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INTRODUCTION
The success of clinical IVF was initially compromised by sub-optimal culture Conditions, resulting in impaired embryo development and a Subsequent loss of viability. However, research during the past 10-15 years has resulted in the development of more physiological and effective culture Media capable of maintaining the viability of the developing embryo.
This in turn has resulted in an increase in implantation rates and a decrease in the number of pregnancies lost. Furthermore, more suitable culture Conditions produce embryos more able to survive cryopreservation. Therefore, improvements in embryo culture technology have significantly contributed to the increase in the overall success rates of human assisted
Conception.
Types of Media for Embryo Culture
Culture media employed for clinical IVF vary greatly in their composition, yet there appears to be little difference between media in their ability to support development of the human embryo in vitro for up to 48 hours or in subsequent pregnancy rates after transfer . This has led to a great deal of confusion concerning the formulation of embryo culture media and the role of individual components in embryo development. An understanding of the role of culture media and their components has been hampered by the routine inclusion of serum in human embryo culture media. Serum has the ability to both mask potential embryo toxins and suppress the beneficial effects of other medium components. In light of this, there has been considerable research into the development of serum-free embryo culture media. Such studies have been invaluable in our understanding of the embryo’s requirements during the prelim plantation period.
Media used to culture the mammalian prelim plantation embryo generally fall into one of four types.
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Simple Salt Solutions with Added Energy Substrates
These media were originally formulated to support the development of zygotes from certain inbred strains of mice and their F1 hybrids. Examples of this type of media used in clinical IVF are M16, T6, Earle’s, CZB, and KSOM. Derived from such types of media were human tubal fluid (HTF) medium, and P1.there has been little change in the formulation of these media over the past 30 years. Such ‘‘simple’’ media are usually supplemented with either whole serum or serum albumin, and are used for the cleavage stage embryo only, i.e., pronucleate oocyte to the 8-cell stage.
Complex Tissue Culture Media
These media are commercially available and are designed to support the growth of somatic cells in culture, e.g., Ham’s F-10. Such media are far more complex, containing amino acids, vitamins, nucleic acid precursors, and transitional metals, and are usually supplemented with 5–20% serum. Importantly, such media were not formulated with the specific needs of the human embryo in mind, and they contain components which are now known to be detrimental to the developing embryo.
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Simplex Optimized Media
This approach to formulate culture media depended on a computer program to generate successive media formulations based on the response of mouse embryos in culture. Once a specific medium was formulated, tested, and blastocyst development analyzed, the computer program would then generate several more media formulations for use in the next series of
cultures. This procedure was performed several times to generate media that supported high rates of blastocyst development of embryos derived from the oocytes of outbreed mice (CF1) crossed with the sperm of an F1 hybrid male, and were termed SOM and KSOM. Such media were subsequently modified by another laboratory to include amino acids (KSOMAA). This last phase of medium development was based on previous studies on the mouse embryo and did not involve the simplex procedure. This single medium formulation, KSOMAA, has been used to produce human blastocysts in culture. In such types of media, the embryo therefore has to adapt to its surroundings as it develops and differentiates.
Sequential Media
The approach taken in our laboratory has not only been to learn from the environment to which embryos are exposed in vivo, but also to study the physiology and metabolism of the embryo in culture, in order to determine what causes intracellular stress to the embryo. By being able to identify and monitor such stress, we have been able to develop stage specific culture media that substantially reduce culture-induced trauma. The development and characterization of such sequential media has been published in detail elsewhere.
Examples of sequential media include G1/G2, universal IVF medium and M3, and P1 together with blastocyst medium. Interestingly, medium M3 is a modification of Ham’s F-10 and F-12, while blastocyst medium is a mosdification of Ham’s F-10.
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COMPOSITION OF EMBRYO CULTURE MEDIA
The composition of embryo culture systems can be broken down into the following components:
- Water
- Ions
- Carbohydrates
- Amino Acids
- Vitamins
- Nucleic Acid Precursors
- Chelators
- Antioxidants
- Antibiotics
- Protein/macromolecules
- Hormones and growth factors
- Buffer system
CULTURE SYSTEM
Optimization of embryo development in vitro is not only dependent upon the composition of the culture medium or media used, but is also affected by physical parameters, such as the incubation environment and gas phase.
Therefore, it is important to consider the ‘‘culture environment’’ when attempting to improve embryo culture systems (Fig. 5).
