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Successful oocyte retrieval and fertilisation after transplantation of cryopreserved ovarian tissue

A combined team from Singapore's Sincere IVF and Gleneagles Hospital, report and describe successful ovarian freeze, transplant and ICSI fertilisation in the Journal of Reproductive Biotechnology and Fertility (Dr Jaffar Ali editor-in-chief). Ovarian tissue freezing is still a novel and complicated technique which can be used clinically for fertility preservation in female children, adolescents and adults with cancer.

The team, led by Professor SC NG, performed the auto-transplantation of cryopreserved ovarian tissue and reestablished a regular menstrual cycle, collecting one egg - and its successful fertilisation showed ovarian function had been restored. In the paper, published early 2019 the authors (from Embryonics International, ACS Clinic for Woman and Sincere IVF) tell that the patient, initially diagnosed with a serous borderline tumour for both ovaries, but with no definite stromal invasion, has, subsequent to the intervention and to date, been well with no evidence of relapse noted. 

The 37-year old woman patient, whilst healthy, had twenty years absence of fertility due to her husband’s azoospermia; she was diagnosed with ovarian cancer and an oopherectomy was laparoscopically performed in 2016 at Gleneagles Hospital, Singapore. The subsequent investigation showed serous borderline tumour for both ovaries but with no stromal invasion. 

Three slices of morphologically normal tissue from the right ovary were collected and immediately delivered to the laboratory where the medullary tissue was removed from the ovarian pieces, and a total of 10 pieces of ovarian cortex tissue approximately 5mm by 5mm and about 1mm in thickness were prepared for cryopreservation (see below). The slow freezing method (Andersen et al., 2008 https://www.ncbi.nlm.nih.gov/pubmed/18603535) was employed to freeze the prepared pieces of the ovarian tissue. 

Ovarian tissue prior to freezingAll ten pieces of ovarian tissue were equilibrated at 4 °C and were then placed into cryo-vials for computer controlled freezing with a Planer Kryo-360. The initial cooling rate was −2 °C/min to −9 °C with manual seeding performed and cooling was continued at different ramps to−140 °C, when the samples were plunged into liquid nitrogen at −196 °C, and transferred to a liquid nitrogen storage tank.

Neither chemotherapy nor radiotherapy was performed on the patient who ten months later underwent heterotopic transplantation of thawed ovarian tissue. Two cryo-vials containing four pieces of ovarian tissue were thawed for transplantation on 16 March 2017. The cryopreserved tissue was placed into a peritoneal pocket in the region of the broad ligament, below the fallopian tube. Menstruation commenced two months later after induction and follicle growth was monitored in the fifth menstruation cycle with one follicle observed in the peritoneal pocket.

The oocyte was successfully retrieved after the fifth cycle, six months after transplantation and fertilised via intracytoplasmic sperm injection (ICSI) using the husband’s frozen-thawed testicular sperm. One 7-cell cleavage stage embryo was then frozen but no embryo transfer has been performed to date. 

Professor Soon-Chye NgPictured here, Professor Soon-Chye Ng, one of the authors mentions "The results of our first ovarian tissue transplant confirmed previous reports and lent support to the objective that ovarian tissue freezing can preserve fertility for cancer patients. This success demonstrated that cryopreservation of ovarian tissue and subsequent transplantation is a viable option for preserving fertility."

Worldwide, it is estimated that several thousand women have had ovarian tissue cryopreserved, but the number of transplantations remains low although it is accepted in an increasing number of countries; to date, more than 100 children have been born from this procedure worldwide (Andersen et al., 2018  https://www.ncbi.nlm.nih.gov/pubmed/29718400).

The authors conclude that although cryopreserving of ovarian tissue for fertility restoration is still regarded as experimental, successful collection of oocytes and fertilisation from auto transplanted ovarian tissue, such as here, show that ovarian tissue freezing can preserve fertility for cancer patients. Goals for the future should include optimising the outcomes of transplantation, with improved tissue quality, reduced follicle loss, shorter transportation times, and longer periods of functionality in cryopreserved ovarian tissue. Such improvement in technique is important as survival rates for cancer patients in their early reproductive life continue to improve with advances in cancer treatment.

