The Upper Egypt Assisted Reproduction Conference 2017, produced some interesting discussions but Dr Mohamed Fawzy of the IVF Laboratory, Egypt pinpointed one in particular: current culture media and incubator designs. He says "Culture media for in vitro human embryo development affects not only the embryo development and the subsequent clinical outcomes but also offspring health and gene expressions. Many brands of culture media are commercially available with encouragement from the manufacturers in usage, to get superior results for IVF outcomes. These brands include two paradigms (sequential media and single-step media). Sequential media paradigm formed of three sequences of media, one for fertilization, the second is to culture from zygote to the cleaved embryo until day 3, and the third is for culturing the embryo from day 3 to 5 or 6. This approach of culture is based on the assumption that each stage of embryo development needs different requirements in regard to nutrition, and others. The single-step approach has been built on the assumption that the embryo should be provided with all the nutrients and to let the embryo choose from them. Both paradigms are basically accepted and supportive to embryo development. The emerging evidence in two meta-analyses suggests that both approaches are equal and there is no preferred medium yet. A recent publication that has presented at UEARS 2017 inferred that adding insulin into single-step culture media is associated with better embryological and clinical outcomes. The culture media effect on gene expression is, to some extent, lacking. For that, a gap exists in the current design of culture media and the culture media need revisiting, however, from different perspectives."
Dr Fawzy goes on: "The incubator is a second crucial piece that can affect the human embryo development and the subsequent outcomes. The incubator is responsible for maintaining a non-stressful regulatory environment for the developing embryo. Inside the incubator, pH, gas exchange, recovery time and humidity should be optimal. According to Armstrong et al. in a Cochrane review, they inferred that there is no evidence to support time-lapse superiority over the conventional incubator. However, there is an anecdotal belief that the Benchtop incubators may offer faster recovery of temperature and gas and this, in turn, could improve the results. To date, there is no agreement on which model of Benchtop is superior. This could notify us that a gap exists in defining the best incubator and leaving us to the designer's wishes." He suggests culture media and incubators should be the first items to be validated through the concept of no harm to the embryo, clinical outcomes, and more importantly the offspring health.
The next UEARC 2018 conference will be held in Cairo on February 21-23, 2018.
Decellularisation of tendon tissue plays a pivotal role in current tissue engineering approaches for in vitro research as well as for the translation of graft based tendon restoration in clinics. Automation of the decellularisation steps such as freeze-thawing is crucial for the development of more standardised decellularisation protocols and commercial graft production under good manufacturing practice (GMP) conditions in the future. Decellularised tissues represent the ideal natural scaffold for many research applications in tissue science and hold great promise as possible transplants for medical applications. Decellularisation of tissues can be performed using physical, chemical and enzymatic methods but to achieve good results for the removal of cells with the conservation of the extracellular matrix, a combination of techniques needs to be explored for the different tissue types to find the optimal method.
Researchers at the Translational Centre for Regenerative Medicine at the University of Leipzig in Germany recently demonstrated that the application of freeze-thaw cycles prior to treatment with detergents enhanced the effectiveness of the decellularisation procedure. A new paper published in BMC Biotechnology by Susanne Roth, Sina Glauche, Janina Burk and others shows that using a controlled rate freezer gives precision and repeatability to the process.
Automated freeze-thaw cycles performed by the Planer Kryo 360 liquid nitrogen based controlled rate freezer were effective for the freeze-thaw procedures for decellularisation of equine superficial digital flexor tendons. The automation of this key procedure in decellularisation of large tendon samples is an important step towards the processing of large sample quantities under standardised and GMP conditions with a view to the production of commercially available tendon graft-based materials for application in human and veterinary medicine.
