Optimal human embryo culture.

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Optimal Human Embryo Culture Jason E. Swain, PhD, HCLD1,2 1 National Foundation for Fertility Research, Colorado Center for

Reproductive Medicine, Lone Tree, Colorado 2 Fertility Lab Sciences, Englewood, Colorado

Address for correspondence Jason E. Swain, PhD, HCLD, 10290 RidgeGate Circle, Lone Tree, CO 80124 (e-mail: [email protected]).

Abstract

Keywords

► ► ► ► ►

optimal culture embryo blastocyst IVF

A large contributor to success during in vitro fertilization (IVF) lies in the processes occurring within the IVF laboratory. These processes make up the “culture system.” This system entails numerous procedures and technical steps that must be optimized to produce a competent embryo. Notably, variations exist between programs that include differences in patient population, clinical stimulation, and other factors. Thus, a single “optimal” culture system to be utilized between all laboratories is likely not feasible. Rather, laboratory procedures should be optimized based on an individual laboratory’s performance. That being said, within the scientific literature, there are key components, approaches, and techniques within the culture system that have been shown to be superior to alternatives. These key components important in improving embryo culture are discussed.

As the number of IVF cycles continues to increase worldwide, there is an obvious desire to continue to improve upon the rising success rates, as well as system efficiency. This not only improves the patient’s chance at achieving a successful pregnancy in a timely fashion, but also presumably makes the process more readily available to the wider population. There are multiple components in the IVF process that impact success. These include, but are not limited to, optimizing oocyte quality through appropriate stimulation, sperm quality, and ensuring appropriate uterine receptivity. However, despite these clinical aspects or the inherent properties of patients themselves, immense focus and responsibility is often placed on the laboratory component of the IVF process. It is often viewed that much of what drives IVF success occurs within the confines of the IVF laboratory. Certainly, when embryo development is poor or outcomes are low, one of the first suspected culprits is a suboptimal culture system. Indeed, laboratory conditions are one component of the system than can impact embryo quality. As a result, immense amounts of time and resources are devoted to developing the “optimal” system to culture embryos. The key word here, however, is “system,” as culturing embryos consists of several variables. Refining or optimizing each of these components is required to maximize embryo development and resulting outcomes. Furthermore, many products

Issue Theme Best Practices in In Vitro Fertilization; Guest Editor, Bradley J. Van Voorhis, MD

and approaches are developed using animal models and determining whether a product is “optimized” for human embryos can be difficult. Importantly, within the context of the entire culture system, “optimal” conditions may vary slightly from laboratory to laboratory. However, initial gamete quality aside and accounting for laboratory-specific considerations, there are certain critical components of the culture system that are important to maximizing resulting embryo development.

Gamete Processing As mentioned, embryo quality begins with the gametes. A significant component of gamete quality is inherent and beyond influence of laboratory conditions. Thus, it should be mentioned, that if gamete quality is poor, even an “optimal” culture system is likely unable to overcome inherent abnormalities. That being said, sperm and oocytes are cultured briefly within the laboratory and these brief periods can impact quality. Final maturation of oocytes in vitro prior to insemination/ injection is critical to maximize embryo development. Oocytes are purposely retrieved prior to ovulation at approximately 36 hours post–human chorionic gonadotropin (hCG), and though many may appear mature via presence of the first

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Semin Reprod Med 2015;33:103–117

Optimal Human Embryo Culture

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polar body, nuclear and cytoplasmic maturation are likely still incomplete. Polarized microscopy has demonstrated that in vitro matured oocytes displaying extrusion of the first polar body may still be immature; and actually be at the telophase stage of meiosis. True metaphase II spindle formation, or completion of nuclear maturation, does not occur until approximately 1 hour following polar body extrusion.1 In these in vitro matured eggs, injection less than 1 hour after polar body extrusion yielded significantly lower fertilization rates compared with other timings, with the highest success coming with injection occurring 2 to 4 hours after retrieval.1 This suggests that a brief “final maturation” may be beneficial prior to intracytoplasmic sperm injection (ICSI). Supporting this approach, when examining different injection timings following a 36-hour post-hCG retrieval, it was found that culture of cumulus oocyte complexes (COCs) for 3 to 6 hours prior to injection yielded superior fertilization and cleavage embryo development compared with COCs cultured less than 3 hours.2 Similarly, using patient randomization in a prospective trial, waiting 4 hours to inject oocytes following retrieval yielded superior fertilization and blastocyst development compared with injection immediately after retrieval.3 Thus, maturation of oocytes for 3 to 4 hours following retrieval is often recommended. Though minimal trimming of cumulus cells to remove blood clots is likely warranted at time of retrieval to avoid extended culture in their presence and possible detrimental effects of reactive oxygen species (ROS) or interference with sperm during standard insemination, it does not appear to improve oocyte quality or improve ICSI outcomes4,5 and final maturation of oocytes should occur with cumulus cells largely intact. Maturing oocytes for the final 4 hours following retrieval with cumulus cells intact appeared to yield superior fertilization and blastocyst development compared to those that were denuded immediately after retrieval3 (►Table 1). It should be noted that at least one other study has shown no difference in fertilization or cleavage embryo development following ICSI with when comparing final maturation timings of 1 to 2 hours versus 5 to 6 hours or with cumulus intact versus denuded oocytes.6 It is unknown if waiting any period of time following denuding oocytes before injection is beneficial. Final oocyte maturation following retrieval commonly occurs in a specialized fertilization media with elevated glucose concentrations, commonly formulated based on met-

