Middle East Fertility Society Journal (2010) 15, 179–182

Middle East Fertility Society

Middle East Fertility Society Journal

www.mefsjournal.comwww.sciencedirect.com

ORIGINAL ARTICLE

Shelf life of embryo culture media: Buffering potential

of media apparently not the determining factor

Amjad Hossain *, Collin Osuamkpe, Safina Hossain, John Phelps

Division of Reproductive Endocrinology and Infertility, Department of Obstetrics and Gynecology,University of Texas Medical Branch, 307 University Blvd., Galveston, TX 77555, United States

Received 29 January 2010; accepted 16 March 2010Available online 19 August 2010

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KEYWORDS

Culture media;

Expiration;

Buffering capacity;

Nutritional value;

Shelf life

Corresponding author. Tel.:

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mail address: amhossai@utm

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Abstract Objective: To investigate if the buffering power of embryo culture media is compro-

mised by expiration.

Design: Buffering potential of expired culture media was evaluated by measuring pH values.

Setting: Fertility Laboratory, the University of Texas Medical Branch, USA.

Materials and methods: The pH of expired culture media were measured and compared with those

of unexpired ones in three experimental conditions: (1) pre-incubation, (2) CO2 incubation and (3)

post-incubation. Sequential media, comprising Quinn’s advantage fertilization, cleavage and blas-

tocyst media (Sage assisted reproduction products Inc.), were used. The media were divided into

expired and unexpired groups, based on the manufacturer’s indicated expiration dates. The unex-

pired group was evaluated in their original condition (first time opened). The expired media bottles

underwent more than one opening. The pH of the media was measured sequentially in conditions 1,

2 and 3.

Results: The expired media (30–390 days) displayed significantly (p< 0.05) different pH values

compared to unexpired media in pre-incubation condition. However, expired media were capable

of yielding similar pH values (p P 0.23) to those of the corresponding unexpired controls in CO2

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180 A. Hossain et al.

incubation as well as post-incubation suggesting that the buffering capacity of the media is appar-

ently not affected by expiration.

Conclusion: Time-dependent nutrient depletion, not the weakening of the buffering system, is

probably the determining factor in setting the shelf life of the culture media.

� 2010 Middle East Fertility Society. Production and Hosting by Elsevier B.V. All rights reserved.

1. Introduction

The buffering capacity of the culture media, used in assisted

reproductive procedures, is critically important. It assuresand maintains the required pH in the culture system. The bio-chemical mechanisms involved in the buffering of the culture

media are well documented (1–4). The bicarbonate bufferemerged as the ideal buffering agent for the mammalian cellculture including human embryo culture (5–7). If media underincubator conditions fail to provide the expected pH due to its

suboptimal buffering capacity, optimum embryo growth can-not be expected (8–11). Efficient buffering potential of themedia secures a stress-free environment for optimum embryo

development so that the desired end result of procedure,implantation and pregnancy is achieved (3,12,13). Realizingthe importance of pH in embryo culture media, the laboratory

personnel monitor the pH in the culture system, either directlyor indirectly (3,12,14). In direct monitoring, the pH of themedia is measured by a pH meter (12–14). In indirect monitor-

ing, the CO2 level in the incubator is measured to predict thepH of the media (4,7,12).

The required level of CO2 is empirically determined ineach laboratory setup, taking into consideration that the

CO2 concentration that yields a specific pH value can varygeographically with the altitude of the region where the lab-oratory is located (3,7). Furthermore, different makes of

media may require different concentrations of CO2 to attainthe desired pH at a given altitude (12,14). The bottom lineis that no matter what method is followed for pH monitor-

ing, maintaining an optimum pH in the culture system isabsolutely critical.

Fertility laboratories all over the world rely heavily onthe utilization of commercially available media for assisted

reproductive procedures (12,15). Several media manufactur-ing companies in the United States and in Scandinaviancountries such as Sweden and Denmark have worldwide dis-

tribution centers (15). Currently, a vast majority of fertilityclinics across the globe utilize the media produced by theseinternational media companies (7,12,15). A common charac-

teristic of all these commercial media is that they possess ashort life span, usually expiring in 3–4 weeks. From a regu-latory point of view it is illegal to use expired media in clin-

ical procedures involving patients (16,17). Thus, by settingsuch a short shelf life, the media manufacturers are indi-rectly forcing the media users to discard a considerableamount of the unused media. Two vital issues, depletion

of nutrients and weakening of the buffering capacity as timeprogresses, probably guide the manufacturers to set the shelflife for the safe use of the media. In this study, we investi-

gated whether or not the buffering capacity of commercialmedia is compromised by the expiration dates indicated bythe manufacturers.