For further information

Journal of reproductive biotechnology & fertility

Read Paper  http://www.ivf-hub.net/journal-archive-new/2019-2/
Planer Kryo 360: https://planer.com/products/cryo-freezers/small-crf/kryo-360.html
More from http://www.ivf-hub.net

Preservation of Cellular Therapies - BioCoR Course

Planer Kryo 560 programmable freezer

The Bio-preservation Core Resource at the University of Minnesota is offering a new course on the emerging issues in preservation of cell therapies aligned with the fundamentals of cryo-preservation. The course includes cryo protocol development, the designing of a storage facility plus practical advice on cryo containers and equipment, reagents, regulatory issues along with clinical aspects of cryo-preservation and quality control. 

The presentation will be given in person as a course on May 21st to 22nd, 2019 in Minneapolis with a simultaneous webcast of proceedings. The lecture topics will cover:-

  • Fundamentals of cryo-preservation
  • Protocol development
  • Regulatory issues in cryo-preservation
  • Designing a storage facility
  • Containers, reagents and equipment
  • Emerging issues in preservation of cell therapies
  • Clinical cell cryo-preservation 
  • Quality control

The course material includes both fundamentals on preservation as well as emerging issues and approaches. The course is appropriate for managers of cell therapy laboratories, technicians who preserve cells as a part of their daily routine, scientists involved in the development of cell therapies, companies that produce products for the cell therapy space, repositories that store cells to be used therapeutically and related disciplines.

The class is offered in two different formats -  via class attendance or via a simultaneous webcast of the class for those who cannot attend the in-class lecture.  Lectures are recorded and attendees over the web have the opportunity to review the lectures at their convenience.

For further information
Class Schedule
Biographies of Lecturers
Registration
Recommended Text


New ovarian tissue freezing programme

Paul HardimanFor women diagnosed with cancer, chemotherapy and radiotherapy treatment may cause irreversible ovarian damage. IVF prior to treatment is only available to women with a partner or those willing to use donor sperm and is contra indicated for those with hormone sensitive tumours and, additionally, may use up valuable time.

Ovarian tissue transplantation can overcome these problems and now a new freezing and transplantation service, the first in the UK to be fully funded by the NHS, will enable specialists in cancer and blood disorders to refer patients to the Royal Free London, ahead of their treatments, for ovarian tissue freezing down, cryostorage and subsequent implantation to hopefully restore fertility or prevent early menopause.

For pre-pubertal girls and some women with hormone sensitive tumours (for example some types of breast cancer), ovarian tissue freezing is the only way that they can preserve their fertility. Patients who are cured by their chemo/radiotherapy can have cryopreserved ovary tissue recovered, thawed and surgically placed onto the remaining ovary or into the lining of the abdominal cavity.

The treatment involves the patient undergoing keyhole surgery to remove one ovary which can then be processed in the hospital’s laboratory, carefully control rate frozen down and then stored at a liquid nitrogen temperature of -196 ℃. The controlled freezing facilitates a crucial ice nucleation step using a pre programmed series of cooling rates; once frozen safely the tissue is ready for cryo-storage. When needed the ovarian slices may be thawed and transplanted back to the patient post treatment - after months or years.

Around the world there have been well over a hundred successful births as a result of this treatment and many other women have benefitted from restoration of their menstrual cycle after tissue implantation.

Consultant Paul Hardiman (pictured here, above right) said: “We have modelled our protocols on how it is done at the Rigshospitalet, Copenhagen, one of the largest hospitals in Denmark, where they have been freezing human ovarian tissue since 1999. This is a well-established method in Europe, the US and Japan but the UK has lagged behind.”  

Natalie GetreuNatalie Getreu (pictured left) is part of the team that has enabled this new service and she is also looking to increase the knowledge base and is researching methods previously used to reduce ischemia in other transplanted tissues, to the human ova

The Royal Free London has a unique mix of facilities and expertise in tissue freezing and cell therapy including the services of Professors Barry Fuller and Mark Lowdell, and is also a leading kidney and liver transplant centre and the principle UK centre for cell and tissue medicines. The new service will be offered to patients within the London but is planned to expand across the UK soon.