abolic needs of sperm and/or cumulus-oocyte-complexes. This may be more important for cases involving standard insemination than ICSI. Alternatively, final oocyte maturation can also be performed within embryo culture media. It is unclear whether a specific media system for final oocyte maturation is superior, and differences in media formulation may be one partial explanation for contradictions in the literature regarding the impact of final oocyte maturation and specific time windows. When trying to maintain gamete quality to ensure optimal embryo development within the culture system, care should also be taken when denuding oocytes. It has been shown that mechanical manipulations during denuding can displace the polar body,7,8 thereby making it difficult to determine the location of the meiotic spindle, which can be problematic in cases of ICSI. Therefore, limiting the size of the pipet used to denude oocytes is prudent. Sizes of approximately 140 µm are sufficiently small to remove the majority of cumulus to permit ICSI and other micromanipulation procedures while avoiding undue physical stress to the egg. Additionally, at least in terms of denuding oocytes in cases of ICSI, limiting exposure to hyaluronidase is likely warranted. Common recommendations include 45 to 60 seconds, taking care to wash oocytes immediately and thoroughly afterward. At least one peer reviewed study using a retrospective analysis without sibling oocytes splits indicates that a recombinant hyaluronidase is superior to bovine-derived enzyme, yielding higher fertilization rates and lower oocyte damage.9 A prospective, randomized trial using sibling oocytes splits also showed a recombinant hyaluronidase yielded superior fertilization compared with bovine hyaluronidase,10 while another trial using sibling oocytes splits indicated noninferiority to animal-derived enzyme.11 Detailed discussion regarding sperm preparation and impact on embryo quality is beyond the scope of this review. It is known that separation and processing techniques can impact sperm quality and DNA integrity and likely resulting embryo quality.12,13 Various studies exist comparing common practices such as density gradient separation and swim-up and impact on sperm. A clear consensus as to the superior approach is not clear from the literature, and conflicting results may stem from subtle variations in techniques, such as gradient used, centrifugation speed and time of centrifugation, number of washes, media used, swim-up technique,

Table 1 Impact of final oocyte maturation timing and denuding prior to ICSI and impact on outcomes Endpoint

Immediate Denuding & Injection

Delayed Denuding & Injection

Immediate Denuding & Delayed Injection

# MII Oocytes

319

313

315

a

72.5

b

46a,b

% Fertilization

43.4

% Blastocysts

16.7aþ

42.9bþ

18.8a,b

% Clinical pregnancy

8.7

26.2

11.8

Abbreviation: ICSI, intracytoplasmic sperm injection. Note: Different letter superscripts represent differences between treatment groups. p < 0.05. þ p < 0.06.

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Abbreviation, CPR, clinical pregnancy rate. a Paternot et al (2010) used different handling media, fertilization media between groups.

ISM1 vs. GM501 Campo et al (2010)

83 vs. 84

Day 2/3

26.5 vs. 22.6%

17.8 vs. 20.0


17.8 vs. 20.0

36.8 vs. 57.5 (p < 0.08) Day 5/6 ECM/Multiblast vs. Global Supelveda et al (2009)

38 vs. 40

Sydney IVF cleavage vs. GM501 Paternot et al (2010)

70 vs. 77

Day 3 or 5

52.6 vs. 72.5

36.8 vs. 57.5 (p < 0.08)

19.6 vs. 26%

CPR/transfer (seq vs. single) Transfer day (2/3 vs. 5/6) Total transfers Media compared Study

Table 2 Comparisons of sequential and single-step media using patient randomization

In trying to discern key components of the “optimal” embryo culture system, focus immediately turns to culture media. Unquestionably, culture media plays a significant role in embryo development and quality, and therefore clinical outcomes. However, in terms of identifying or selecting the single “optimal” medium for use within IVF laboratories, this is likely a futile endeavor. Numerous variables present within the laboratory, many discussed later in this article, impact culture media performance.21 Thus, the same medium can perform differently between laboratories. The inability to identity a single optimum media is reflected by examining the numerous studies in the literature comparing one medium against another.22 Indeed, when examining prospective trials using sibling oocytes splits or patient randomization, comparison of different sequential media23–29 and comparisons between single-step culture media and sequential media30–34 fail to identify a superior approach or product (►Tables 2–5). These studies are often underpowered and not rigorously controlled, thus making it difficult to draw definitive conclusions. Indeed, no trial exists comparing every available media against each other in a controlled fashion, which would be required to identify the superior medium. It is also unclear whether changing media at 48- or 72-hour intervals, or utilizing uninterrupted culture yields superior results with modern media formulations. Individual laboratories should perform their own trials/splits to determine which product and methodology performs most successfully under their specific conditions. That being said, when selecting a medium, laboratories can make sure to consider key factors. Carbohydrates: Basal culture media will contain pyruvate, lactate, and glucose in various combinations and concentrations. This will vary from medium to medium and no clear consensus on an optimal concentration or ratio of components exists. Pyruvate can be labile in solution35,36 and may be influenced by alkaline pH, which has led some to adopt a practice of limiting the time frame they will use a media once a bottle has been opened. A proper ratio of lactate to pyruvate is likely important to maintain cellular redox potential and is often recommended that media only contain the metabolizable L-form of lactate, rather than mixture of L- and D-forms, to avoid unnecessary lowering of cellular internal pH.37

