical flow hoods, on the other hand, provide significant protection to the user and the cell culture.
Clean Benches
Horizontal laminar flow or vertical laminar flow “clean benches” are not biosafety cabinets; these pieces of equipment discharge HEPA-filtered air from the back of the cabinet across the work surface toward the user, and they may expose the user to potentially hazardous materials. These devices only provide product protection. Clean benches can be used for certain clean activities, such as the dust-free assembly of sterile equipment or electronic devices, and they should never be used when handling cell culture materials or drug formulations, or when manipulating potentially infectious materials. Recent guidelines suggest that vertical flow cabinets should be used to handle samples from patients with HIV or hepatitis B or C. They should be kept clean at all times and the number of items inside the flow hood should be minimized to ensure that the air flow is not disrupted. The flow cabinets should be serviced annually with filter changes every six months.
Culture Lab Air Cleaner (Coda Air Filtration System)
Awareness about air quality has increased in the last few years. Air contaminants include: volatile organic compounds (VOCs) such as aldehydes; small organic molecules such as nitric oxide, sulphur dioxide and carbon monoxide (from exhaust fumes); and liquids such as floor wax that contains heavy metals. The degree of pollution depends on the location of the IVF unit. Urban laboratories are more likely to have higher pollution. Contaminants in the ambient air will also circulate through the incubators. Cohen et al. (1997) showed that certain adhesives arrested > 90% of mouse embryos at the 2-cell stage with very low blastocyst formation. Brand new incubators emit>100 times higher concentrations of VOCs than used ones (Cohen et al. 1997). Many laboratories have a positive pressure HEPA filtration system to extract inorganic contaminants (e.g. dust, bacteria). Additional absorption systems containing activated carbon and potassium permanganate are required to remove VOCs. Airconditioning systems should recirculate HEPAa-filtered air rather than draw air from the outside.
The incubator filter contains activated carbon and conveniently fits into an incubator compartment. It purifies the air by reducing VOCs, heavy metals and chemical air contaminants. The filters are changed monthly or every time the incubators are cleaned.
The filtration towers incorporate a four-stage filter, which contains a unique blend of activated carbon, alumina impregnated with potassium permanganate for absorption and oxidation of a wide variety of gases and particulate contaminants. It removes up to 99% of all contaminants by filtration through 0.2 μm filters and performs 12 to 15 air exchanges per hour. The number of Coda Towers needed depends on the size of the laboratory (one tower for 300 square feet). 6The filters are changed quarterly with a yearly general service. In line CO2 filters are installed in the tubing between the CO2 tanks and the incubator. This filter is suitable for tri-gas as well as pure CO2. These filters can also be attached to CO2 controlled environmental chambers used for routine embryology.
GenX Coda Aero towers, ZANDAIR and zIVF-AIRe and manymore are available, they are equally good or bad andrequire periodic check for VOC and the HEPA filters.
In Vitro Fertilization (IVF) Workstation
This is a controlled environmental chamber which is mobile and is specifically designed to maintain ideal temperature and pH during the handling of gametes and embryos. The working chamber is maintained at a temperature of 37°C with 5 to 6% CO2 in air. Temperature is monitored by a thermostat and CO2 levels by an infrared sensor. A dissecting microscope is needed for egg collections inseminations, fertilization checks, denuding eggs for ICSI (intracytoplasmic sperm injection), normal evaluation and grading of embryos and embryo transfers.
Several studies have shown that prolonged temperature fluctuations can be very detrimental to the meiotic spindle in human oocytes. This can lead to chromosomal aberrations in the resulting embryo with a reduced chance of implantation. The IVF chamber is ideal for training embryologists as temperature fluctuations are minimized if the manipulation takes longer than expected.
Heated stages for oocytes and embryo viewing are necessary. Prolonged exposure of oocytes and embryos to temperatures below 37°C can disrupt their cytoskeleton. All microscopes used for gametes and embryos should have heated stages. Heated stages can be installed on most brands of dissecting microscopes (Nikon, Hunter Scientific). A heated surface with a wide working area can also be incorporated in a Class II flow cabinet.