Figure 5 A holistic analysis of human IVF. This figure serves to illustrate the complex and interdependent nature of human IVF treatment. For example, the stimulation regimen used not only impacts oocyte quality (hence embryo physiology and viability), but can also affect subsequent endometrial receptivity. Furthermore, the health and dietary status of the patient can have a profound effect on the subsequent developmental capacity of the oocyte and embryo. The dietary status of patients attending IVF is typically not considered as a compounding variable, but growing data would indicate otherwise. In this schematic, the laboratory has been broken down into its core components, only one of which is the culture system. The culture system has in turn been broken down into its components, only one of which is the culture media. Therefore, it would appear rather simplistic to assume that by changing only one part of the culture system (i.e., culture media), that one is going to mimic the results of a given laboratory or clinic. A major determinant of the success of a laboratory and culture system is the level of quality control and quality assurance in place. For example, one should never assume that anything coming into the laboratory that has not been pre-tested with a relevant bioassay (for example, mouse embryo assay) is safe merely because a previous lot has performed satisfactorily. Only a small percentage of the contact supplies and tissue culture ware used in IVF come suitably tested. Therefore, it is essential to assume that everything entering the IVF laboratory without a suitable pretest is embryo toxic until proven otherwise. In our program, the 1-cell mouse embryo assay (MEA) is employed to prescreen every lot of tissue culture ware that enters the program, i.e., plastics that are approved for tissue culture. Around 25% of all such materials fail the 1-cell MEA (in a simple medium lacking protein after the first 24 hours). Therefore, if one does not perform QC to this level, one in four of all contact supplies used clinically could compromise embryo development. In reality, many programs cannot allocate the resources required for this level of QC; and when embryo quality is compromised in the laboratory, it is the media that are held responsible, when in fact the tissue culture ware is more often the culprit. Abbreviations: IVF, In vitro fertilization; MEA, mouse embryo assay; QC, quality control; QA, quality assurance.
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Oxygen
The concentration of oxygen in the lumen of the rabbit oviduct is reported to be 2–6% . It was subsequently determined that the oxygen concentration in the oviduct of hamster, rabbit and rhesus monkey was similar at 8% . However, the oxygen concentration in the uterus was significantly lower than in the oviduct ranging from 5% in the hamster and rabbit to 1.5% in the rhesus monkey.
Studies on several types of mammalian embryo have demonstrated that culture at a reduced oxygen concentration results in enhanced development in vitro . In the mouse, culture at oxygen concentrations as low as 1% were sufficient to support embryo development to the blastocyst stage. Furthermore, several other studies have shown that a reduced oxygen concentration, between 5% and 8%, enhances development to the blastocyst stage in the mouse. Similarly, studies on the rabbit and on domestic animal species such as sheep, goats , and cows have also demonstrated that an oxygen concentration of 7% results in increased development in vitro compared to 20% oxygen. Furthermore, it has been observed that human blastocysts cultured in a low oxygen environment (5%) have significantly more cells than those cultured in a high oxygen environment (20%) (Gardner DK. Unpublished Data. 1998). Even a transient exposure for one hour to 20% oxygen reduced mouse embryo development in vitro . It has subsequently been determined that equilibrating culture dishes at 20% oxygen for five hours prior to culture in 7% oxygen decreases mouse zygote development to the blastocyst stage and resultant blastocyst cell numbers. This inhibition can be attributed to the fact that it takes more than five hours for the oxygen concentration to fall
to embryo-safe levels.
Interestingly however, human and mouse embryos can grow at elevated oxygen concentrations and this has lead to some confusion regarding the optimal concentration for embryo culture. The physiology of the reproductive tract and the beneficial effects of using a reduced oxygen concentration as determined in controlled studies indicate that to employ low
An oxygen concentration appears prudent.