Implantation rate (total)

Culture Media

20 vs. 28%

Implantation rate day 3 (seq vs. single)

Implantation rate day 5/6 (seq vs. single)

and other variables.14 Appropriate sperm concentrations should be used for standard insemination cases, with 50,000 to 100,000 motile sperm/mL as standard accepted concentrations. Elevated sperm concentrations could be detrimental due to increased ROS, lowered pH, or other media alterations. In regard to ICSI, at a minimum, normal morphology sperm should be utilized for injection with sperm assessment at 400x. Use of higher magnification approaches, such as intracytoplasmic morphological sperm injection (IMSI) do not appear overly beneficial, though this may depend on patient population.15–18 Emerging selection approaches, such as those use hyaluronan binding to identify/select sperm, may also be useful,19 though this likely depends on the patient population and semen characteristics.20

20 vs. 43%a



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17 vs. 25 (31 had embryos from both)



No. of transfers for each media

Day 3

Day 3–5

Day 3 or 5

Transfer day (3 vs. 4/5)

58.8 vs. 48%

0 vs. 40% (0/1 vs. 2/5)

CPR D3 (seq vs. single) a

66.6 vs. 58.3%

CPR day 4/5 (seq vs. single)a

38.2 vs. 42%

0 vs. 28.6%

Implantation rate D3 (seq vs. single)

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P1 þ 20% SSS vs. IVF-50 þ 10 mg/mL HSA

P1 þ 20% SSS vs. Cook IVF þ 1% HSA

G1.1/G1.2 vs. Sydney IVF Cleavage/Blastocyst

G1.2/G2.2 vs. GIII series

G1.2/G2.2 vs. BlastAssist M1/M2

Mauri et al (2001)a

Ben-Yosef et al (2004)b

Van Langendonckt et al (2001)

Balaban and Urman (2005)c

Zollner et al (2004)d

182 (196 cycle) vs. 167 (179 cycles)

91 each media

Patients/cycles

Day 3–5

Day 3 or 5

Day 3 or 5/6

Day2/3

Day 3

Transfer day (2/3 vs. 4/5/6)

Overall CPR: 31.0 vs. 28.1%

37.8 vs. 50.3 (p < 0.01)

48.6 vs. 48.7

19.4 vs. 13.8

37.0 vs. 33.7

CPR/transfer day 3

51.4 vs. 69.4

48.6 vs. 48.7

CPR/transfer day 5/6

Overall implantation: 13.2 vs. 13.0%

14.5 vs. 25.7 (p < 0.05)

18.9 vs. 16.6

9.9 vs. 6.0

17.4 vs. 15.8

Implantation rate day 3 transfer


Abbreviation, CPR, clinical pregnancy rate. a Mauri et al (2001) also changed sperm prep media, PVP, HEPES-media, and oil for each of the treatment groups. b Ben-Yosef et al (2004) also changed sperm preparation and oocyte incubation media between treatment groups. c Balaban and Urman (2005) changed handling media and other preparatory steps between treatment groups. d Zollner et al (2004) did not report CPR or implantation based on day of transfer, but did note that no statistically significant differences existed between day of transfer or media used.

Media compared

Study

Table 4 Comparison of different sequential media systems using patient randomization

29.0 vs. 45.1 (p < 0.05)

43.1 vs. 36.1

Implantation rate day 5/6 transfer

57.1 vs. 52.6%

Implantation date D4/5 (seq vs. single)

Clinical pregnancy rate (CPR) and implantation rates were calculated using only cycles in which all embryos were transferred from one media group. Single-step media of Reed et al did not have any media refreshment during day 5 culture. The study did not permit reporting of CPR or implantation rates for single versus sequential media.