It is suggested that the stage temperature is set slightly higher than 37°C so that the drops of media with the embryos or oocytes are maintained at 37°C. It is advisable to experiment with a dish without embryos (in the system that you intend to use) to determine the ideal temperature setting.
Good microscopes are required for routine embryological and andrological procedures. A dissecting microscope is essential to score oocyte cumulii complex, pronuclei and embryos for transfer. The range of its magnification should be between 20 and 160x.
A phase contrast microscope is needed to score detailed semen parameters such as morphology. Some clinics use the computer aided semen analysis system (CASA) to reduce interobserver variation.
A micromanipulator is necessary for ICSI and embryo biopsy for preimplantation genetic diagnosis. In addition, a good inverted microscope with a heated stage and a magnification of up to 400x is needed. The controls can be either pneumatic or hydraulic. Although hydraulic 7syringes have more effective control and movement, they can be fairly time-consuming and messy to prime the tubing with oil every time there is trapped air within the system. The micromanipulation system should be placed in a vibration-free environment. Hydraulically controlled antivibration tables are commercially available. If there is limited air filtration in the laboratory, the micromanipulator system can be placed within a class II flow hood. When choosing the manipulators, it is very important to consider the ease of manoeuvrability.
Some systems have automated and complicated electrical controls whereas some are more manual. The simpler the system, the easier it is to rectify problems in an emergency without the need to call in a technician. For units performing embryo biopsy, a triple tool holder may be necessary to breach the zona. However, if an infrared laser system is installed then a dual tool holder should be sufficient to perform he procedure. For teaching purposes, it is advisable to have a video-linked camera mounted on the micromanipulator. This is also useful to record embryo biopsies and look back to detect any contamination of cumulus cells. A commonly used micromanipulator systems are Research Instruments, Narashige, and Eppendorf.
Disposable micropipettes are available commercially from Cook, Humagen, RI etc. The Integra Ti™ by Research Instruments is currently leader in pack. It is accurate, simple to use and reliable. It has been designed to offer the ultimate in user control. Simple pipette set-up and angle adjustment reduce the time of ICSI procedures, therefore optimizing the results.
Ovum Aspiration Pump
It creates a negative pressure and is used for ovum pickups. It has foot operated switch with vacuum gauze variable from 0 to 500 mm Hg. Fluid trap prevents aspiration of fluid into vacuum unit.
Rocket Pumps are reliable and available many years ago, Cook has come with new pump. Both are equally good.
Controlled Rate Freezing System/Cryosystems
CryoMed Freezers for IVF are designed for human invitro fertilization with precise control, ease of setup and operation, and convenience. Chamber, sample temperature and operation status are continuously displayed on the control panel. These IVF models provide convenient front and top access for easy sample “seeding”.
They are hardly used with vetrification in vogue nowadays. While different cryosystems are available having different instruments, we are using open system McGill Cryoleaf by Origio.
General Equipment
Centrifuge is used for density gradient semen preparation. Nowadays good centrifuges are available which can be programmed to give accurate g force. REMI centrifuges are used by our lab.8
It is used to store culture media and other media used for embryology and andrology. Culture media are very temperature sensitive. Certain amino acids and proteins in the media can be inactivated by extreme temperatures and hence should be kept at 2 to 8°C.
Dry Heat Oven for Sterilizing
It is used for drying and sterilizing glass pipettes and other nondisposable items such as tweezers, spatulas and media-making glassware.
A pH meter and an osmometer are designed for particular laboratories making their own media.
All laboratory equipment should be easy to use and to maintain, besides having good service and repair facility. A log of servicing and maintenance should be kept.
Makler's Chamber/Phase Contrast Microscope
These are inescapable requirement for andrology lab and you should go for the best available. Indian equipment is equally good and we have used them for package.
Laser-Assisted System for PGD and Hatching
Saturn 3 by RI or XY Clone by Hamilton Throne are available and can be integrated into the ICSI micromanipulator. The software and accuracy of Saturn system is slightly better than that of Hamilton.