Carbon dioxide
Carbon dioxide is not only required to maintain the pH of bicarbonate buffered medium, but is readily incorporated into protein and nucleic acids by the mouse embryo at all stages prior to implantation. Culture systems for the preimplantation mammalian embryo routinely employ a carbon dioxide concentration of 5% coupled with a bicarbonate concentration of around 25mM. However, analysis of the uterine environment in the guinea pig on day 4 of pregnancy determined the carbon dioxide concentration to be 10% . Studies on the hamster 8-cell embryo showed that development to the blastocyst stage is increased by a carbon dioxide concentration of 10%. Similarly, rabbit zygote development to the hatching blastocyst stage is increased in a carbon dioxide concentration of 10% compared to 5%. It has been proposed that the beneficial effects of a high carbon dioxide concentration is due to a decrease in the pH of the medium as the beneficial effects of increased carbon dioxide could be replaced with a weak acid . The pH of media containing 25mM bicarbonate that is equilibrated in a CO2 environment can be calculated using the Henderson–Hasselbach equation. Interestingly, at 5% CO2 the pH of medium containing 25mM bicarbonate is 7.45, while a CO2 concentration
Of 6% is required to maintain the external pH of the medium at around 7.4.
The optimal concentration of carbon dioxide for human embryo development has yet to be determined; however the CO2 concentration is related to pH of the medium which also needs to be considered. Typically, Fyrite has been employed to quantitate CO2 within the chamber used. However, the accuracy of such a procedure is rather low. Fortunately, there are
Now available new hand-held infra red (IR) CO2 analysis meters with very high accuracy (to 0.1%)
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PH
The pH of fluid collected from the reproductive tract of rhesus monkey was reported to alter in parallel with changes in the bicarbonate concentration increasing from 7.1 to 7.3 during the follicular phase of the estrous cycle to 7.5–8.0 at the time of ovulation and during luteal phase. Dale et al. recently reported that the pH of the uterine fluid is lower than that of the oviduct. External pH of culture media formulated for preimplantation embryos is commonly between 7.3 and 7.4 (depending on the CO2 concentration used). Studies on the mouse embryo determined that development from the 2-cell stage to the blastocyst stage could occur in media with a pH range from 5.9 to 7.8 . Similarly, hamster 8-cell embryos could develop to the blastocyst stage in medium with an external pH range from 6.4 to 7.4 . In contrast however, it has recently been shown that a transient exposure of zygotes and 2-cell mouse embryos to medium with elevated pH significantly reduced subsequent development to the blastocyst stage. Although these studies demonstrated that some embryos could still develop to the blastocyst stage in a wide range of external pH’s, the subsequent viability of these embryos following transfer is unknown.
Subsequently, there has been considerable work in the area of intracellular pH (pHi) and its role in regulating embryo development. It has been established that even relatively small fluctuations in pHi can significantly retard subsequent developmental competence. Fluctuations in either the acidic or the alkaline range can drastically reduce development.
Even more significantly, it has been determined that mammalian oocytes and embryos for around six hours following fertilization lack any functional transport systems to regulate pHi in either the acid or the alkaline ranges. Therefore, care should be taken to avoid fluctuations
in the pH of media during embryo manipulation and culture. This is especially relevant for oocytes that are stripped of their cumulus before an ICSI procedure. Immediately following the denudation procedure these oocytes and embryos cannot regulate their ionic homeostasis. Amino acids are also known to increase the intrinsic buffering capacity of the embryo and reduce fluctuations in intracellular pH of the embryo. Therefore, as discussed
Earlier, addition of amino acids to the culture or handling medium can also help to reduce pHi fluctuations . It is also important to consider the relationship between the pH of the culture medium (pHo) and the pHi of the embryo; over a pHo range of 7.0–7.6, the pHi of the mouse zygote remained at 7.17. Rather than pHo affecting pHi directly, the presence of weak acids or bases in the culture medium dramatically affects pHi (84). The pH of a CO2/bicarbonate buffered medium is not easy to quantitate. A pH electrode can be used, but one must be quick and the same technician must take all readings to ensure consistency. Solid state probes are now available with a higher degree of accuracy. An alternative approach is to take samples of media and measure the pH with a blood-gas analyzer.
A simple and reliable method of checking the pH of a medium is to use color standards. The color standards use 0.067M solutions of potassium phosphate and sodium phosphate. These solutions are then added together in varying quantities to produce solutions of the required pH. The preparation of such standards is shown in Table 7. However, this approach requires the medium to contain phenol red.
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Temperature
Temperature fluctuations at the early cleavage stages have been demonstrated to decrease the subsequent development potential of embryos in vitro. Exposure of mouse zygotes to room temperature for just five minutes reduced cleavage rates. Increasing the exposure time to 10 and 15 minutes further decreased cleavage rates and reduced development to the blastocyst stage such that blastocyst development was half of the control after 15 minutes at room temperature. Exposure of rabbit cleavage stage embryos to room temperature for three hours also decreased cleavage rates as assessed by thymidine incorporation and development to morulae
and blastocyst stages . Exposure of human oocytes to room temperature has been reported to induce damage to the meiotic spindle. It would therefore seem advisable to maintain a constant temperature of 37_C when handling human oocytes and embryos.