73

49

80

No. of patients (total transfers)


b

a

ECM/multiblast vs. SSM

Ciray et al (2012)

Sage vs. Global

G5 Series vs. Global

Reed et al (2009)b

Basile et al (2013)

Media compared

Study

Table 3 Comparison of sequential and single-step media using sibling embryo split

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Table 5 Comparison of sequential media systems using sibling embryo splits Study

Media compared

No. of patients (total transfers)

No. of transfers for each media

Transfer day (2/3 vs. 5/6)

CPR/transfera

Implantation rate (seq vs. single)

Hambiliki et al (2011)b

G1.5 vs. EmbryoAssist

108


Day 2/3

46.4 vs. 36.4

40.9 vs. 37.5

Sifer et al (2009)

GIII vs. ISM1


Day 3

44.2 vs. 35.3

32.2 vs. 28.4

Glucose can be present or absent for early cleavage development38,39 but is required for later stages to promote compaction and blastocyst formation. Amino acids: Amino acids support numerous cellular processes, including acting as metabolites, osmolytes, antioxidants, and buffers, and likely help alleviate stress in the embryo culture system.40 Brief period of culture with no amino acids present can impair mouse embryo development.41 Thus, all embryo culture media should contain some assortment of amino acids. While animal studies have given some insight,42–44 it is likely impossible to determine the optimal mixture and concentrations of specific amino acids for human embryo culture. Studies have identified glycine, taurine, and glutamine as important amino acids for human embryos.45–47 If glutamine is present, modern embryo culture media should include the dipeptide form of the amino acid. Use of the stable dipeptide-glutamine helps avoid ammonium accumulation and the associated detrimental effects on human embryo development, and appears superior to glutamine.48–51 No clear consensus exists regarding whether glycyl-glutamine or alanyl-glutamine is the superior dipeptide for human embryo culture. Antibiotic: Though not required, an antibiotic of some sort should likely be included in embryo culture media. If antibiotic is included, gentamicin, rather than penicillin-streptomycin (pen-strep), should be used. Pen-strep–resistant cases of contamination in IVF52 were 91%, and pen-strep was detrimental to human and hamster embryo development,53,54 possibly via damage to embryo chromatin, gene expression, and increased apoptosis.55 Additionally, gentamicin is heat and pH stable, and of 70 bacterial strains in contaminated embryo media, all were sensitive to gentamicin.52 Protein: Protein supplementation to culture media can have a dramatic impact of embryo development and resulting outcomes. The macromolecule supplementation can act as a surfactant, as a nitrogen source, to stabilize membranes, to modulate physical microenvironment, as a carrier molecule for other compounds, or even to bind trace elements/toxins. A recent report even indicates that protein in culture media can impact human birthweight,56 though results of the study should be interpreted with caution due to lack of control over other system variables. Regardless, it is irrefutable that protein supplements are one of the least defined components of

the culture system, and thus a large source of potential variation. For example, stabilizers/preservatives used when making protein solutions, such as octanoic acid, may be embryotoxic.57 In addition, as mentioned, other proteins, growth factors, and hormone “contaminants” may be present at varying levels in protein preparation.58–60 Some clinical IVF media currently include growth factors, though limited data exist to support their purposeful use61–63 and this remains a controversial topic.64 Thus, to optimize performance of the culture system, specific lots of protein should be screened for efficacy prior to clinical implementation Primary protein supplements used in clinical embryo culture media include human serum albumin (HSA), as well as complex protein supplements containing HSA and a combination of α and β globulins.65,66 Recombinant albumin is also available, as well as a dextran-based macromolecular supplement. Data suggest that a complex protein supplement with globulins is superior to HSA for animal embryos,65,67 human embryo development,68,69 and even live birth.70 This may be due to growth hormones and other components in the complex protein products.59 Recombinant albumin gave comparable embryo development to HSA in a prospective randomized trial and may reduce media variability.71 Optimal concentrations of protein are unknown, though 5% of albumin or 10% of complex protein products are commonly used and higher concentrations of up to 20% of a globulin supplement may be beneficial in certain circumstances.72 Oil: Oil overlay is commonly used during embryo culture to prevent media evaporation, which can result in media osmolality increase and compromised embryo development. Oil is required if using modern benchtop incubators with no humidification. However, open culture, or culture without oil overlay, may also be used if humidification is present and a sufficient volume of media is used. Similar to protein, despite improvement in refinement and testing, oil overlay products can differ significantly due to contaminants and are a large source of variation within the culture system. Washing of oil with either water or culture media can help alleviate some toxicity concerns73 and a good practice to ensure that all oil is washed. Additionally, proper storage is required to avoid peroxidation of oil, which can lead to embryo toxicity.73–76 Recommendations often include keeping oil out of direct light and storing at 4°C. Seminars in Reproductive Medicine

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Note: Limitations often exist with media comparisons, including the changing of several variables, making it very difficult to determine impact of the media itself. a Clinical pregnancy rate (CPR) and implantation rates were calculated using only cycles where all embryos were transferred from one media group. b Hambiliki et al (2011) used different fertilization media and different proteins for each group.


