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Cryo safety for the embryologist

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 (, 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 1:790, 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 ( 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.

Further information

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

Cryosafety in the Embryology Laboratory: Download full text PDF

BCGA code on LN2 50 litre dewars



Andrologists with a good idea

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.

See the full details of how to apply in the PDF attached here or contact Prof Holt at the BAS




 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 !

Freezer installed in the Moscow Institute of Poliomyelitis for freezing bacteria

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.

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IVF in Namibia

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.

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Cryopreservation of ovarian tissue for fertility preservation in young girls

Following on the news of the birth in London to a 24 year old woman from implanted cryopreserved ovarian tissue taken when she was nine years old - a world first - a paper has just been published in Human Fertility on the background needs for such preservation. The study, by Jensen, Yding Andersen (pictured here) and others, looks at the need for ovarian tissue cryopreservation in girls undergoing radiation treatment and concludes it should be recommended to all young girls, who present a high risk of developing ovarian insufficiency or infertility following high doses of chemotherapy and/or irradiation. The cohort study examined a total of 176 Danish girls under 18 years of age who had received ovarian tissue cryopreservation performed over a period of 15 years

The latest birth in London was to a Mrs Al Matrooshi who had needed chemotherapy before receiving a bone marrow transplant. As a precautionary measure her right ovary was removed in an operation in Leeds and the tissue frozen by Professor Helen Picton and her team. The procedure to store the tissue in 2001 was done before any birth from ovarian tissue preservation had occurred, the first such birth being in Belgium 2004. Both the first and the latest births from frozen ovarian tissue used Planer programmable freezers.

Prof Picton, who oversaw the tissue-freezing at Leeds University, reported to the BBC, that in Europe alone, several thousand girls and young women have had ovarian tissue frozen and stored. The paper from the Copenhagen researchers concludes that "Although it cannot be predicted which girls will become infertile or develop premature ovarian insufficiency following intensive chemotherapy or irradiation, patients who are at high risk should be offered ovarian tissue cryopreservation. This includes girls who are planned to receive either high doses of alkylating agents, conditioning regimen before stem cell transplantation, total body irradiation or high radiation doses to the craniospinal, abdominal or pelvic area."

Worldwide more than 60 babies have been born from women who had their fertility restored, but Moaza is the first case from pre-pubertal freezing and the first from a patient who had treatment for beta thalassaemia.

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Dr Ye Yinghui - part of the IVF team at the Women's Hospital, Hangzhou

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.  


Unique birth from ovarian tissue frozen in 2001

Kryo 360 Controlled Rate FreezerMrs Moaza Al Matrooshi, from Dubai, gave birth in London on 13 December, after surgeons implanted ovarian tissue taken when she was nine years old - before puberty - and this appears to be a world first. Mrs Al Matrooshi was born with an inherited blood disorder and needed chemotherapy before receiving a bone marrow transplant at Great Ormond Street Hospital, London. This chemotherapy would render her infertile and as a precautionary measure her right ovary was removed in 2001 in an operation in Leeds and the tissue frozen by Professor Helen Picton and her team.

As with many such ovarian preservations, the tissue was frozen down in a Planer controlled rate freezer before being stored at liquid nitrogen temperatures. It was mixed with cryopreservatives and controlled rate frozen so that the sample stayed viable and undamaged after the liquid nitrogen storage temperatures experienced.

Prof Picton, Scientific Director of the Leeds Centre For Reproductive Medicine, says that the procedure to store the tissue in 2001 was done before any birth from ovarian tissue preservation. The first such birth occurred in Louvain, Belgium under Prof Donnez in 2004 - again using Planer programmable freezers. Prof Picton, who oversaw the tissue-freezing at Leeds University, reported to the BBC, that in Europe alone, several thousand girls and young women have now had ovarian tissue frozen and stored. 

Mrs Al Matrooshi's ovary remained frozen until 2015 when it was sent to Denmark, where the transplant took place. The surgeons there transplanted five slivers of the ovarian tissue back into her body – four were stitched on to a failed left ovary and one on to the side of the uterus. After the transplant hormone levels returned to normal Mrs Al Matrooshi began ovulating and fertility was restored. IVF was undertaken at the Portland hospital to maximise the chance of pregnancy, resulting in the production of three embryos, two of which were implanted. Worldwide more than sixty babies have been born from women who had their fertility restored, but Moaza is the first case from pre-pubertal freezing and the first from a patient who had treatment for beta thalassaemia.

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Forty years on....Planer freezers

In the 1950s fowl sperm was first cryopreserved by a UK team led by Christopher Polge; the process was repeated in humans giving successful pregnancies. But the rapid immersion in liquid nitrogen gave viability problems for some samples – at that time embryos and bone marrow. As understanding of the freezing mechanism grew the importance of controlled or slow freezing for post thaw viability developed. In 1963, Peter Mazur, in the U.S., showed that damaging intracellular ice could be avoided if cooling was slow enough to permit sufficient water to leave the cell during freezing. Cryoprotectants, initially glycerol, protected the cells from freezing injury – both direct damage from ice crystal formation and from the increase in concentration of solutes. The controlled cooling process let these biological samples equilibrate osmotically in cryoprotectant via a series of cooling stages before their transfer to liquid nitrogen for long term storage; the first birth from frozen bovine embryo was reported in 1972 - using an early Planer machine.

Working with Prof David Pegg, Planer introduced controlled rate freezing equipment in the 1970s, first for animal embryos, with Professors Rawson and Wilmut pioneering the area, and then after 1984 in human IVF. In that year Drs Alan Trounson and Carl Wood in Melbourne helped Zoe Leyland into the world via a Planer freezer and she became the first human born from a frozen embryo. Two years later, April 1986, the first successful attempt at freezing and thawing of a human oocyte was reported in the Lancet by Dr Christopher Chen et al: a twin pregnancy was achieved using DMSO protectant and freezing in a Planer machine down from -7C to -36C followed by rapid freezing to -196C before storage in liquid nitrogen.  Research into freezing other material continued and in 1991 arterial graft material was frozen successfully for the first time and in 2004 the longest successful storage of sperm was reported by doctors from St. Mary's Hospital and Christie Hospital in Manchester, UK who worked with a couple who conceived using sperm frozen down in a Planer controlled rate freezer, and then stored for twenty one years.

In 1997 a patient, Ms Touirat, in Belgium had parts of her ovaries removed and controlled frozen in a Planer machine before beginning treatment for Hodgkin's lymphoma which would necessarily leave her infertile. In 2003, free of the cancer, a Belgian team under Prof Donnez thawed and reimplanted and she became the first woman in the world to give birth to a healthy baby following such a graft.  More lately, in 2014, Prof Bruce Campbell of Nottingham, UK, reported (in ‘Human Reproduction’) he had successfully frozen and transplanted a whole ovary - the first frozen organ from an adult large mammal successfully transplanted.

There are some 10,000 Planer temperature control machines that have been sold around the world since the company started in 1974. Used for freezing precious samples in use for human, animal and cell biology – samples like oocytes, skin, blood products, bone marrow, embryos, sperm, stem cells, tissue they are found in hospitals, veterinary practices and research laboratories everywhere.