Incubator/Incubation Chamber
As discussed above, for optimal development of human embryos in vitro it is important to maintain both the pH of the medium and temperature. The choice and use of incubators is therefore paramount for the success of an IVF program. Several studies have determined that embryo development in vitro is increased by restricting the opening of an incubator. Furthermore, the use of IR sensor incubators which restore CO2 levels within two to three minutes helps to alleviate the detrimental effect of repeated opening of the incubator. Alternatively, the use of modular incubator chambers (MICs) inside the incubator enables the gas phase that embryos are cultured in to remain constant. Mouse embryo development in MIC’s at the same gas phase as the incubator (5% CO2 in air) was significantly increased compared to development in the main chamber of the incubator. The use of incubators that have multiple chambers within the single incubator chamber can also reduce fluctuations in temperature and CO2 levels induced by frequent door openings.
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Light
Several studies have investigated the effect of exposure to visible light on the Development of mammalian preimplantation embryos in vitro. Collection and culture of embryos from the hamster, and rabbit under low illumination increased development and cleavage in vitro. Exposure of hamster oocytes for one hour to visible light prior to insemination disrupted the completion of meiosis and fertilization. In the human, implementation of an oocyte collection system employing low light at low oxygen concentration resulted in significantly increased rates of blastocyst formation of spare embryos and increased pregnancy and live birth weights when embryos were transferred on days 2–4 of culture. Bedford and Dobrenis reported that a 20–30 minute exposure of rabbit oocytes to light did not affect subsequent fertilization in vivo or resultant pregnancy rates. However, these oocytes were immediately transferred to the reproductive tract of a recipient and not maintained in culture. Therefore, it would seem prudent to perform all oocyte and embryo collections and manipulations under low illumination.
Incubation Volume/Embryo Grouping
Within the lumen of the female reproductive tract, the developing embryo is exposed to microliter volumes of fluid. In contrast, the embryo grown in vitro is subject to relatively large volumes of medium up to 1 mL. Consequently, any autocrine factor(s) produced by the developing embryo will be diluted and may therefore become ineffectual. It has been demonstrated in the mouse that cleavage rate and blastocyst formation increase when embryos are grown in groups (up to 10) or reduced volumes (around 20 ML). Of greatest significance is the observation that decreasing the incubation volume significantly increases embryo viability due to an increase in ICM development. Similar results have been obtained with sheep and cow embryos. It is therefore apparent that the preimplantation mammalian embryo produces a factor(s) capable of stimulating development of both itself and surrounding embryos. Once the identity of the factor(s) responsible for this stimulation of development has been determined, it can be added to the culture medium to optimize the embryo’s response. In order to culture in such reduced volumes (20–50 ml), an oil overlay is required. Although the use of an oil overlay is time-consuming, it prevents the evaporation of media, thereby reducing the harmful effects of increases in osmolality, and reduces changes in pH caused by a loss of CO2 from the medium when culture dishes are taken out of the incubator for embryo examination. Furthermore, it has been proposed that an overlay of mineral oil can actually act as a trap for potential embryo toxins. If oil is to be used then paraffin or light mineral oils are recommended. Unfortunately paraffin oil has the disadvantage of being highly labile and therefore has a relatively short shelf-life. However, in a busy laboratory the oil is used before it has a chance to become toxic. It is advisable to purchase paraffin oil in small bottles (100–500 mL) to ensure rapid use once it is opened. It is essential that each bottle of oil purchased is tested using the mouse bioassay prior to clinical use. The use of silicone oil has also been advocated for embryo culture, however it appears that embryo-toxic zinc can be a contaminant of this oil.
The washing of oil with medium is commonly recommended. However, the washing of oil has no effect on embryo development provided the oil has passed the quality control (mouse embryo bioassay; see below). Therefore, an oil which was borderline in the quality control can be made to support higher rates of embryo development after washing. However, such bottles of oil should not normally be accepted in the first place.
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WHAT STAGE SHOULD THE HUMAN EMBRYO BE
TRANSFERRED?