For further information
London gets new ovarian tissue freezing programme
Planer Kryo 560 controlled rate freezer

 

New DATAssure sensors added to the range



DATAssure™ combined temperature and level sensor released
Since its introduction in 2017 the DATAssure™ wireless and monitoring system has proven itself as the ‘go to’ monitoring system for laboratories around the world.

The easy to use and intuitive system provides a wide array of different sensors, many of which are designed by Planer for use within our customers’ specific applications, and has become a huge success.

One of the specialised probes that had previously been available on the DATAcentre system (the predecessor to DATAssure) was the combined temperature and level sensor for liquid nitrogen sample storage dewars - unique to the Planer system.

We are pleased to announce that the combined temperature and level sensor is now available for the DATAssure system, both 433MHz and 2.4GHz versions.

Part numbers for the new sensors can be found in the table below.

Description Part Number Detailed Description
Temperature & Level
Dewar - up to 50 litres
GDDA-TL-DEWAR-433 Premium combined temperature and level sensor for 433 MHZ DATAssure systems, formable to dewar shape to ensure no interference when entering or exiting samples
Temperature & Level
Dewar - up to 50 litres
GDDA-TL-DEWAR-2400 Premium combined temperature and level sensor for 2.4GHz DATAssure systems, formable to dewar shape to ensure no interference when entering or exiting samples


Range of "standard" sensors now available
Many of the premium sensor solutions, like the temperature level sensor, Planer has developed are bespoke sensors designed to provide the optimal solution to monitoring a specific parameter or a specific piece of equipment.

However we are frequently asked for more cost effective solutions. Therefore, we are also pleased to announce that as well as the premium sensor we already provide, we are adding a new range of standard sensors which will give more cost effective, although not optimal, solutions to monitoring requirements.

Details for all the new sensors can be found in the table below:

Description Part Number Detailed Description
For 433 MHz Systems    
Temperature - Benchtop Incubator - Standard GDDA-T-BT-BAS433 Standard solution provides a flat ribbon cable sensor which can be stuck down inside the chamber and the cable ran out through the chamber seal. Requires one per chamber
Temperature - Dewar - Straight Sensor - Standard GDDA-T-DEWSTR-BAS433 Standard solution incorporating stiff 370mm temperature sensor which can be inserted through the dewar lid to monitor the temperature inside the dewar chamber (requires hole to be drilled through dewar lid for installation)
Temperature - Dewar - Curved Sensor - Standard GDDA-T-DEWCUR-BAS433 Standard solution incorporating flexible formable 1500mm temperature sensor which can be inserted through the dewar lid to monitor the temperature inside the dewar chamber (requires modification to dewar lid for installation)
Temperature - Fridge -45 to +45 - Standard GDDA-TEMP-45-BAS433 Standard solution using NTC 5K sensor with round artic grade cable which will need to be inserted around the door seal. Will measure down to -45 °C.
Temperature - minus 40 Freezer - Standard GDDA-TEMP-50-BAS433 Standard solution using NTC 5K sensor with round artic grade cable which will need to be inserted around the door seal. Will measure down to -50 °C.
Temperature - minus 80 Freezer - Standard GDDA-TEMP-100-BAS433 Standard solution using PT1000 sensor with round artic grade cable which will need to be inserted around the door seal. Will measure down to -100 °C.
For 2.4 GHz Systems     
Temperature - Benchtop Incubator - Standard  GDDA-T-BT-BAS2400 Standard solution provides a flat ribbon cable sensor which can be stuck down inside the chamber and the cable ran out through the chamber seal. Requires one per chamber.
Temperature - Dewar - Straight Sensor - Standard  GDDA-T-DEWSTR-BAS2400 Standard solution incorporating stiff 370mm temperature sensor which can be inserted through the dewar lid to monitor the temperature inside the dewar chamber (requires hole to be drilled through dewar lid for installation)
Temperature - Dewar - Curved Sensor - Standard  GDDA-T-DEWCUR-BAS2400 Standard solution incorporating flexible formable 1500mm temperature sensor which can be inserted through the dewar lid to monitor the temperature inside the dewar chamber (requires modification to dewar lid for installation)
Temperature - Fridge -45 to +45 - Standard GDDA-TEMP-45-BAS2400 Standard solution using NTC 5K sensor with round artic grade cable which will need to be inserted around the door seal. Will measure down to -45 °C.
Temperature - minus 40 Freezer - Standard  GDDA-TEMP-50-BAS2400 Standard solution using NTC 5K sensor with round artic grade cable which will need to be inserted around the door seal. Will measure down to -50 °C.
Temperature - minus 80 Freezer - Standard  GDDA-TEMP-100-BAS2400 Standard solution using PT1000 sensor with round artic grade cable which will need to be inserted around the door seal. Will measure down to -100 °C.

