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Fertility preservation

Gene Expression and Aneuploidy In Human Oocytes

We welcome you to a selection of talks from "Oocytes, Ovary, and Transplantation: New Discoveries Applied To Fertility Preservation." We are honored to present to you recognized experts who are innovating advances in in vitro maturation, oocyte and organ cryopreservation, transplantation technology, and stem cell applications. Thank you for participating in this self-study course.

Brief Description of Dr. Well's presentation: Many aspects of oocyte biology are currently poorly understood. The cellular processes occurring during normal oocyte maturation are not yet fully characterized; the impact on the oocyte of infertile pathologies, such as polycystic ovary syndrome, are not satisfactorily defined at the molecular level; similarly, the effect on the oocyte of different ovarian stimulation regimens and in vitro maturation protocols remain unknown.

A useful approach for gaining insight into the processes occurring in a biological sample is the examination of gene expression. The activity of individual genes is constantly changing, fluctuating in response to the changing needs of the cell. Variation in environmental conditions will induce characteristic changes in gene expression, as will factors such as altered metabolic requirements, the presence of aneuploidy, and progress through the cell cycle. It is clear that the analysis of gene expression has great potential to enhance understanding of oocyte development, revealing new markers of oocyte quality, assisting in the optimization of ovarian stimulation and IVM protocols, and revealing the underlying basis of a variety of infertile pathologies.

Impact Of Human Oocyte In Vitro Maturation and Cryopreservation On Oocyte-Granulosa Crosstalk

Brief Description of Dr. Albertini's presentation: Advancements in the application of cell culture and cryobiological methods to gametes for the production of human embryos for clinical use are moving at an alarming rate. Limited in many cases by access to human materials, most studies seeking to deploy oocyte freezing and/or in vitro maturation (IVM) in the clinic must rely upon pregnancy or live-birth outcomes to ascertain measures of efficacy and safety. As evidence accumulates to indicate that ovarian somatic cell support is vital for the oocyte to achieve and maintain its developmental potential after fertilization, the need to better understand the mechanisms that underlie oocyte-granulosa crosstalk has grown. In particular, methodology is available that allows for the analysis of crosstalk in metaphase-2 human oocytes following either IVM or cryopreservation. In the case of IVM, it is now clear that unless cumulus-oocyte complexes (COCs) are harvested in a medium that stabilizes oocyte-granulosa contacts via transzonal projections (TZPs), the pace of meiotic progression will be accelerated and a loss of coordination between nuclear and cytoplasmic maturation will occur. Thus, two key challenges in this field will be to design cell culture and cryobiological strategies that both preserve and/or allow for the re-establishment of oocyte-granulosa crosstalk in order to maintain oocyte quality.

Human Oocyte Cryopreservation By Slow Freezing

Brief Description of Dr. Borini's presentation: The first protocol used to freeze oocytes was the same one applied to embryo cryopreservation and is based on a two-solution procedure. Borin’s lab developed a novel protocol for oocyte cryopreservation and describes that protocol here. This protocol was developed based on the rationale that a sucrose gradient between the freezing and the thawing procedure would reduce the osmotic stress that the egg has to undergo during this delicate procedure. With this protocol in place, Borini has succeeded in improving both survival and pregnancy rates to the point that they are finally comparable to cycles using fresh, never frozen eggs. With frozen eggs Borini reports now being able to achieve 6 live births out of 100 oocytes cryopreserved.

Optimal Cooling Conditions and Permeating Parameters Of Ovarian Tissue

Brief Description of Dr. Arav's presentation: A whole ovary is a large voluminous sample that creates problems of heat and mass transfer during freezing. In order to evaluate the permeability of ethylene glycol (EG), Arav has perfused radio labeled EG into sheep ovaries through an artery and measured the time it takes to reach the ovarian cortex. He found that 3 to 6 minutes are sufficient to reach saturation in the cortical area of the ovary. Heat transfer of such a large organ is problematic; freezing propagates from the exterior to the interior of the organ resulting in a non-homogeneous cooling condition. In addition, a massive latent heat is released, which can cause melting of the frozen area, leading to prolonged isothermal process that damage the tissue.