It has been an accepted global practice to transfer embryos on day 2 (around the 4-cell stage) or on day 3 (around the 8-cell stage) of development. However, such
cleavage stage embryos reside in the fallopian tube and not in the
Figure 6 Effect of incubation volume and embryo grouping on embryo development and differentiation. (A) A single embryo cultured in a 4-well plate or test tube, any factor produced by the embryo will become ineffectual as a result of dilution. (B) Culture of embryos in reduced volumes and/or groups increases the effective concentration of embryo-derived factors, facilitating their action in either a paracrine or autocrine manner. (C) Effect of embryo grouping on bovine blastocyst development and differentiation. Bovine embryos were cultured either individually or in groups of two or four in 50 ml drops of medium. Like pairs are significantly different (P<0.05). Abbreviation: ICM, inner cell mass.
Uterus. The significance of this observation is that in other mammalian species the transfer of cleavage stage embryos to the uterus results in lower pregnancy rates than are attained by the transfer of post compaction or blastocyst stage embryos.
With the development of more effective culture media, it has become possible to culture human embryos to the blastocyst stage as a matter of routine.
The potential advantages of blastocyst culture and transfer in human IVF include:
1. The synchronization of the embryo with the female tract, leading to increased implantation rates, therefore reducing the need for multiple embryo transfers.
2. The ability to assess embryo development and viability over extended culture. This can be achieved by both the identification of those embryos with little developmental potential, as manifest by slow development or degeneration in culture, and by the introduction of non-invasive tests of developmental potential to select the most viable embryos from within a cohort for transfer.
3. Minimizing the exposure of the embryo to a hyper stimulated uterine Environment.
4. Culture for an extra 2–3 days increases the time available between cleavage stage embryo biopsy and the time of transfer. This is of particular importance where the biopsied material has to be sent to a separate locale for analysis.
5. Assessment of true embryo viability, i.e., assessing the embryo post genome activation.
6. Reduced uterine contractions on day 5 of embryo development, minimizing the chance that an embryo will be expelled from the uterus.
7. Increased ability to undergo cryopreservation.
8. The generation of blastocysts will facilitate the introduction of Trophectoderm biopsy for the screening of genetic diseases. Trophectoderm biopsy represents the earliest form of genetic diagnosis of non-embryonic material.
9. Reduced pregnancy loss.
From such a list, it would appear that blastocyst transfer on day 5 offers several advantages over the convention of transferring early embryos on days 1 to 3 of development. However, many clinics around the world have yet to adopt extended culture and blastocyst transfer. In a review on blastocyst vs. cleavage stage transfer, it was noted that in most all papers published on blastocyst transfer, very little information regarding the culture system, save the medium type, was ever reported. This in turn makes comparing various studies rather problematic. An analysis of 16 prospective randomized trials revealed that 8 studies found a positive outcome when blastocysts were transferred compared to transfers on day 2 or day 3, with only one finding a negative outcome. The remaining seven demonstrated equivalency between days of transfer. However, more and more reports are now leaning to blastocyst transfer, especially with regard to single embryo transfer. Furthermore, it appears that cryopreservation of human embryos is most effective at the blastocyst stage.
Consequently, extended culture, facilitated by the culture systems outlined in this chapter, may represent a way to increase the overall efficiency of human IVF. Figure 7 shows the morphology of human blastocysts following culture in sequential media, together with an alpha-numeric scoring system (Fig. 8). It is evident that blastocyst score is correlated with subsequent implantation potential, with implantation rates of >60% being attainable.
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Figure 7 Photomicrographs of a human blastocyst. (A) Human blastocyst on the morning of day 5 after culture in medium G 1 for 48 hours from the pronucleate stage, followed by culture in medium G 2 for 48 hours from the 8-cell stage. Note the thinning of the zona pellucida. A dense group of cells, the inner cell mass, can be seen in the middle of the blastocyst. The embryo would be scored as 4AA. (B) Human blastocyst cultured under the same conditions as the embryo in (A). This blastocyst has commenced hatching and would be scored as 5AA.
