Ovarian Tissue Transplantation Options Explored
By Tommaso Falcone, MD
Over the past few decades, considerable attention has been given to the long-term reproductive function of females undergoing cancer treatment. Many treatments damage the ovaries and induce premature ovarian failure.
There are several options for these patients. Some are routinely available clinically, such as in vitro fertilization (IVF) and embryo freezing. Others, such as oocyte and ovarian tissue freezing, are considered experimental. Oocyte freezing still requires IVF and therefore sufficient time for ovarian stimulation. Furthermore, this approach is not possible in prepubertal children.
Ovarian tissue can be removed surgically and frozen. Usually the cortex is cut into
small pieces and the tissue cryopreserved and subsequently transplanted back.
However, since this is an avascular transplant, ischemic necrosis of the graft occurs
before neovascularization can occur. As an alternative, we proposed removing,
freezing and subsequently thawing and transplanting an ovary with a vascular
pedicle to try to diminish the ischemia. We chose a heterotopic site, the inferior
epigastric vessels that could theoretically provide an easier access. A sheep model
was used. The ovary was removed from its initial site with its vascular pedicle, cryopreserved, thawed and transplanted to the inferior epigastric vessels.
Our first experiments showed successful autotransplantation of frozen/thawed
ovaries with their vascular pedicle. This was manifested by restoration of selected
ovarian function including in vitro maturation of collected oocytes, presence of
follicles from several stages of folliculogenesis and blood vessels expressing
specific markers of vascularization, and proliferation and apoptosis of ovarian
cells. Thus, heterotopic autotransplantation of a whole frozen/thawed ovary
allows for development of preovulatory follicles, oocyte growth, and restoration
of vascularization and cellular function. However, there was some necrosis of the
vessels (above), probably due to the differential freezing characteristics of different
Additional improvements are required to enhance the efficiency of auto transplantation
of frozen/thawed ovaries to produce more oocytes. Freeze-thaw protocols still require further optimization and depletion of the primordial follicle reserve will need to be addressed. But current evidence suggests that whole frozen-thawed ovary transplantation may be successful in humans in the future.
Dr. Falcone is Professor and Chairman of the Ob/Gyn & Women's Health Institute. Physicians may reach him at 216.444.1758 or firstname.lastname@example.org.
New Horizons in Embryo and Oocyte Cryopreservation
By Nina Desai, PhD, HCLD
In vitro fertilization (IVF) is one of the most exciting and dynamic areas in medicine. In the past two decades, tremendous strides have been made with IVF techniques. Clinical pregnancy outcomes have risen dramatically, from 20 percent to almost 60 percent in younger patients. With this increase has come a movement to reduce the number of embryos being created and also to avoid high order multiple pregnancies by reducing the
number of embryos being transferred.
Effective techniques for cryopreservation of embryos and unfertilized oocytes for a future cycle have been the focus of research in laboratories all over the world. Traditional cryopreservation technology has involved the use of cryoprotectant agents, dehydration
of the cell and programmed slow cooling at rates of -0.3 ºC a minute down to -36 ºC, before plunging in liquid nitrogen. The entire process can take as long as two hours. The most frequent cause of damage is intracellular ice crystal formation leading to cellular disruption on thaw. Oocyte cryopreservation has been even more challenging than embryo freezing due to the large volume of the egg and the susceptibility of the meiotic spindle to depolymerization as temperatures are reduced. Disruption of the meiotic spindle and aberrant chromosome segregation during oocyte freezing can lead to an increase in embryos with aneuploidy.
A new cryopreservation technique known as “vitrification” shows tremendous promise and has been adopted by the Cleveland Clinic’s IVF program for cryopreservation of embryos at all stages, as well as oocyte cryopreservation. Vitrification involves ultra rapid freezing of the oocyte/embryo from 37 ºC to -196 ºC at a rate of -20,000 to -30,000 ºC per minute. High concentrations of cryoprotectants and high cooling rates are necessary to transition the embryos into a “glass-like state” to avoid cryoinjury. To achieve such high cooling rates, the oocyte/embryo is frozen in miniscule fluid volumes of less than one microliter.
Cleveland Clinic’s IVF laboratory uses a novel carrier called the “cryoloop” for vitrification. The cryoloop is a fine nylon loop measuring no larger than the eye of a sewing needle. The loop is dipped in cryoprotectant and the embryo/oocyte is placed on the fluid film (<1ul) using a micropipette. The cryoloop is then immediately immersed directly in liquid nitrogen instantaneously freezing the sample.