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There are several oil products for embryo culture, including paraffin oil and light mineral oil. Products are sold in plastic or glass bottles. There is no clear consensus on a superior oil type, and preferences of a particular laboratory may be based more on oil viscosity and impact of dish preparation techniques. Regardless of the oil, testing using an approved bioassay is recommended prior to use. pH: pH of culture media is of special concern, as this parameter is directly controlled by and can vary significantly between laboratories. Media pH is primarily determined by the bicarbonate concentration in the media itself and the CO2 concentration of the culture incubator. However, factors such as laboratory elevation, protein supplementation as well as different media types all impact pH and prevent a single incubator CO2 value from being used throughout the field. Thus, this parameter should be measured and set by each laboratory to account for their particular conditions. Though embryos can develop over a range of media pH, it appears it may impact embryo development. It is known that changes in internal pH can impact embryo metabolism, development, and even fetal growth in rodents.77–79 Certain cell types, such as the denuded mature oocyte or thawed embryos, lack robust internal pH regulation and may be more susceptible to changes in media pH.77,80,81 Preliminary data suggest media pH may impact human embryo development.82,83 However, it is difficult to isolate pH as the sole variable as both bicarbonate or CO2 values change to alter media pH and both can influence embryo biosynthetic pathways. Regardless, maintaining tight control over media pH is likely prudent as part of a rigorous QC program. Unfortunately, no optimal pH has been identified for human embryo culture.84 Phenol red is a common media component added to media to help visually monitor pH changes. This may be useful to some to gauge media age or incubator function, etc. Some media have chosen to exclude this indicator due to possible negative impact on embryo development. It has been suggested that possible estrogenic effects of phenol red could be problematic. However, estrogenic activity appears to be due to the contaminants in certain preparations, not due to the phenol red itself.85 In addition, it has been suggested that phenol red use may lead to ROS generation following light exposure,86 though this has not been shown using modern human embryo culture media or under conditions utilized for modern human embryos. Therefore, it cannot be concluded that phenol red conveys a negative impact, and its use may be warranted in a selected commercial media if a visual pH indicator is useful for a particular laboratory. Related to pH, use of media that include synthetic zwitterionic buffers to stabilize pH for procedures outside the laboratory incubator may impact embryo development. These buffered media are often used for procedures such as oocyte retrieval, sperm preparation, ICSI, embryo transfer, and cryopreservation. Common buffers include HEPES, MOPS, or their combination. Despite poorly designed studies indicating buffers, such as HEPES, may be toxic for human gametes or embryos following ICSI,87 numerous studies have demonstrated safety of both HEPES and MOPS and there Seminars in Reproductive Medicine

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is no clear advantage of one over the other in terms of embryo development.88,89 When other variables are controlled, including pH, gas atmosphere, bicarbonate levels, and substrate composition, no detrimental effect of HEPES or MOPS have been observed.88–91 Importantly, phosphate buffered saline (PBS) should not be used for procedures involving gametes and embryos, as concentrations of phosphate required to provide adequate buffering have consistently been shown to impair embryo development.89,91 Of note, if care is taken in the laboratory and proper equipment is used, including gassed isolette incubator chambers, use of these specialized buffered media can be avoided for oocyte retrieval or embryo transfer and may help reduce the number of variables in the culture system. Osmolality: Osmolality is a component of culture media that can significantly impact embryo development. Several studies exist demonstrating that high osmolality can result in developmental arrest of rodent embryos.92–96 The impact of osmolality on embryo development depends on media composition, as components, such as key amino acids, can act as osmolytes and help cells regulate volume. Though osmolality is set by the commercial media manufacturer, conditions in the laboratory can alter osmolality and thereby impact embryo development. Factors impacting evaporation can result in media osmolality shifts and are present during media/dish preparation.97 Time, media volume, airflow, and temperature can all result in detrimental osmolality shifts and compromise embryo development. Thus, a strict regime must be followed when performing daily media preparation that limits these variables to avoid altering media osmolality. Additionally, evaporation within the incubator must also be considered. This is especially important considering modern incubators that are devoid of humidification.

Incubators Minimizing environmentally induced stress within the IVF laboratory is crucial in creating a culture system optimized for embryo development and to achieve maximal assisted reproductive outcomes. Key environmental variables to consider within the culture system include properties of culture media, such as the aforementioned pH and osmolality, as well as variables such temperature and air quality. Importantly, all of these potential stressors can be impacted by the laboratory incubator. As a result, the incubator is arguably the most important piece of equipment in the laboratory. Thus, incubator selection and management is critical to ensuring optimization of the culture system. Multiple incubator types exist with varying capabilities and differing methods of regulating their internal environment. Box-type incubators, initially developed to hold multiple flasks of somatic cells, have been long used for clinical IVF. These were later adapted to smaller box-type incubators. Most recently, a variety of embryo-specific benchtop incubators have been developed. These newer incubators tend to have small chambers and direct heat to try to speed environmental recovery following incubator openings/closings. As a result, selection of an appropriate culture incubator for the


