Q From U: Human IVF Culture Medium

From a reader this week:

I was wondering about the culture that is used to put the embryos in. I know nothing about it and got curious. My clinic had to get some particular culture/medium in so they could do day 3 vitrification and it got me thinking. What is the culture? What is it made from. What are the different types? Why do you use one sort as opposed to another?

Where to start? Human IVF medium has evolved considerably in the decades since the first IVF birth. Human IVF medium was based on simple culture medias used to grow other types of animal cells. Early culture medium, based on research studies in mice, rabbits and domesticated species was often a simple pH-controlled fluid consisting of water, salts, and some simple energy source like glucose.

Small changes in media like the substitution of calcium lactate for calcium carbonate in mouse culture medium let mouse embryos grow past their prior “two-cell block” to later stages of development. Lactate and pyruvate became the preferred energy substrates for early embryo culture. In contrast, the simpler sugar glucose was found to be essential for later stages of culture when the embryo’s own genetic transcripts became active. Culture media was further improved and made more tolerable for embryos by the addition of  serum and later isolated proteins derived from bovine serum. For a while, it was popular to use the patients own serum for co-culture although this didn’t provide consistently good results.

With concerns about mad cow disease and AIDS, undefined whole serum was replaced by specific purified protein molecules, like albumin, isolated from serum. Eventually, recombinant proteins (serum substitutes) became available which entirely eliminated the need for serum proteins. Early culture media could only support embryo cell culture for a few days. As our scientific understanding improved regarding the metabolic needs of human embryos, medium composition became more complex and well-defined, allowing human embryos to be cultured for five days in culture to the pre-implantation blastocyst stage. A typical human IVF medium contains at minimum: Calcium Chloride,  Gentamicin sulphate (an antibiotic), Glucose, Human Albumin, Magnesium Sulfate, purified water, Phenol Red (a pH indicator), Potassium Chloride,  Sodium Bicarbonate, Sodium Chloride,  Sodium phosphate , Sodium Pyruvate and a synthetic serum replacement protein source.

Culture for 5 days to the blastocyst stage usually resulted in some differentiation among embryos in the same cohort in regards to their “thrivability” , allowing technicians to identify  (and select) the most progressive embryo(s) for transfer. Being able to chose the most progressive or “best’ embryo meant that fewer embryos could be transferred while preserving a high pregnancy rate.

Sequential media was the term used to describe a type of culture medium that required one medium for days 1-3 of culture and a slightly modified medium for continued embryo culture to day 5 and 6.  Unfortunately, sequential media were somewhat more challenging to use and added expense to the procedure because at least two types of media were required, making many programs resistant to adoption of day 5 culture methods. In fact, sequential media became even more complex when some companies offered slightly different formulations of the base media for each step of the IVF procedure. For instance Vitrolife offers media for egg recovery, micromanipulation techniques, fertilization, sperm preparation, and embryo culture including :

  • G-RINSE™ for “For rinsing of contact materials and washing of the cervix before oocyte aspiration and embryo transfer.”
  • G-MOPS™, handling medium for handling oocytes and embryos outside of the incubator, available with or wiothout protein. Vitrolife also offers another medium for handling oocytes and sperm (G-Gamete) but apparently not ideal for embryos.
  • G-IVF™, fertilization media, to be used for coincubating sperm and egg during the fertilization phase
  • G-1™, culture for days 1-3
  • G-2™, extended culture
  • G-PGD™, handling medium to maintain pH of embryos during biopsy
  • EmbryoGlue®, hyalarunon containing medium promoted as beneficial for increasing implantation rate
  • HSA-solution™, human serum albumin solution to be added to any of the above media.
  • G-MM™, recombinant protein supplement for all medium

Every few years, new media becomes popular, displacing older media. Perhaps in a reaction to the extreme specialization (and expense) of needing a specific medium for every step, at least one company has replaced sequential media with a simplified medium that can support the entire embryo culture period. Irvine Scientific offers a medium called Continuous Single Culture which claims equally high embryo development and pregnancy rates from sequential medium without all the fuss of stage-specific media. In fact, Irvine’s promotional materials suggest that media changes are not necessary with their formulation, allowing the embryo to remain mostly undisturbed during the culture period.

This newer trend toward not disturbing the embryo with repeated handling and moving to fresh medium is aligned with increasing interest in a technology called microfluidics which has the potential to radically change how IVF is performed. Microfluidics would allow embryos to stay in a single culture unit throughout all aspects of IVF from removal of cumulus cells surrounding the egg, introduction of sperm, removal of excess sperm, fertilization check and embryo culture. Steps would be mediated by altering the composition or flow rate of individual streams of culture medium bathing the embryo. You can read a recent post about microfluidics here.

Today there  are many competing embryo culture media vendors including : VitrolifeIrvine Scientific , IVF on-line, LLC., Cooper Surgical , Origio and others. If you get a group of embryologists together, you will find animated discussion, but little consensus, on which medium is best. Any of these media can provide good results.

IVF programs typically choose a media based on several factors, the most important of which is how well it works in their system. Other factors besides ease of use include the quality of the technical support from the vendor and of course, the expense of the media.  Culture medium for embryos needs to be “equilibrated” by placing it in the incubator for to achieve the desired temperature and gas environment before the embryos are exposed to the medium. Even the best media can be mishandled or used past it’s “use by” date, thus providing sub-optimal results.

I don’t have any commercial interest in any of these media or vendors and have had good results with many of them.

Typically, when a program wants to introduce a new IVF medium, the new medium is tested against the old by splitting cases, culturing half the embryos in the old and half in the new. After some test period, results are compared to see if adequate or superior fertilization rates and embryo progression is achieved with the new medium relative to the old. If the new media delivers superior results at a good price and is easily introduced into the daily workflow of the lab, it is adopted for use.

My reader also asked about vitrification medias. These might have the same basic composition as embryo culture medium but have high concentrations of cryoprotectants added which make them great for short term exposures before plunging into liquid nitrogen but totally unsuitable for extended exposure. When embryos are received from another institution, it is important that the accompanying documentation include information about the freezing media used so that complementary thaw media and methods can be used.  Below are some references for more reading.

References

Basic principles of culture medium  by Michael L. Reed, PhD

Human embryo culture medium

© 2012, Carole. All rights reserved.

©2012 Fertility Lab Insider. All Rights Reserved.

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