Figure 8 Scoring systems for human blastocysts. Initially blastocysts are given a numerical score from 1 to 6 based upon their degree of expansion and hatching status: (1) early blastocyst; the blastocoel being less than half the volume of the embryo; (2) blastocyst; the blastocoel being greater than or equal to half of the volume of the embryo; (3) full blastocyst; the blastocoel completely fills the embryo; (4) expanded blastocyst; the blastocoel volume is now larger than that of the early embryo and the zona is thinning; (5) hatching blastocyst; the trophectoderm has started to herniated through the zona; (6) hatched blastocyst; the blastocyst has completely escaped from the zona. The initial phase of the assessment can be performed on a dissection microscope. The second step in scoring the blastocysts should be performed on an inverted microscope. For blastocysts graded as 3 to 6 (i.e., full blastocysts onwards) the development of the inner cell mass (ICM) and trophectoderm can then be assessed. ICM grading: A—tightly packed, many cells; B—loosely grouped, several cells; C—very few cells. Trophectoderm grading: A—many cells forming a tightly knit epithelium; B—few cells; C—very few cells forming a loose epithelium.
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SUMMARY
In contrast to the female reproductive tract, most conventional embryo culture systems are static, employing a single medium for the entire preimplantation period. In vivo, the embryo is exposed to a constantly changing environment as it passes along the oviduct to the uterus. Concomitantly, the embryo itself exhibits a changing nutrient preference, reflecting the changes in physiology and energy metabolism which occur between fertilization and the blastocyst. Furthermore, there is the added problem of in vitro induced artifacts, such as the build up of potential toxins in the medium over time, if it is not renewed.
It is therefore argued that optimal conditions for IVF and the subsequent development of the mammalian zygote to the blastocyst stage in culture requires more than one culture medium, rather than letting the embryo adapt to a static environment as its requirements change during development and differentiation.
The role of the cumulus in early embryo development has all but been ignored. However, it is now evident that the cumulus cells provide many important functions not just for the oocyte but also for the very early embryo undergoing fertilization, i.e., pronuclei formation, polar body extrusions, organelle redistribution, and cytoskeletal rearrangements. The cumulus not only provides a microenvironment that consists of low levels of glucose and high lactate concentrations, but also provides homeostatic regulation for the oocyte and early embryo. Furthermore, analysis of cumulus physiology and gene expression may assist in the diagnosis of a competent oocyte. In procedures such as ICSI, where the cumulus is stripped from the oocyte, the oocyte lacks any system to regulate the intracellular environment. Therefore, extra care must be taken in the handling of the oocyte before and after ICSI, and all media must contain amino acids in order to prevent homeostatic stress.
Finally, optimal culture conditions for the mammalian embryo depend on more than just media composition. Figure 5 attempts to highlight the interconnectedness of all components of an IVF cycle. It is hoped that this model will assist not only in improving the performance of a given laboratory, but will help in the troubleshooting.
EMBRYO CULTURE PROTOCOL
The protocol described below is based on the assumption that the media in question will be renewed after 48 hours. As discussed above, this is paramount whether one is using a biphasic or non-sequential system. It has been validated using the media and consumables listed. Any change to the protocol, whether it be a different source of oil or media needs to be validated carefully. When using the sequential media G1 and G2, embryos should be cultured in a gas phase of 5% O2 and 6% CO2. Remember, human embryos will grow at 20% O2, but development is superior at lower oxygen tensions.
Pronucleate Stage Embryos to Day 3 Culture
All manipulations of oocytes and embryos should be performed using a pulled Pasteur pipette, glass capillary or a displacement pipette. It is important to use a pipette with the appropriate size tip. For example, once the cumulus is removed (day 1 to 3), a bore of around 175–200 mM is required.
Using the appropriate size tip minimizes the volumes of culture medium moved with each embryo, which typically should be less than a microliter. Such volume manipulation is a pre-requisite for successful culture. Around 4 PM on the day of oocyte retrieval, label 60mm Falcon Primaria dishes with the patient’s name. Using a single-wrapped tip, first rinse the tip, then place 6_25 ml drops of G1 into the plate. Four drops should be at the 3, 6, 9, and 12 o0 clock positions (for embryo culture), the fifth and sixth drops should be in the middle of the dish (wash drops). Immediately cover drops with 9mL of tested oil (such as Ovoil, Vitrolife). Prepare no more than 2 plates at one time. Using a new tip for each drop, first rinse the tip and then add a further 25 ml of medium to each original drop. Place the dish in the incubator at 5% O2 and 6% CO2. Gently remove the lid of the dish and set at an angle on the side of the plate. Dishes must gas in the incubator for a minimum of four hours (this is the minimal measured time for the media to reach correct pH under oil). For each patient, set up a wash dish at the same time as the culture dishes. Place 1mL of medium G1 into the center of an organ well dish, place 2mL of medium into the outer well, and then place the dish in the incubator. If working outside an isolette, use a MOPS or HEPES buffered medium with amino acids. This should not be placed in a CO2 incubator, but rather warmed on a heated stage. Following removal of the cumulus cells, embryos are transferred to the organ well dish and washed in the center well drop of medium in the culture dish. Washing involves picking up the embryo 2–3 times and moving it around within the well. Embryos should then be washed in the two centers drops in the culture dish and up to 5 embryos placed in each drop of G1.