Arav developed a new freezing method in which he can achieve a slow freezing process in which latent heat is efficiently removed, minimizing the isothermal period, resulting in a homogeneous cooling/freezing process through the entire tissue. The method is based on directional freezing where the sample is moved at a constant velocity through a predetermined thermal gradient. A very slow movement at a velocity of 0.01mm/sec produced a cooling rate of 0.3ºC/min. Using this method, Arav's lab was able to successfully freeze whole sheep and cow ovaries.

Whole Human Ovary Cryopreservation

Brief Description of Dr. Patrizio's presentation: One of the options for the preservation of future fertility is represented by cryopreservation of ovarian cortical strips. Unfortunately, the survival time after thawing and re-transplantation is rather limited, being about 2 to 3 years at the most due to the ischemic damages of the ovarian tissue. To overcome this limitation Patrizio began to study cryopreservation of whole human ovaries using a novel multi-gradient freezing device. He reports on the histological survival after freezing/thawing for different time periods. The preliminary work shows excellent histological survival after cryopreservation and thawing of whole human ovaries. With the advances in whole ovary transplantation surgeries, the availability of an effective cryopreservation methodology is paramount.

History Of Ovarian Banking and Transplantation

Brief Description of Dr. Gosden's presentation: Transplantation of human ovarian tissue was pioneered by Robert Morris in New York City over a century ago. He claimed success with allogeneic transplants, but transplantation antigens were unknown then and cryopreservation technology was still over 50 years away. In recent decades, two converging factors have led to novel applications, namely 1) rising numbers of young, long-term survivors of cancer and other diseases requiring cytotoxic treatment, and 2) development of effective tissue banking methods. In this presentation Dr. Gosden discussess the history of ovarian banking and transplantation.

Breast Cancer and Assisted Reproduction

Brief Description of Dr. Fabian's presentation: In 2007, approximately 178,00 women were diagnosed with invasive breast cancer. Approximately 5% were under the age of 40 at the time of diagnosis, and many were advised to have cytotoxic chemotherapy with alkalating agents and anthracyclines as part of their treatment. The majority (81%) of these young women will be alive and free of disease at 5 years. The likelihood of infertility after treatment is dependent on age, dose of alkalating agent, and number of cycles of treatment. Approximately 30% of these women or 3,000 women per year will not yet have had children at the time of diagnosis and will desire a later option of childbearing. Currently, 5% or fewer women diagnosed with breast cancer under 40 go on to deliver a live child. Although emergency fertility preservation services are available, up to 6 weeks lead time prior to initiation of planned chemotherapy may be needed to obtain embryos for cryopreservation. Other procedures requiring less lead time are generally associated with a lower proportion of live births. Barriers to emergency fertility preservation procedures include 1) lack of immediate access to services and co-ordination of care with treating oncologists, 2) concerns on the part of the treating oncologist that elevated hormone levels after gonadotrophins will increase the risk of reoccurrence in women with hormone receptor positive tumors, and 3) increased use of neo-adjuvant chemotherapy (prior to definitive surgery) in patients needing systemic treatment, which reduces or eliminates the lead time for ovarian stimulation and egg harvest. Incorporating a fertility specialist into the mainstream breast/cancer center clinical framework with appropriate supportive infrastructure and protocols for immediate referral and consultation reduces these barriers.

Successful Animal Uterine Transplant

Brief Description of Dr. Brännström's presentation: Uterine transplantation is developed as a possible future treatment for patients with absolute uterus factor infertility. Patients with the Mayer-Rokitansky-Kuster-Hauser (MRKH) syndrome, patients having had hysterectomy for benign or malignant uterine/cervical diseases, and patients with intrauterine adhesions are the major groups of patients that could benefit from this procedure.

Several uterine transplantation animal models have been developed by Brännström's research group to examine various aspects of uterus transplantation and to optimize the uterus transplantation procedure for human use. It is predicted that uterus transplantation may reach a clinical stage within 3 to 5 years, in the event of a continuous high research activity within this field.

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