 

 

Our contingency plans for Brexit

BrexitOn the 29th March 2019 the UK is due to leave the European Union. At the current time negotiations on the new relationship between the UK and the rest of the EU are in a state of flux but a period of adjustment after that date seems likely.

However the unexpected can happen and so we, at Planer, have contingency plans in place for an unmanaged departure. In that event:

(1) Our interactions and transactions with the UK and countries outside the EU will remain unchanged.

(2) As we have exported to 85 countries since 1973 we are well versed in tariff and customs procedures so changes regarding shipments to and from the EU under any new rules should be routinely accommodated.  We have a Customs Deferment facility under which we can import goods from around the world. Our products and components generally have low or no duties (import and export), being medical equipment.

(3)  Some of our products are registered as medical devices across the world and will of course continue to comply with UK, US, Chinese, Russian, Asian and other areas as they currently do. When Britain leaves the EU we anticipate a period of equivalence with Europe. 

Brexit contingency planning(4) None of our staff will be impacted by any potential changes to immigration policy therefore we will be working as normal here at our Sunbury headquarters, ensuring product and services are available when required.

(5) Like many UK companies we have ordered extra stocks of parts that we import from any EU suppliers and we have sufficient to meet customer demand for quite a long period. We do not anticipate problems with components or products shipped into the UK. Extra staff will be available to minimise any impact and we are working with our freight partners to make sure goods can be shipped in the most efficient way.

We hope that a satisfactory agreement between the EU and UK will be achieved and that none of our contingency plans will be necessary.  I would like to reassure all our customers in the UK, in the European Union and worldwide that the team here at Planer are confident products will be available globally just as they have been for the last 45 years.

Adrian Fuller,
Managing Director,
Planer plc

 

Optimising Cryopreservation of Human Testicular Tissues

While adolescent and adult men have the option to cryopreserve sperm prior to chemotherapy or radiation in cancer treatment, this is not available to pre-pubertal boys. Researchers from the University of Pittsburgh School of Medicine and the Magee-Womens Research Institute write that several IVF centres worldwide are preserving testicular biopsies for these patients in anticipation that spermatogonial stem cells (SSCs) in the tissue can be used to achieve fertility in the future. In order to maximise the use of these tissues, the authors Valli-Pulaski et al, compared the cryopreservation of a cell suspension to an intact piece of tissue to discover the optimal cryopreservation method.

In the experiment the efficiency of each technique was analysed by immuno-cytochemistry for spermatogonia markers and human-to-nude mouse xenotransplantation. The average immuno-cytochemistry tested positive cells were highest in the fresh tissue; but from the cryopreserved groups, large tissue pieces and small tissue pieces had similar numbers of positive cells while cell suspension had the least positive cells and was significantly worse than cryopreserved intact tissue pieces. The authors conclude that based on their results, slow freezing of small piece of tissue is the most efficient technique to cryopreserve human testicular tissue. 