This will result in no more than 20 embryos per dish. Return the dish to the incubator immediately. It is advisable to culture embryos in groups of at least 2. Therefore for example, for a patient with 6 embryos, it is best to culture in 2 groups of 3 and not 4 and 2 or 5 and 1. On day 3, embryos can be transferred to the uterus in a hyaluronan enriched medium.
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Day 3 Embryos to the Blastocyst Stage
On day 3, before 8:30 am, label a 60mm dish with the patient’s name. Using a single-wrapped tip, rinse the tip, then place 6_25 ml drops of G2 into the plate. Immediately cover with 9mL of oil. Never prepare more than 2 plates at one time. Using a new tip for each drop, rinse the tip and then add a further 25 ml of medium to each original drop. Place the dish in the incubator and gently remove the lid and set on the side of the plate. For each patient, set up one wash dish per 10 embryos. Place 1mL of medium G2 into the center of an organ well dish. Place 2mL of medium into the outer well. Place into the incubator. Dishes must gas in the incubator for a minimum of 4 hours. If working outside an isolette, use MOPS or HEPES buffered medium with amino acids. This should not be placed in a CO2 incubator, but rather warmed on a heated stage. Set up one sorting dish before 8:30 AM. Place 1mL of medium G2 into the center of an organ well dish. Place 2mL of medium into the outer well. Place immediately into the incubator.
If working outside an isolette, use HEPES/MOPS buffered medium with amino acids. This should not be placed in a CO2 incubator, but rather warmed on a heated stage.
Moving embryos from G1 to G2 should occur between 10:00 AM and 2:00 PM Wash embryos in the organ well. Washing entails picking up the embryo 2–3 times and moving it around within the well. Transfer the embryos to the sorting dish and group like stage and quality embryos together. Rinse through the wash drops of medium and again place up to 5 embryos in each drop of G2. Return the dish to the incubator immediately.
If working outside an isolette, use HEPES/MOPS buffered medium with amino acids in the sorting dish. This should not be placed in a CO2 incubator, but rather warmed on a heated stage. The morning of day 5, embryos should be scored (Fig. 8) and the top
one or two scoring embryos selected for transfer. Manipulation of blastocysts requires the use of a capillary bore of 275–300 mm. Transfers should be performed in a hyaluronan enriched medium . Any blastocysts not transferred can be cryopreserved. Should an embryo not have formed a blastocyst by day 5, it should be cultured in a fresh drop of G2 for 24 hours and assessed on day 6.
QUALITY CONTROL
Mouse Bioassay
The preimplantation mouse embryo is the most widely used bioassay for medium components, culture media, and equipment used in clinical IVF. Using mice for testing media for human embryos has been the focus of much discussion due to conflicting reports in the literature of its suitability as a bioassay . Fukuda et al. reported that for the mouse, in vitro fertilization and the development of zygotes and 2-cell embryos in culture was positively correlated with the purity of the water source used in the preparation of media. In contrast, George et al. and Silverman et al.
Found that media prepared with tap water could support adequate development
of both 1-cell and 2-cell mouse embryos to the blastocyst stage respectively,
Compared to media prepared with ultrapure water. The apparent Contradiction of these studies can be resolved by taking into account the different Stages of development used at the start of culture, the types of media used, and the supplementation of medium with protein. Fukada et al. used BWW, a ‘‘simple-type’’ medium, whereas Silverman et al. (314) used Ham’s F-10. The latter medium contains amino acids, which may chelate any possible toxins present in the tap water, e.g., heavy metals. George et al. included high levels of BSA in their zygote cultures to the blastocyst.