2. Materials and methods

The pH of expired culture media were measured and compared

with those of unexpired ones in three different experimentalconditions to get an answer to the question raised above.Sequential media, comprising Quinn’s advantage fertilization

(Q-fert), Quinn’s advantage cleavage (Q-cleve) and Quinn’sadvantage blastocyst (Q-blast) obtained from SAGE assistedreproduction products Inc. (Cooper Surgical Company, Trum-bull, CT), were used in the study. The media were divided into

expired and unexpired groups, based on the manufacturer’sindicated expiration dates. The media bottles were stored inthe refrigerator at 4 �C keeping the caps properly tightened

during use and also after expiration. Media type, lot number,expiration date, and the volume remaining in the bottles weredocumented prior to pH measurement. The unexpired group

was evaluated in their original condition (first time opened).The expired media bottles underwent more than one opening.Along with the removal of a variable amount of media for use,

the rest were stored before they were brought under investiga-tion. In both cases, the media bottles were opened as neededbut for only short periods of time (615 s in each incident).

The pH of the media was measured under the following

three conditions. Condition 1: media bottles with caps tight-ened were kept out of the refrigerator until the media reachedambient temperature (23–24 �C). Condition 2: media volume

of 5 mL in Falcon tubes was kept in the incubator (37 �Cand 6% CO2) with caps loosened for 26 h. Condition 3: incu-bated media (condition 2) were stored in the refrigerator with

caps tightened for 72 h and then brought back to ambient tem-perature. The pH measurement was performed sequentially inconditions 1, 2 and 3, respectively, following the manufac-turer’s instructions to use the Orion 410+ pH meter (Thermo

electronic corporation). A total of 10 unexpired and 85 expiredmedia bottles were analyzed in condition 1. For conditions 2and 3, 10 media bottles in each media type (Q-fert, Q-cleve

and Q-blast) reflecting P30 days of post-expiration were se-lected from 85 expired media for comparison with the unex-pired controls. Data were expressed as the mean ± SD.

Analysis of variance, which included t-test and correlationcoefficient, was used for statistical analysis with Sigma Plotsoftware, version 11.0 for Windows. p < 0.05 was considered

statistically significant.

3. Results

The pH values recorded for unexpired and expired media areshown in Table 1. The expired media, in condition 1, exhibitedsignificantly (p 6 0.05) higher pH compared to that of unex-pired ones. The Q-fert, Q-cleve and Q-blast revealed pH of

7.77 ± 0.15, 7.75 ± 0.15 and 7.72 ± 0.12 in the expired state,and 7.29 ± 0.03, 7.23 ± 0.04 and 7.27 ± 0.04 in the unexpired

Table 1 Comparison of pH of Q-fert, Q-cleve and Q-blast media under three different treatment conditions.

Media type Media group Treatment conditions

Condition 1 Condition 2 Condition 3

Q-fert Unexpired 7.29 ± 0.03 7.35 ± 0.04 7.41 ± 0.05

Expired 7.77 ± 0.15 7.38 ± 0.05 7.44 ± 0.06

Q-cleve Unexpired 7.23 ± 0.04 7.29 ± 0.05 7.38 ± 0.04

Expired 7.75 ± 0.15 7.35 ± 0.07 7.40 ± 0.05

Q-blast Unexpired 7.27 ± 0.04 7.32 ± 0.05 7.41 ± 0.04

Expired 7.72 ± 0.12 7.35 ± 0.06 7.43 ± 0.04

Treatment conditions were defined in Section 2. The media groups (unexpired vs. expired) in each media type were significantly different in

condition 1 (p 6 0.05) but not in conditions 2 and 3 (p P 0.23).

Shelf life of embryo culture media: Buffering potential of media apparently not the determining factor 181

state, respectively. The pH differences between the expired and

unexpired groups in all three media types (Q-fert, Q-cleve andQ-blast) were statistically significant (Table 1). There was aconsiderable variation in the volume of the media that re-mained in the expired bottles: Q-fert 35.8 ± 14.3 mL, Q-cleve

32.6 ± 16.1 mL and Q-blast 37.3 ± 11.1 mL. The expiredgroup also represented wide expiration durations ranging from30 to 390 days. A significant negative correlation showing

lower volume and higher pH was seen in all the threemedia (Q-fert: r = �0.51, p = 0.005; Q-cleve: r = �0.63,p = 0.001; Q-blast: r= �0.50, p = 0.003) in condition 1.

Also, there was a significant (p < 0.05) positive correlation (rvalues for Q-fert, Q-cleve and Q-blast: 0.77, 0.63 and 0.63,respectively) relating longer expiration duration and higherpH.

Under CO2 incubation (condition 2), the expired Q-fert,Q-cleve and Q-blast produced identical pH values (p P 0.23)when compared to that of the respective unexpired counter-

parts (Table 1). Further, the CO2 equilibrated expired mediastored for 72 h (condition 3) revealed the same pattern of pHchanges compared to that of the corresponding unexpired con-

trols (Table 1).