108

IVF laboratory has become a complex process. To date, there is no clear consensus as to a superior incubator type, as efficacy and environmental stability rely heavily on incubator use and management.98 Thus, an appropriate number of incubators are required to avoid excessive openings/closings. Reduced incubator opening appears to benefit mouse embryo development,41,99 as well as improve human blastocyst development.100 A mix of incubator types within an individual laboratory permits a wide variety of uses to accommodate different dish or test tube types, as well as emerging culture technology. That being said, there are certain environmental variables that should be utilized within any incubator type. Atmosphere: Today’s optimal culture system requires the use of low oxygen within the incubator. Numerous animal studies; including of the mouse, cat, sheep, pig, cow, and rat, have repeatedly shown the benefit of using reduced oxygen concentrations compared with atmospheric levels. Similarly, use of low oxygen has also been studied extensively in the human and the majority of studies demonstrate an improvement in embryo development.101–112 Admittedly, confounding variables in some experimental designs can sometimes make direct comparisons difficult. That being said, no studies indicate a detriment of using low oxygen. Importantly, use of low oxygen throughout the entire culture period through day 5/6 appears to be required to see the most benefit.107,109,113 In one of the better designed studies, a prospective RCT in a high performing program showed that use of low oxygen significantly improved pregnancy, implantation, and live birth rates (►Table 6).113 Similar improvements in live birth have been reported by others.114 The exact mechanism of the benefit of low oxygen use for embryo culture is unknown. Use of low oxygen may be superior due to improved embryo metabolism, reduced ROS, or perhaps even improved air quality. It is known that air quality within the incubator is another component of the culture system that can impact embryo development.115 The presence of volatile organic compound (VOCs), such as aldehydes or toluene, within the air of IVF laboratories has been well-documented throughout the literature,116–118 and it is well accepted that poor air quality can compromise the culture system, whereas measures to improve air quality result in improved outcomes,119 presumably due to the detrimental effects of this VOCs on the preimplantation embryo.116,118,120,121 Low oxygen incubators use approximately 90% nitrogen or premixed cylinders of gas in place of room air. This may provide purer gas quality within the laboratory incubator as compared to those that use high

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oxygen/room air. However, this may not always be the case, as gas cylinders can be the source of harmful VOCs, such as benzenes or alcohols.116,117 Thus, inclusion of specialized inline filters from the gas supply or filters placed within the incubator can remove VOCs and improve outcomes,121–124 though this is not always the case122,125,126 and may depend on the existing air quality in the laboratory/ incubator. Temperature: Temperature is another variable of the culture system that may impact efficacy. It is well known that temperature can impact various aspects of gamete and embryo function, most notably, meiotic spindle stability127–129 and possibly embryo metabolism.130 Studies have reported that maintaining media temperature around 35 to 37°C compared with 25 to 30°C during retrieval is beneficial for bovine embryo development and mouse embryo gene expression.131,132 Maintaining temperature stability around 37°C during human oocyte injection also improved fertilization rates and embryo development.128 However, the optimal temperature at which to culture embryos remains unknown. Commonly, 37°C is used to culture embryos and is based on the estimate of human core body temperature. However, body temperature varies and is likely not exactly 37°C. Temperature can vary based on time of day, sex, and between individuals. Furthermore, temperature of the female reproductive tract may actually be slightly less than core body temperature. The temperature of the human follicle is approximately 2.3°C cooler than core body temperature, and in animal models the fallopian tube is approximately 1.5°C cooler than core body temperature.133–138 As a result, the question has been posed “whether human IVF and related procedures should be carried out, at, say, 35.5 to 36°C rather than at 37°C.”130 Use of a 1.5°C lower temperature would presumably lower the metabolic rate of the embryo approximately 15%, which may result in a more “quiet” metabolism, thereby possibly benefiting embryo development.130 However, this hypothesis has not been proven. Interestingly, retrospective, observational data on incubator performance indicated that an increased pregnancy rate was obtained from incubators with a temperature of 36.96° C 0.13 compared with those with a temperature of 37.03° C 0.13.122 However, this study was not well controlled and results cannot be attributed to temperature alone. In addition, biologic explanation for how such a small temperature difference (0.07), which falls into the acceptable variation range of many thermometers, could result in observed developmental differences in unknown. However, this has led some

Table 6 Effects of reduced oxygen concentration in a predominantly blastocyst transfer program Endpoint

21% O2

5% O2

p value

Clinical pregnancy rate

56/115 (48.7%)

74/115 (64.3%)

0.027

Implantation

95/267 (35.6%)

122/247 (49.4%

0.003

Live birth

49/115 (42.6%)

66/115 (57.4%)

0.043

Note: When examining all patients in a prospective randomized trial, extended culture in low oxygen significantly improved clinical pregnancy, implantation, and live birth. Source: Adapted from Meintjes et al.70,113 Seminars in Reproductive Medicine

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laboratories to culture embryos around 36.5 to 36.8°C in order to avoid the possibility of temperature rising above 37°C. It has been shown that overheating oocytes to a temperature of 39°C or greater is detrimental to formation of the meiotic spindle during maturation in vitro.127 However, there is no proof that 37°C is too high for human embryo culture or that this may be detrimental. In fact, animal embryos have mechanisms that help convey thermotolerance to deal with slight elevations in temperature,139–142 and the same may be true for human. Recently, a prospective randomized controlled trial utilized patient embryo splits and examined the effect of culture at either 37 or 36°C.143 Controlling for temperature variations, incubator type, and pH, the study demonstrated that culture of human embryos at 37°C yielded higher average cell numbers at day 3 of development, higher blastocyst formation, and higher useable blastocyst rates compared with 36°C. No differences were observed in rates of aneuploidy or implantation (►Fig. 1). More detailed analysis of the optimal temperature for IVF is required, but the best practice at the moment appears to be maintaining temperature around 37°C. There is no clear advantage of one incubator type over another in terms of temperature regulation, though some types may recover temperature faster or maintain temperature better than others under various circumstances.98