Albumins can chelate potential embryotoxins and thereby mask the effect of any present in the culture medium. Furthermore, all studies used blastocyst development as the sole criterion for assessing embryo development. Blastocyst development is a poor indicator of embryo quality and does not reflect developmental potential. A far more sensitive and quantitative parameter is blastocyst cell number.
The sensitivity of mouse embryos to their environment is inversely proportional to the age of the embryo at recovery, i.e., 1-cell embryos are more susceptible to toxins in the medium than embryos collected at the 2-cell stage. Removal of the zona pellucida from the zygote may further increases the sensitivity of the embryo to the culture conditions, and endotoxins. Furthermore, the type of mouse bioassay performed depends upon the strain of mice used. Inbred strains and their F1
hybrids are less sensitive to their environment and, therefore, the embryos are collected at the zygote. Embryos from outbred strains of mice are more sensitive to environmental factors, however, such embryos exhibit the 2-cell block in culture media such as HTF and therefore are collected at the 2- cell stage. However, with the recent development of more optimized culture conditions, it is now possible to routinely culture such embryos from the zygote stage.
A practical mouse embryo bioassay is to culture the zygote for 96 hours in protein-free medium. The rationale for using protein-free medium is that serum or serum albumin can chelate toxins, such as heavy metal ions, present in the medium. The presence of proteins would therefore hide any potential detrimental effects of the medium. Multiple ovulations are induced by injecting four to six week old virgin females with 5–10 i.u. pregnant mares' serum (PMS), followed 48 hours later with 5–10 i.u. human chorionic gonadotrophin (HCG). Females are placed with males immediately following the second injection and mating assessed the following morning by the presence of a vaginal plug. Embryos are cultured in groups of 10 in 20 ml drops of medium under an oil overlay at 37_C. Culture dishes should be set-up and allowed to equilibrate overnight in a 6% CO2 atmosphere. To overcome any donor variation, embryos from each female should be allocated equally to each treatment group. Around 10 AM on the day of plug, females are sacrificed and the oviducts excised and placed into warm collecting medium in a petri dish. The ampullary region of the oviduct is torn open close to the cumulus mass, which is then expelled under positive pressure into the medium. The cumulus is disaggregated by the addition of hyaluronidase (1 mg/mL) in collecting medium. After around 1 min, the cumulus disperses leaving denuded zygotes. The embryos are then washed twice in collecting medium and once in the culture medium and placed into culture. Zygotes are cultured in a protein free medium and cultured for 96 hours. Rather than a single end point of blastocyst development, it is important to determine appropriate on time development at set time points, and that there is no signs of necrosis in the blastocyst (this can be visualized easily on an inverted but not a stereo microscope,. Furthermore, it is most important that the cell number of the resulting blastocysts is determined.
One can readily obtain 80% blastocyst development on day 5, but it is important also to note how embryos formed blastocysts in the afternoon of day 4 (i.e., on time development), and what the cell number is of the resultant blastocysts. There is a significant difference in the viability of blastocysts with 40 versus 80 cells.
Using such an approach to testing, one can pick up subtle problems that can exist with any media, or more typically with any contact supplies. It is important to note that just because a lot number of culture dishes has been approved safe for use in somatic cell tissue culture, does not automatically mean that it can support gametes or embryos.
With regards to alternative assays, such as hybridoma cell lines, although such cells in culture can be particularly sensitive to toxins in the medium, they are not embryonic cells and may therefore not detect potential embryo toxins. Although the mouse embryo bioassay does have a role in clinical IVF, at best it is only a test of the ability of the mouse embryo to develop. There is no guarantee that factors that do not affect the mouse will not be detrimental to the human. The ultimate quality control on all media for IVF is their ability to support human embryo growth. It is obviously unethical to use human embryos for this purpose. A possible solution would be the use of triploid embryos as an assay of media quality, as these cannot be replaced in the prospective mother. Therefore vigilant monitoring of embryo development within the IVF laboratory is not only essential, but should be considered as part of the laboratory’s overall quality control system.
Sperm Bioassay
An alternative method to the mouse embryo bioassay is to use sperm motility as a method to detect potential embryo toxins. Hamster sperm bioassay has been used to assay media and medium components for potential toxins. This assay utilizes both the number of motile sperm and degree of motility to determine the suitability of media to support embryo development. An advantage of this test is that it can be performed in four to six hours as opposed to the several days for the embryo bioassay.
Unfortunately, however, there is little clinical data on the applicability of such tests.
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