For further information
Optimising Cryopreservation of Human Testicular Tissues
Reproductive Biomedicine Online
Volume 37, Supplement 2, Page e4

 

Treating acute liver failure: a new scaffold for the culture of liver-organoids shows promise

The development of an artificial liver has been of prominent interest, as liver disease is the fifth biggest cause of deaths in the UK. Acute liver failure (ALF) in children is a life-threatening condition. The prognosis of ALF is generally poor and medical intervention relies on a liver or hepatocyte transplantation if the native liver is unable to recover. The major limitation of the technique today is the availability of donor organs. There have been limited clinical advancements in the treatment of ALF utilising novel regenerative techniques, such as the use of embryonic stem cell techniques to induced pluripotent stem cell-derived hepatocytes. The use of human bio-compatible scaffolds to grow liver type/functional cells is increasing and the main challenges in translating advances in basic science of cell therapy into the clinic has been determining the best route of delivery, the rapid elimination of transplanted cells by the recipient, poor engraftment and proliferation of transplanted cells within the liver.

Researchers at Kings College London, Anil Chandrashekran (pictured right) and Anil Dhawan (pictured below), examined the use of methylcellulose as a scaffold to obtain liver-organoids. They cultured hepatocyte and MSCs in the presence of methylcellulose and growth factors and tested the ability of organoids to produce albumin and detoxify ammonium chloride, in optimally cultured conditions describing their research in BioInsights December 2018.

Mesenchymal stem/stromal cells (MSCs) can be easily cultured from adult bone marrow and full-term umbilical cord (blood or the Wharton’s jelly) and have been isolated from muscle connective tissue, adipose tissue and in some circumstances peripheral blood. Currently novel scaffolds are under scrutiny for the unmet need to use organoid-based product suitable for transplantation in ALF, this until the patient receives a liver transplant or recovers. 

Hepatocytes fractions were collected, treated and cells were frozen in cryovials and cryobags using a Planer controlled rate freezer before storage at -180°C vapour phase nitrogen freezers. The MSCs were isolated from Wharton’s jelly of umbilical cords obtained following caesarean section deliveries. Once cell cultures were established, they were expanded, quality controlled and cryopreserved

The study showed that methylcellulose, an inert semi-solid media more suitable for clinical use than current products, could be utilised as a scaffold to establish liver-organoids that resemble liver structure and function. And they say the suitability of using methylcellulose towards clinical grade expansion of organoids is highly compelling. Further optimisation and scaling up of the process is underway. 

Improvements to hepatocyte transplantation have been made by encapsulating hepatocytes with alginate beads which eliminates the need for immunosuppressants. More recently, bio-fabrication techniques have been developed using 3D plotting with methylcellulose and alginate. MSCs cultured in these 3D scaffolds retained viability and differentiation properties and so taking these techniques together it should be feasible to establish organoids in methylcellulose, as done here and then encapsulating the resulting organoids in alginate-methylcellulose 3D scaffolds.  The organoid culture system established is thought highly applicable in the treatment of ALF but might also be well suited to drug screening and disease modelling.

For further information
Read article: Methylcellulose as a scaffold in the culture of liver-organoids for the potential of treating acute liver failure
Planer freezers: Medium sized control rate freezers

UEARS

UEARS - Upper Egypt Assisted Reproductive ConferenceThe 4th Upper Egypt Assisted Reproductive Conference (UEARS), will take place in Cairo from the 20th – 22nd February 2019 and is aimed at gynaecologists, andrologists, embryologists, lab directors, medical field researchers and those involved in Quality Control and Total Quality Management of the ivf laboratory. 

This year, UEARS hopes to welcome over 2000 delegates and will offer a cutting-edge programme, with an international line up of speakers. UEARS’ goal is to foster innovation and collaboration and to fill the gap between science and practice.

Planer will be supporting our local distributor, Modern BioSystems. Come and see us on stand 7 to find out more about our CT37stax™ benchtop incubatorDATAssure™ laboratory wireless alarm and monitory system and our range of cryogenic freezers.

To find out more:-
UEARS - Upper Egypt Assisted Reproductive Conference
CT37stax™ benchtop incubator

 

News Stories - 2018

News Stories - 2017