Group Culture Embryo density: It is known that embryos secrete various proteins during their culture, and research analyzing the

embryo secretome has become an active area of research.144–148 In addition, using sensitive chemical microprobes, gradients of dissolved oxygen, potassium, and calcium have been shown to extend around the embryo(s) that are measurable beyond 50µm from mouse embryos cultured in vitro.149–151 Thus, embryos may modify their own microenvironment and this may impact embryo development. Considering the ability of embryos to modify their microenvironment, group culture has long been known to be beneficial for animal embryos, from both mono- and polyovulatory species. Various studies have shown a benefit of group culture of embryos from the rodent152–156 as well as domestic species such as the cow, sheep, and pig.157–164 Various factors involved in group embryo culture are likely important, and they include the ratio of media volume used to the number of embryos (embryo density), embryo spacing, as well as quality of the companion embryos.165 While detailed analysis on factors such as embryo density, spacing, or identification of zones created around human embryos is largely lacking, there are studies indicating beneficial effects of group embryo culture. Group culture of embryos significantly enhanced cleavage rates but not embryo morphology grades as compared with embryos grown individually in one study of 55 patients.166 Another small study found significantly improved clinical pregnancy rates with group culture as compared with individually cultured embryos (43 vs. 24%).167 Rebollar-Lazaro and Matson168 retrospectively analyzed patients who had embryos cultured from days 1 to 3, either individually or in groups of three to five embryos. Culturing in groups for the first 3 days had no impact on pregnancy or implantation rates compared with

Fig. 1 Impact of different culture temperature on human embryo development. (a) A sibling embryo split design was used, and incubator type, temperature variation and pH were accounted for as confounding variables. (b) Data indicate that 37°C is superior to 36°C in terms of embryo development. Different superscripts within a column represent statistically significant differences between temperature treatments p < 0.05 (Adapted from Hong et al, 2014.) Seminars in Reproductive Medicine

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Table 7 Impact of group culture on human embryo development Individual culture Compaction

98 (38.9%)

a a

Group culture with no contact

Group culture with contact

116 (38.2%)

a

188 (49.5%)b

124 (40.8%)

a

212 (55.8%)b

Blastocyst formation

114 (42.5%)

Blastocyst quality

44/68 (64.7%)a

66/96 (68.6%)a,b

122/154 (79.2%)b

Live birth

5 (38.5%)

5 (41.7%)

28 (62.2%)

those embryos cultured individually, but did result in more usable blastocysts. This is similar to a prospective report that indicated no benefit of group culture of human embryos prior to day 2 or 3 transfer,169 suggesting extended culture may be required to observe the full benefits of communal culture. Finally, an interesting prospective trial of 72 patients compared outcomes of embryos split between three treatment groups: individually cultured embryos, embryos cultured in groups in which embryos were physically separated but able to exchange media, or embryos cultured in groups without physical separation. Importantly, all treatments were included within the same culture dish to help control for variables. Group culture of embryos without physical separation resulted in greater rates of compaction on day 4 and blastocyst formation on day 5 compared with group culture with physical separation and embryos cultured individually. There was a trend toward a higher live birth rate with group culture of embryos without physical separation compared with individual culture (►Table 7). This indicates that extended group culture of embryos may be beneficial for human preimplantation embryo development, and that physical contact or proximity of embryos seems to be an important factor.

Culture Dishes Importantly, new micro- or nanovolume culture dishes are being developed, and use of these dishes to optimize the microenvironment may help further improve the benefits of group embryo culture, as discussed previously. Additionally, these dishes could perhaps improve individual embryo culture or be used for study of embryo metabolomics, time-lapse imaging, or embryo tracking for genetic diagnosis.171–173 At least one preliminary study indicates that a microvolume approach using the well-of-a-well (WOW) dish may be superior for human embryo development compared with larger volume culture.174 This WOW approach uses microwells to house individual embryos, but all embryos share a common media overlay and reside in close proximity to each other. Currently, there is no consensus on a superior culture dish or volume of media used for culture. Directly related to optimizing the culture system is the need to ensure that the culture dish and other contact materials used are nontoxic. Though the importance of oil and avoiding toxicity has been discussed, this also includes culture dishes and other contact materials that could