Labware and Disposables
All plasticware that is used in the laboratory should be proven to be nonembryotoxic. The most commonly used plasticware in IVF laboratories are supplied by equipment companies. All the plastic ware should be disposable and not re-used under any circumstances. All new batches should undergo in-house quality control testing before use. Batches that fail in-house checks should be returned. If possible, new packs of dishes should be opened for each patient or each working day to keep the contents sterile. Resealing opened packs should be kept to a minimum. It is strongly recommended to use individually wrapped sterile pipette tips and plastic pipettes for inseminations and sperm preparation. This will minimize the potential risk of any cross-contamination.
Glass pipettes used for routine embryology should be borosilicate and not the cheaper soda glass since these are thought to leach minerals into the culture media. The pipettes should be double washed in sterile water before being plugged with cotton wool and sterilized at 100°C. Glass pipettes should be stored in special metal canisters and the number of pipettes in each should be kept to a minimum so that a new sterile can is opened each day. All pipettes should be flame polished before use. Presterilized (gamma irradiated) and plugged pipettes are also commercially available.
9Sterile nontoxic powder-free latex gloves should be worn during embryo transfer. The powder can be embryotoxic if it enters the dish. Gloves minimize the introduction of contaminants and skin flora, into the incubator or culture system.
Ovum Pickup Needles
Ovum pickup needle is used for ovum aspiration with help of TV probe and aspiration pump. It is designed for precise recovery with minimal trauma. It is available with single and double lumen systems with a choice of 16G or 17G SS needle. Ecomarking maximizes ultrasound visibility.
We generally use Wallace or Cooks single lumen pickup needles.
Embryo Transfer Catheter
This catheter is used for transvaginal transfer of embryos into uterus along with tissue culture medium with ultrasound guided marking and available in different sizes. Being designed to avoid embryo damage, the inner catheter is made of medical grade polyurethane which is known to be non embryotoxic. The hand finished round tip prevents traumatic introduction and graduation on the inner catheter allows alignment with the outer sheath, producing a smooth radius for minimal trauma during insertion.
The choice of embryo transfer catheter is a joint decision between the clinician and the embryologist. Several factors have be taken into account when deciding which catheter to use. These are:
  • The material used in making the catheter (embryo safe)
  • Packaging/sterility
  • The lumen diameter of the inner sheath and the ease of loading the embryos
  • Embryos should be released in a minimum amount of medium
  • The ease of cannulating the cervix
  • Cost
  • Any clinical trial result.
Oocyte Preparation Pipettes
It is used for the transfer of the oocyte or embryos from dish to dish. The tip is well rounded by fire polishing. Available in different IDs as per application.
Denudation Pipettes
It is used for the denudation of the oocyte before the microinjection by ICSI. Cumulus mass and corona tissues are removed by repeated aspiration and expiration of the oocyte. Available in different ID as per application.
Holding Pipettes
Used for ICSI, assisted hatching and blastomere biopsy. The well shaped fire polished tip allows secure fixation of oocyte and avoids damage of cell. Maximal contact surface, 95 micron or 120 micron OD, bend angle available from 5 to 35 degree.10
Injection Pipette
Used to perform intracytoplasmic sperm injection into oocytes. It is available with/without heat formed spike having different angles, spiked or hypodermic bevel. Four to six micron ID, bend angles available 5 to 35 degree.
Apart from the above the labware used is as under
  • Conical tube 15 mL (Falcon ref. 3215/3217)
  • Centrifuge tube 4 mL (Falcon ref. 2003)
  • Plate with center well 60 mm (Falcon ref. 3037)
  • Plate without center well 60 mm (Falcon ref. 3002)
  • Plate 35 mm (Falcon ref. 3001)
  • Plate 50 mm (Falcon ref. 1006)
  • Transfer pipette 3 mL (Falcon ref. 7575)
  • Pipette 10 mL (Falcon ref. 7551)
  • Pipette 5 mL (Falcon ref. 7543)
  • Flask 50 mL (Falcon ref. 3013)
  • Flask 200 mL (Falcon ref. 3024)
  • 4 well plates (Nunc ref. 176740)
  • Glass mark pen (ref. 750 – Glass mark pen)
  • Stripper (MID Atlantic Diagnosis Inc.)
  • Stripper Tips 200 μm (ref. MXL3-200); 150 μm (ref. MXLE-150 ); 125 μm (ref. MXL3-125).