Following the recent death of a police officer investigating a leakage of liquid nitrogen (LN2) in Atlanta, Georgia, USA, cryo safety has become a topical issue. A survey of the risks associated with it by Mathew Tomlinson and David Morroll, reported in Human Fertility (https://www.ncbi.nlm.nih.gov/pubmed/18320438, concluded that incidents involving liquid nitrogen were more frequent than expected, and that training and awareness of risks associated with cryo stores was generally lacking. Steve Fleming and Alex Varghese have edited a book with a useful chapter on ‘Cryobank Management’, (by John Ryan) called the Organization and Management of IVF Units, published by Springer Science.
Steve (pictured here) and Alex say that an important starting point in liquid nitrogen cryo safety is the actual design of the cryo store where most handling of liquid nitrogen will occur. Because liquid nitrogen expands rapidly as it boils at -196 ºC, the liquid:gas expansion ratio being approximately 1:700, it can dramatically reduce the concentration of oxygen in the atmosphere within an enclosed or poorly ventilated space, leading to asphyxiation without any warning symptoms. Therefore, it is essential to install an oxygen monitor within a cryo store to provide early visible and audible warning.
It is also well known that liquid nitrogen and its vapour can potentially inflict burns or frostbite, if mishandled. So staff working within a cryo store will need special gloves and safety glasses along with face shields, aprons, fully enclosed shoes and long sleeved clothing. It will be necessary to educate staff in the variety of hazards associated with handling and storing liquid nitrogen. Instructions should include written standard operating procedures (SOPs) and prominently displayed notices within the cryo store, including evacuation maps. In addition to burns and frostbite, the typical hazards that staff may be exposed to include eye injury and/or infection resulting from explosion of straws or cryovials due to rapidly expanding nitrogen upon warming of any liquid nitrogen that may have seeped into them during cryo storage.
In many countries, provision of a licence to provide a clinical embryology service is contingent upon having in place appropriate monitoring, alarms, safety equipment, policies and procedures necessary to deal with unexpected leaks or spills of liquid nitrogen from bulk liquid nitrogen supply tanks or storage dewars. From a European perspective, the European Society of Human Reproduction guidelines for good practice (https://www.eshre.eu/Guidelines-and-Legal.aspx) cover cryo safety in embryology laboratories in considerable detail. However, not all countries where cryo storage of gametes and embryos is performed have a similar regulatory framework for protection of staff. Under such circumstances, it is very important that cryo store working areas remain well ventilated when in use and that staff do not work alone or investigate any low oxygen alarms on their own.
In an ideal world, nobody would suffer any injuries from working with liquid nitrogen, but in the real world tragedies such as those that occurred recently can happen so we must insist on best practice when handling liquid nitrogen to minimise the risks to ourselves and our colleagues working within embryology laboratories.
Steven D. Fleming & Alex C. Varghese, Editors, Organization and Management of IVF Units
A Practical Guide for the Clinician Springer Science ISBN 978-3-319-29371-4
BCGA code on LN2 50 litre dewars http://www.bcga.co.uk/pages/index.cfm?page_id=72
If you are looking for some modest financial support for a pet research project in andrology, the British Andrology Society now has a limited grant fund for small awards (up to £5000) towards projects relating to any aspect of male-related clinical infertility and/or basic reproductive biology.
Consideration will be given to applications that are carefully compiled, scientifically convincing and have well defined objectives. Applicants, who must be UK citizens or employed in a UK-based organization, can apply for either the November or April deadlines.
The topics can be fairly wide ranging, from Feasibility studies prior to an award-granting body, developing or improving a technique or other science communication. Would-be applicants should note that applications for travel or attending a conference would not be eligible and applications for undergraduate vacation scholarships would be considered, but would be capped at £2500. Winners will be expected to become BAS members and to provide a brief report on their research.
And in case you didn't guess what the photo is - it is ram sperm frozen in ice (-20oC) on a cryomicroscope which was sponsored by Planer back in the 1990s !
