Do you have any knowledge about only basic medium usage for IVM? Because it is well known that the factor, called oocyte maturation inhibiting (OMI) factor prevent nuclear maturation of oocyte in follicle. When we aspirate or remove cumulus oocyte complexes (COCs) from this condition, is maturation spontaneously induced in COCs?
Hi Navid,
I do not have any problem about maturation medium. Also I am not working on IVM at the moment. Nevertheless I am thinking about maturation and fertilization. In our studies on IVF, we have had high maturation rates, but we had low cleavage and blastocyte rates. We routinely use TCM199 with estradiol, LH, FSH, and EGF to induce maturation. There is no problem about maturation rates but we obtained low cleavage and blastocyte rates...
Thank you for your interest
Maybe this paper from Kim et al, will help you
Fertility and Sterility Vol. 95, No. 2, February 2011
http://madang.ajou.ac.kr/~minc/data/Comparison%20of%20in%20vitro%20maturation%20media%20of%20immature.pdf
Dear UMUT
There are various comercial media for IVM, which give approx 80% of maturation.
However, these media were designed for OCCs aspirated from patients induced (or not) for IVM cycles. In other words: patients are monititored , and OCCs are aspirated before ovulation might occured. Follicles are generally smaller.
Else there are patients who are induced with low doses of Ovitrel (hCG) and OCCs are aspirated before ovulatiion time. In those cases comertial media are effective.
There is a big difference in the behaviour of those OCCs in culture, in comparison to GVs aspirated in a conventional cycle; where most of the cohort underwent maturation; those which are still in the GV stage will generally will not undergo maturation. It is likely that those OCCs have some kind of deffect (receptors, whatever...) and this might be a reason why they are still immature, or they were aspirated from follicles that are still immature and not in the antral stage.
Yes the answer is that cumulus enclosed oocytes should undergo spontaneous maturatoin. This occurs in IVM media (might contain LH+FSH)', butgenerally not in OCCs from a conventional cycle.
You can try using ionophor. More information you can find in the works of the group of Prof Thomas Ebner
Dear Dr Omut,
Your medium composition is suitable, but there are some more additives we use in our protocol:
Pyruvate + ITS (Insulin-transferrin-selenium) + 10%FBS (fetal bovine serum)
Dear Islam,
We routinely use TCM199 with LH, FSH, EGF (epidermal growth factor), Na-Pyruvate and %10 or 20 FCS for IVM. However, I hear first the ITS (I think this is a mix of insuline, transferrin and selenium). ITS may be used an other time. Can you send me the detailed medium you routinely use in your IVF stages (I mean IVM, IVF and IVC/D). And Can you say your success in IVF stages (maturation rates, cleavage rates, percentage of morula and blastocyste)?
Thank you for your information.
Dear Dr Umut
Please see the details in our article (attached).
Regards.
Dear Umut,
I think your problems are IVF and IVF method.
In our Lab we use The TALP- medium for IVF and IVC.
In the first 72 hours of the IVC, we cultured zygote in the TALP-medium contained with Glutamine 0.05 mM without Citrate and Glucose.
Then, choosing the embryo at 4 cells stage for cultured in TALP medium containing with citrate 0.05 mM and Glucose 1.5 mM without Glutamine, culture from hour at 72 to 144.
After that, selected the early-Blastocyst embryo for culture in IVM medium (TCM-199; No hormone added) throughout until hatch.
Keep in your mind, do rapidly and stability in the temperature.
Good Luck
S. Denpong
Dear Denpong,
I am used to use TCM199(with FSH,LH,E2,EGF,Na-pyruvate, %10FCS or 6mg/ml BSA) for IVM, SP-TALP (with heparine and hepes) for swim-up sperm-separation, IVF-TALP for fertilization and TCM199 again for IVC/D. I had not faced exact problem in routine IVF until OPU. Nevertheless I think your mediums are more suitable for IVF stages. Because TCM199 is more expensive and sometimes unreachable compared with TALP. Do you add phenol-red to TALP during IVC to determine pH changes?
I faced an other problem in transvaginal OPU in cattle. We did not gained any fertilization and cleavege. I think the root of the problem is changes in temperature. Because we had to carry out our OPU in cold (minus zero and sometimes -20C) place and in my opinion this temperature detrimentally affected our result in spite of warm water bath for collection tubes. Because our 2m Teflon tubes we used to collect oocytes was directly exposed this temperature. We did not use any isolation for this Teflon cord. Do you have any precautions for isolation the Teflon tube or cord in cold condition or places?
Thank you for your information.
Umut
Dear Umut,
sorry for a late answer.
1) The pH of IVM medium is very serious for me. I had added phenol-red in the medium, the result was fail. So, I adjusted the pH using pH meter in the range of 7.36-7.38.
IVM medium
We useTCM-199 (M0650, Sigma); PVA1 g/L; HEPES 7.5 mM; Pyruvate 0.45 mM; Cysteamine 0.2 mM; Cysteine 0.2 mM; Glutamine 0.25 mM; NaHCO3 25 mM; Antibiotics, and Deionized water
Adding FSH and Incubate at 38.5 oC 5%CO2 for 3 hrs before use.
2) I don't have an experience to perform the OPU in the cool weather. The mean of weather in our country is 32-35 oC.
Denpong
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