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#	Title	Date Added	Hits	Replies
1	Gas Concentrations in Trigas Incubators used in IVF	07 November 2008	30	1
	These days trigas incubators are being marketed with success. Will somebody explain me the concentration of CO2, O2 and N2 in these incubators in IVF programmes? Furthermore I would also like to know if they are superior to those using the pre-mixed air. Noreen [ Reply here ]
2	CO2 Incubators	06 November 2008	37	1
	I wonder, Do we need any required concentration of O2 for culture of Human embryos and gametes in CO2 incubators? I am asking this question because most of the modern CO2 incubators have the options of concentration of O2 from 1-21% and 5-90%. Therefore, for an IVF lab. which concentration of O2 wi be most suitable. Noreen Noreen Fatima [ Reply here ]

Replies

Re: CO2 Incubators
The question regarding the best oxygen concentration for human IVF has been approached, essentially, in two ways.

First, using atmospheric oxygen is easy, and there are pregnancies, therefore it is ok to use approximately 20 to 21% atmospheric oxygen. Second, using physiological oxygen is less easy, but it is logical to assume that lower oxygen would be better for the embryos, as in the tissues, the embryos are exposed to say, around 5% or so oxygen in the oviduct and perhaps a bit higher in the uterus.

As to what the best concentration of oxygen is, there are no studies in the human where defined oxygen concentration increments, e.g. in 1% intervals from 0 to atmospheric (or higher), have been studied. From an ethical standpoint, one could start at 5% and work up to atmospheric oxygen easily enough, but lower and higher end studies, that might not be easily approved in the human.

I've found two animal references, that did study increments of oxygen tension on development:

Cattle embryos were cultured using 2.5% to 20% oxygen, and 5 to 10% O2 yielded a higher proportion of morulae, with the best development at 5% O2. Additionally, mean cell numbers per blastocyst were higher under 5% O2, compared to 20% O2 or co-culture. Takahashi et al 1996 J Vet Med Sci 58:897-902.

Rabbit embryos were cultured under conditions of 1% to 20% O2 (steps of 5%). 5% O2 was found optimal for the proportion of hatching blastocysts, and cell numbers per embryo. And, interestingly, the embryos grown in 1% O2 were not substantially different from those grown in 5% O2. Li and Foote 1993 J Reprod Fertil 98:163-167.

There are a number of very recent papers that support the use of lower oxygen tension (5%O2) for human IVF, in particular with extended embryo culture to day five. And to me, more importantly, there are a number of animal papers that demonstrate that gene expression patterns can be altered by oxygen tension, and that reduced oxygen culture conditions yield patterns of expression more similar to that found with in vivo embryos.

Of course, we still need the CO2 until someone develops a culture medium that can replace, successfully, the majority (not all) of the bicarbonate with some other buffer, that can maintain embryo friendly pH.

Oocyte maturation, it appears that maturation/competency might be more successful at higher oxygen tensions until time for fertilization, and as for sperm, I'd love to hear from andrologists on that one, as I don't have too many sperm incubation papers where oxygen has been looked at, other than those talking in terms of ROS effects.

Mike
Michael L. Reed Center for Reproductive Medicine of New Mexico

Re: Gas Concentrations in Trigas Incubators used in IVF
Ok, incubators part two. This is a great question as well. There are several methods used to get to 'triple' mix incubation.

Incubators that use nitrogen to purge the atmosphere to control O2, concomitant to injection of CO2, use a whole lot of nitrogen. This is one of the downside aspects of triple mix incubators. Nitrogen can be delivered to the incubator from either a nitrogen generator system, from standard pressurized gas cylinders, or from the vapor bleedoff of pressurized liquid nitrogen storage tanks.

The upside to this type of system is the extreme flexibility, where you can set whatever triple mix you want to meet low O2 and CO2 needs.

And, there are folks that are using less nitrogen by reducing O2 to one-half atmospheric levels (10%), rather than working at one fourth atmospheric levels (5%). Reducing O2 is a good thing, and balancing O2 reduction against nitrogen use something each lab can determine for themselves.

Another system is to use 'pre-mixed' pressurized cylinders that are filled by weight and analyzed by gas chromatography. This gas mix is not flexible, in that you can only have one mix per tank. Using this type of mix for continuous flow through a small incubator chamber uses a lot of gas, however if you use small sealable enclosures, e.g. a glass desiccator jar or a Billups-Rothberg style chamber, you can conserve gas quite nicely. The other downside to using small chambers is that they take up a bit more room in an incubator. I use glass jars myself, and I can fit three jars in a standard incubator, where one patient is placed in each jar. This is also good to eliminate door opening issues, as no matter how many times the incubator door is opened, the sealed jars protect the embryos from change. Another benefit to using a pre-mixed gas is that you have a very consistent product within a cylinder - no variations to worry about, and you have a very clean product that is not vuneralbe to envrironmental conditions.

Mike

Re: Embryo fragmentation
Dear Antonia Gilligan, here are the answers of Your questions: - We use a VOC meter of Research Instruments (RI) company, which should detect any VOC if it is present in the lab air.I believe that there is nothing in the lab, that should provide aldehydes in the air. - I clean the incubators with a spray of the PromoKine Company - Pharmacidal, which is especially designed for cleaning of CO incubators and for IVF. For the incubator water bath we use another chemical of the same company - it si called Aquaguard-1, especially for water desinfection of CO incubators. - About comparing the lots - we haven't, because we order small amount of media but frequently just to be sure that we use media from different lots. - I know that the best think to do is to use mouse embryo to resolve the problem.Unfortunately we do not have the possibility to do it. - Maybe the problem could come from the fact that our patients from those last IVF cycles are between 36 and 42, they have a lot of problems - endometriosys, PCOS etc.
Thans You for Your answer. Maybe we will try with desiacatios as You suggest.
Mya

Re: Embryo fragmentation
Hello, I think your problem comes from the cleaning procedure for the incubators, therefore I want to ask you not to clean the incubators every two weeks, even you are using a safe chemicals but with time it will concentrate much more in the incubators, for that after each cleaning you should wash the incubators very well with sterile distal water then run the outosterilization if you have, then remove the water used in sterilization and replace it with a fresh sterile distal water as recommended in your incubators, and do not use the incubators directly, I mean keep open and close the incubators doors for three days or the suitable time you found it is enough, until all the air inside is circulating much times taking out any smell or remaining chemicals. And this is what we do in our lab. I hope this will help.
Mutaz Alahmad Maternity and child hospital AbhaKSA

Re: Embryo fragmentation
Embryo fragmentation is basically because of toxicity in the environment. check the chemicals you use for cleaning and sterilization. all the best
alok teotia

Re: Co2 and Temperature analyser
I\'ve heard from many customers they are very happy with the handheld meters offered by Vaisala which you can purchase on their website I believe.

On the other hand a continuous monitoring and alarm system which can be used to measure your CO2, Temp, Humidity etc may be more useful to you. Planer offer one for example called Assure24seven which can be used for all types of incubators (including the MINC)which would record the information 24/7 to your PC/PC's or server.

I hope this is helpful.
Ravi Dattani