Based outside Moscow, the Federal State Scientific Institute Chumakov produces many vaccines, such as for tick-borne encephalitis, Measles, Rabies, Yellow fever, Canine distemper and more. And our distributor Cryogentech recently installed a freezer there for the controlled rate freezing of bacteria. The Institute is a leading one on a world stage. Back in 1988, it joined the World Health Organization Program for Poliomyelitis eradication worldwide and by 2002 Russia was certified free of the polio virus. During just one generation the eradication of an infectious disease – Polio (infantile paralysis) - had been successfully carried out.
This state scientific institution (FSUE) named Chumakov after the vaccine pioneer scientist (pictured here), who gave so much of his life to it, was established in 1957 as a part of the USSR Institute for the Study of Poliomyelitis. Its aims were to specialise in monitored quality production of Polio vaccine to supply the then USSR and other countries with anti-viral medication for mass immunisation. The production facilities have since been renovated and re-equipped and brought to international GMP standards. This has reaped rewards and in April 2009, the WHO certified the Yellow Fever vaccine production and put FSUE on the list of the four global manufacturers of this vaccine.
Current staff at the enterprise number about 350 employees and the company's activities have been rewarded with numerous international, national and regional awards.
When Namibia is mentioned, perhaps the first thing that comes to mind is a beautiful rather remote African country. With a population of just 2.1 million, and containing the large Namib Desert, the country is one of the least densely populated in the world, but it enjoys high political, economic and social stability. So we were proud to discover that a new IVF laboratory there, based within the Mediclinic complex in the capital Windhoek, has two of our BT37 units.
The Windhoek IVF clinic opened its doors in January 2017 with state of the art equipment in the IVF lab section. The personnel consist of top quality surgeons and scientists and it has a close liaison with the Aevitas Clinic in Cape Town, so its patients benefit from having access to the combined knowledge and expertise of the whole team for a collective opinion. The clinic started from a concept of Prof Igno Siebert and Dr Riaan van der Colf (pictured here) a few years ago, recognising that such an assisted fertility service did not exist in Namibia. Prof Siebert is a part of the well-known Aevitas Clinic in Cape Town, who also use Planer's BT37s. Prof Igno Siebert started visiting Windhoek four times a year, consulting and performing laparoscopic surgery locally. Dr Riaan van der Colf started in a private O&G practice in 1990 in Windhoek after completing his postgraduate studies at University Stellenbosch, RSA.
Dr Ye Yinghui was part of the team at the Women's Hospital, Hangzhou, China who carried out an evaluation study on two different formulations of slow-freezing media for cleavage stage embryo cryopreservation. The work was funded by the China Natural Science Foundation and China National Key Technology Support Program. Dr Ye graduated from Zhejiang University School of Medicine in 1995, got her PhD in 2008 and studied gamete and early stage embryo development in Japan as a research fellow in Akita University via a medical scholarship. Currently she works on IVF-ET, ICSI, embryo freezing and Recovery, PGD etc and she uses a Planer Kryo 360 freezer.
Some IVF labs prefer to cryopreserve pronuclear stage and cleavage stage embryos using conventional slow-freezing. Dr Ye and her co authors say that in their clinic, they consider slow freezing more practical. Additionally, using the new Vitrolife media especially formulated for slow freezing, their study found that embryo survival rate increased as did the clinical pregnancy rate per embryo transfer. Freezing and thawing cleavage stage embryos using the modified protocol and new formula - MOPS-buffered media containing amino acids - the researchers found that they could achieve the same high survival rates with traditional slow-freezing method as with vitrification. The improved level of dehydration combined with the cooling and warming rates of their protocol resulted in a combination which gave excellent survival.
Dr Ye and the team conclude " ... we can continue using our freezing machine and can spend time on other things in the lab while the machine is running. For the time being, we find slow freezing with this modified method very effective" and it frees up time during busy working days.
Dr Ye is also a committee member of the Medical Genetic Study Group of China Medical Association, the Zhejiang Medical Genetic Study Association, and Specialist of the Zhejiang ART Quality Control Society. She has authored and co-authored more than ten SCI articles.