Culture media for embryo development must meet the metabolic needs of preimplantation embryos by addressing amino acid and energy requirements based on the specific developmental stage of the embryo.
Culture media for embryo growth was first described in 1912 for the growth of an embryo of a rabbit. Later on in 1949, mouse embryos were grown in culture media from the 8-cells stage to blastocysts. These culture media, like Earle, Ham's F10, Tyrode's T6 and Whitten's WM1 were based on different salts and were constructed to support the development of somatic cells and cell lines in culture. These culture media, known as physiological salt solutions were used by Robert Edwards for his first successful IVF attempt. These media were formulated for use with or without serum supplementation, depending on the cell type being cultured. The Ham's Nutrient Mixtures were originally developed to support growth of several clones of Chinese hamster ovary (CHO) cells, as well as clones of HeLa and mouse L-cells.
In 1984 to 1985, special media were developed for human IVF. Menezo and his colleagues published a paper, in 1984, describing a new concept in Human Embryo culture. They suggested adding serum albumin as a source for amino acids. The serum protein ensures that oocytes and embryos do not adhere to the glass surface of the pipette used to manipulate them. The medium entitled B2, is still in use today. In 1985, Quinn et al. published in the journal Fertility and Sterility a formula entitled Human Tubal Fluid (HTF), which mimics the in vivo environment to which the embryo is exposed. The formulation of HTF was based on the known chemical 11composition of the fluids in human fallopian tubes as known at that time. This medium is based on a simple balanced salt solution without amino acids; however, the concentration of potassium was adjusted to that measured in the human female reproductive tract. This medium was found to be better compared with earlier media developed.
The supplementation of the HTF medium with either whole serum or with serum albumin became a gold standard for the production of culture medium for human embryos transferred on day 2 or day 3 of culture.
Over the years, further basic research on the metabolism of preimplantation embryos revealed that there are specific needs depending on the developmental stage of the embryo. Energy source requirements evolve from a pyruvate-lactate preference while the embryos, up to the 8-cell stage, are under maternal genetic control, to a glucose based metabolism after activation of the embryonic genome that supports their development from 8-cells to blastocysts.
The above observation lead to the development of the first commercial media. The culture media developed was based on HTF: both media were free of inorganic phosphate, glucose and amino acids. Pool and his colleagues formulated HTF which was free of glucose and phosphate.
Later experiments performed by Gardner and his colleagues supported these findings, further demonstrating the changing metabolic needs of the embryo between its cleavage stages up to 8-cells and later stages up to blastocysts. Cleaving embryos use pyrovate and lactate as energy sources and non-essential amino acids (NEAA) for protein metabolism. From the 8-cell stage the major energy source is glucose and for protein metabolism the embryos use essential amino acids (EAA). Gardner et al. also showed significant differences in the concentrations of various metabolites between the fallopian tube and the uterus.
These findings lead Gardner and his colleagues to formulate the composition of two culture media G1 and G2 that are to be used in sequence. G1 supports the in-vitro development of the fertilized oocyte, the zygote, to the 8-cell stage, and G2 from 8-cells to blastocyst. Several modifications to these media were formulated also by other groups. Sequential media are now being used successfully in IVF treatment all over the world.
Culture media containing a phosphate buffer or Hepes organic buffer are used for procedures that involve handling of gametes outside of the incubator, flushing of follicles and micromanipulation.
pH and Osmolality
Most culture media utilize a bicarbonate/CO2 buffer system to keep pH in the range of 7.2 to 7.4. The osmolarity of the culture medium must be in the range of 275 to 290 mosmol/kg.
The human oocyte is temperature-sensitive and a humidified incubator with a temperature setting of 37.0 to 37.5°C must be used for oocyte fertilization and embryo culture.12
Culture Conditions
Embryos should be cultured under paraffin oil, which prevents evaporation of the medium preserving a constant osmolarity. The oil also minimizes fluctuations of pH and temperature when embryos are taken out of the incubator for microscopic assessment. Paraffin oil can be toxic to gametes and embryos; therefore, batches of oil must be screened and tested on mouse embryos before use in culture of human embryos.