inadvertently alter media characteristics and growth conditions to impair embryo development. Contact material testing is one of the most important aspects of laboratory QC/QA and is paramount for optimizing the culture system. It is well documented that not all products, despite being packaged or sold as sterile, are inert in terms of impact on embryo development.175,176 Thus, verification of material safety is required prior to clinical use. This is usually performed using a relevant bioassay, such as the mouse embryo assay (MEA) or the human sperm survival assay (HSSA), also known as the human sperm motility assay (HSMA). Comparisons and the merits of these two assays have been discussed elsewhere177–183 and each can be useful in their own respect, with factors such as cost and availability warranting attention. Perhaps more relevant, the sensitivity of the bioassay is important and approaches can be modified to increase this sensitivity to ensure that subtle material toxicity can be detected. Examples of approaches used to increase sensitivity are use of the one- versus the two-cell MEA, outbred versus inbred versus hybrid mouse strains, blastocyst cell counts versus simple blastocyst formation, exclusion of protein from media versus protein inclusion, and use of a simple media versus a more robust complex media.184–186 Importantly, thresholds must be set for an assay to “pass,” and these thresholds should be rigorous. Each laboratory can determine which assay and threshold suits their needs regarding sensitivity and cost. A commonly used and often recommended sensitive assay includes using the one-cell MEA, noting time-appropriate embryo development with rate of expanded or hatching blastocyst of greater than70% at 96 hours (4 days) of culture. Real-time assessment of morphokinetics may also be useful in helping improve sensitivity of the MEA.187 One interesting point is that many materials from manufacturers can now be purchased as “pretested,” having passed some manner of bioassay. However, these manufacturer’s bioassays may not meet the sensitivity criteria of individual laboratories. Furthermore, factors impacting material quality can convey detrimental effects at a point following initial successful passing of the bioassay. For example, improper storage conditions in warehouses or delayed effects from prolonged storage can cause a “prescreened” item to later become toxic once received in the laboratory. A common example of this appears to be toxicity associated with mineral oil.73,175,187 Also, potential toxicity from a single item could be very minor, but when used in the context of several Seminars in Reproductive Medicine

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Note: Use of sibling embryos indicates that group culture improves embryo development following extended culture. Different superscripts represent statistically significant differences between culture treatments, p < 0.05. Source: Adapted from Ebner et al.170


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sequential items used in a particular laboratory culture system, this toxicity could be compounded due to interactions with other system components. This has been noted anecdotally across several laboratories, in which “tested” materials are later found to have a detrimental impact. Therefore, more stringent laboratories often retest certain prescreened materials, testing them under their own laboratory conditions before releasing them for clinical use.

4 Ebner T, Moser M, Shebl O, Sommergruber M, Yaman C, Tews G.

Conclusion

7

It is evident that there are numerous variables to consider when optimizing an embryo culture system. Though most variables are consistent between laboratories, small differences in laboratory conditions will affect what is the “optimal” embryo culture system for a given program. Of course, even if a culture system is optimized, embryo selection remains a critically important factor to maximize outcomes. In fact, if a system is optimized to maximize embryo development, selection becomes even more difficult, as there are more “good-quality” embryos from which to choose. The best embryo selection method to use then becomes critically important and is the topic of much active research. That being said, the following key, evidenced-based factors should be included in a highly successful culture system: 1. Human eggs should be cultured for 3 to 4 hours with cumulus cells intact following retrieval to allow for optimal egg maturation prior to ICSI. 2. Use of low oxygen should be followed for all embryo culture, with the most benefit occurring when used for extended culture to the blastocyst stage. 3. Careful attention to detail is required when preparing culture dishes to avoid increased in media osmolality, which can compromise embryo development. 4. Culture media should be selected based on an individual laboratories performance, but media should include some assortment of amino acids, and if containing glutamine, should contain a dipeptide form. 5. Careful control of incubator workflow is required to maximize embryo development, making sure that sufficient numbers of incubators are available to help limit openings and closings to keep strict control of atmosphere (pH) and temperature. 6. Maternal testing and an appropriate biossay should be in place to avoid toxicity.

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Composition of protein supplements used for human embryo culture.

Composition of commercial media used for human embryo culture.

Embryo culture media for human IVF: which possibilities exist?

Mammalian preimplantation embryo culture.

Whole embryo culture and teratogenesis.

Human embryo research.

Supplementation of bovine embryo culture medium with L-arginine improves embryo quality via nitric oxide production.

The ART of studying early embryo development: progress and challenges in ruminant embryo culture.

Contribution of human somatomedin activity to the serum growth requirement of human skin fibroblasts and chick embryo fibroblasts in culture.

Hybridization in the genus Lens by means of embryo culture.

Commercially available equipment suitable for post-implantation embryo culture.

Reduction of embryotoxicity by protein in embryo culture media.

Whole embryo culture, teratogenesis, and the estimation of teratologic risk.

Does embryo culture at low oxygen tension improve ART outcomes?

Interspecific hybridization of perennial Medicago species using ovule-embryo culture.

Optimization of mouse embryo culture media using simplex methods.

Survival of chick embryo sympathetic neurons in cell culture.

Techniques for assessment of teratologic effects: embryo culture.

Preparation of rat serum suitable for mammalian whole embryo culture.

Two-stage carcinogenesis with rat embryo cells in tissue culture.

Observations on the fusion of chick embryo myoblasts in culture.

Co-culture for embryo development: is it really necessary?

Organ culture of the mammalian and avian embryo otocyst.

Parameters determining isotretinoin teratogenicity in rat embryo culture.