4. Discussion

The media pH was measured under three experimentalconditions: condition 1 represented the original state of therespective media bottles; condition 2 provided incubator envi-

ronment (temperature and CO2) to see if the expired media be-have the same way as the unexpired ones in maintaining pH inthe culture; and condition 3 allowed us to see how the unex-pired and expired media perform after equilibration with

CO2. The unidirectional pH changes confirming a shift to-wards alkalinity that is seen in all the expired media is a knownfact. In other words, the expired media becoming alkaline is

not unexpected. It is the characteristic of all bicarbonate-buf-fered media to turn alkaline if exposed to ambient air(4,6,12). However, in this study we showed that the degree of

alkalinity of the expired media is affected by the expirationlength as well as the volume of the media.

More importantly, it was interesting to find that the expired

media, irrespective of volume and expiration duration, werecapable of yielding a pH value at the same level as that ofthe unexpired ones (controls) in the incubator environment.As seen, in CO2 incubation (condition 2), the pH of expired

and unexpired media were within the manufacturer’s indicated

expected range (7.20–7.40) and, more importantly, were not

different. Further, the CO2-equilibrated expired media in72 h storage (condition 3) produced identical pH readingscompared to that of the corresponding unexpired controls.These findings, in experimental conditions 2 and 3, suggest

that the buffering capacity of the media probably was not af-fected by expiration, given the fact that the pH of the expiredmedia would not be identical to that of the unexpired ones un-

der incubation and post-incubation conditions.Fertility laboratories are obliged to stop using the media or

reagents if they cross the expiration date (16,17). The laborato-

ries, serving infertile patients by ART procedures, thus end updiscarding a large amount of unused media due to failure ofutilization within the manufacturer’s recommended set time.The clinics dealing with large IVF loads benefit in utilizing

as much of the purchased media as possible. However, sincethe majority of clinics are either small or modest in size, theyoften fail to utilize media in their entirety before expiration

due to low procedure loads. The cost of media incurred by fer-tility laboratories can be controlled if such unwanted mediawaste is prevented.

Our study found that the short shelf life set by the mediamanufacturers was not due to the possible weakening of thebuffering system of the media. The other possible cause of

the short shelf life is that the nutritional value of the mediadepletes faster than the deteriorating of its buffering capac-ity. The time-dependent instability of the amino acids thatare added in the media is reported in the literature (3,12).

The media manufacturers thus probably emphasize the nutri-tional strength of the media more so than its buffering po-tential in setting its shelf life. It is obvious that the

nutrients present in a media will deplete gradually over time,but the depleted nutrients may still be sufficient in support-ing full embryo growth. Therefore, it is important to know

whether the depleted nutrient pool of an aged (X day old)media can still support the embryo growth on the same levelas a media with no nutrient depletion (0 day old). In otherwords, the cut-off date of a media should not be based on

how depleted of nutrients the media is, rather it should bebased on whether or not the media’s capacity of supportingembryo growth is diminished. The media manufacturers have

limitations in addressing these issues due to the fact thatmouse embryos are used in determining the quality of themedia to be used for human embryo growth (12,18). Mouse

bioassays may not truly accomplish this task (19–21). Thelimitations of mouse embryos in evaluating the quality of

182 A. Hossain et al.

culture media have been repeatedly reported in previous liter-atures (18–21).

The longer expiration duration of media certainly can be

economically beneficial for the media users but gaining suchfinancial benefit should not be at the cost of diminished poten-tial of the media. Precise determination of shelf life requires the

information on the dynamics of nutritional depletion in mediaand its impact on human embryos, not mouse embryos. Thejoint investigative venture of both media manufacturers (pro-

duction plants) and media users (fertility laboratories) will per-form the task better than the manufacturers alone. In vitrofertilization procedures are not in their infancy any more.Much, if not all, is now known on the in vitro requirements

of human embryos and the factors that affect them. Proceduralflexibilities that do not compromise the outcomes have beendemonstrated (7,12). The way the new information on human

embryo culture is emerging, the time will come when the hu-man embryo culture system itself will be safely utilized indetermining the shelf life of the media to be used for them.

5. Conclusion

We analyzed the pH of expired media and found that the buf-

fering capacity of the embryo culture media is not affected byprolonged expiration. The nutritional value of the media is ex-pected to diminish over time, thus it may be the sole determin-

ing factor in setting the shelf life of the culture media. Mediausers will benefit from knowing the dynamics of the depletionof nutrients in the media and its impact on human embryogrowth. The joint effort of media manufacturers and media

users can lend to a better approach in determining the shelf lifeof media more accurately.

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