The medium is composed of 99% water. Purity of the water is crucial, and is achieved by ultrafiltration.
Protein Source
Albumin or synthetic serum are added in concentrations of 5 to 20% (w/v or v/v, respectively). Today, the commercial media includes synthetic serum in which the composition is well known.
Sources for Protein Supplements (serves today for research only)
  • Human serum
  • Human cord serum (HCS) (difficult to obtain)
  • Human serum albumin (HSA)
  • Fetal calf serum (FCS)
  • Bovine serum albumin (BSA).
Commercial IVF Media
  • Synthetic serum
  • Recombinant albumin.
Salt Solution in MTF
NaCl, KCl, KH2PO4, CaCl2. 2H2O, Mgso4. 7H2O, NaHCO3
Carbohydrates are present in the female reproductive tract. Their concentrations vary throughout the length of the oviduct and in the uterus, and are also dependent on the time of the cycle.
Together with the amino acids they are the main energy source for the embryo. Culture media that support the development of zygotes up to 8-cells contain pyruvate and lactate. Some commercial media are glucose free, while others add a very low concentration of glucose to supply the needs of the sperm during conventional insemination.
Media that support the development of 8-cell embryos up to the blastocyst stage contain pyruvate and lactate in low concentrations and a higher concentration of glucose.
Amino Acids
Supplement of the culture medium with amino acids is necessary for embryo development. Media that support the development of zygotes up to 8-cells are supplemented with non essential amino acids. Proline, serine, alanine, aspargine, aspartate, glycine, glutamate.
Media that support the development of 8-cell embryos up to the blastocyst stage are supplemented with essential amino acids: Cystine, histadine, isolucine, leucine, lysine, methionine, valine, argentine, glutamine, phenylalanine, therionine, tryptophane.
Their role in the culture medium is unclear.
The majority of ART laboratories use culture media containing antibiotics to minimize the risks of microbial growth. The most commonly used antibiotics being Penicillin (β-lactam Gram-positive bacteria disturbs cell wall integrity) and Streptomycin (Aminoglycoside Gram-negative bacteria disturbs protein synthesis). The antibacterial effect of penicillin is attributed to its ability to inhibit the synthesis of peptidoglycan, unique glycoproteins of the bacterial cell wall. Streptomycin and gentamycin belong to the aminoglycoside group of antibiotics which exert their antibacterial effect by inhibiting bacterial protein synthesis. The use of gentamicin is still controversial and it is not being used by every laboratory.
EDTA is used as a chelator in medium that supports the embryo from the zygote stage to 8-cells and prevents abnormal glycolysis.
HTF Hepes
Designed for oocyte retrieval and follical flushing. It is modified HTF formulated with EDTA and alanyl glutamine buffered with bicarbonate and hepes. It contains needed antibiotics.
Enhance HTF
Designed for in vitro procedure involving the culture of early cleavage stage embryo. Modified HTF formulated with EDTA, low phosphate, alanyl glutamine.
Enhance Day 1
Designed for culture of embryo from day 1 through the morula stage, day 3 or 4. It is glucose and phosphate free with the addition of EDTA and alanyl glutathione.
Enhance Day 3
Designed for culture of embryo for day 3 transfers or for Blastocyst culture. It is low glucose and phosphate free media formulated with EDTA, taurine, glutathime and alanyl glutamine.14
Enhance Day 5
Designed for culture of embryo to blastocyst and day 5 embryo transfers. It is phosphate free and having elevated levels of glucose supplemented with vitamins, essential and non essential amino acids formulated with glutathione and alanyl glutamine.
Human Serum Albumin (HSA)
It is the protein of choice for use as a tissue culture supplement in those applications requiring protein supplementation. It contains 100 mg/mL total protein (wt/volume) in normal saline.
Mineral Oil
It is the recommended oil for overlaying during fertilization of embryo culture and micromanipulation.
Polyvinyl Pyrrolidone (PVP)
It is the recommended media to decrease sperm motility for ICSI procedure prevent sperm sticking to the glass pipette.
It is the recommended medium for removal of cumulas cells from the oocyte after egg retrieval and prior to ICSI.