Vitrification has had a tremendous impact on our embryo cryopreservation program. We were one of the first laboratories in the United States to successfully apply this methodology for cryopreservation of eight-cell embryos (Desai et al, 2007). To date, the post-thaw survival rate has been 95 percent. We have achieved a clinical pregnancy rate of 45 percent (based on fetal heart by ultrasound). Vitrification technology has completely replaced conventional methods for freezing in our laboratory.
The existence of excess cryopreserved embryos, together with steady improvements in clinical outcomes, often present an ethical dilemma for patients who become pregnant and no longer need their frozen embryos. Many have difficulties consenting to
embryo disposal or donation for a variety of reasons, including religious beliefs. Oocyte cryopreservation has the potential to resolve some of these issues. While vitrification appears to have increased the efficacy of the procedure, oocyte freezing is still in its infancy. This new technology is very appealing and offers women the ability to bank their gametes in much the same way as men bank sperm. Cleveland Clinic now offers oocyte cryopreservation on an experimental basis to a select group of patients who may benefit from this procedure, including cancer patients who can freeze oocytes before undergoing radiation and chemotherapy.
The process of oocyte cryopreservation is much more difficult than embryo freezing because of the large volume of the egg and the susceptibility of meiotic spindle disruption during the freezing process, resulting in embryos with chromosomal abnormalities. Using the cryoloop, Cleveland Clinic’s IVF laboratory has been able to successfully freeze oocytes in fluid volumes of less than one microliter.
Dr. Desai is the Director of IVF and Clinical Research. For more information about oocyte cryopreservation, contact her at 216.839.2907, or email@example.com.
Trophectoderm Biopsy for PGD Offers Greater Diagnostic Confidence, Increases Potential for Successful Pregnancy Outcome
Cleveland Fertility Center now provides patients access to a cutting-edge technique for genetic screening of patient embryos that offers several advantages over traditional PGD techniques that remove a single cell on day three. This powerful new technique, known as Trophectoderm Biopsy, allows the removal of multiple cells at the blastocyst stage. Embryos can be screened for inherited genetic disorders as well chromosomal abnormalities. By selecting only normal embryos for transfer, the patient's opportunity for a pregnancy with a healthy baby is increased.
Trophectoderm biopsy is performed on day five, at the 16- to 32-cell blastocyst stage when the embryo has the highest implant potential. A laser is used to open the zona pellucida and excise five or six cells from the trophectoderm as it begins to herniate. The inner cell mass is unaffected. The cells are analyzed using a highly valid and rapid microarray technology offered by Genesis Genetics.
This technology examines all 24 chromosomes for abnormalities by comparing the sample against a known normal control. A computer-generated chromosome map is produced for each embryo sample. Embryos found to be abnormal are discarded and only the chromosomally normal embryos are implanted. The screen is completed within 24 hours, allowing patients to have fresh embryo transfers in the same in vitro fertilization (IVF) cycle.
Because trophectoderm biopsy looks at multiple cells at a point in which the embryo has shown developmental potential, it offers greater diagnostic confidence and a higher likelihood for a successful implantation and healthy pregnancy outcome. The technique is especially powerful for patients of advanced maternal age who have a high rate of aneuploid embryos, patients with naturally recurring miscarriages as well as unsuccessful IVF cycles, patients with unexplained infertility, and those concerned about passing on known inherited genetic mutations.
For more information about this procedure, contact the Cleveland Clinic Fertility Center at 216.444.8360.
Richard E. Jacobs Health Center Now Open in Avon
Cleveland Clinic care is now available in western Cuyahoga County and beyond with the opening of the Richard E. Jacobs Health Center. Located at 33100 Cleveland Clinic Drive, the new facility features not only fully equipped medical offices for routine and specialty care for the whole family, but services to help meet the needs of those seeking infertility advice and treatment.
The 190,000-square-foot building includes a pharmacy, on-site laboratory and full conference facilities. Four operating rooms, four procedure rooms and 23-hour overnight stay capabilities round out the facility. A 24-hour emergency department with helipad will open in the future. Internal medicine and family medicine specialists and obstetricians and gynecologists now see patients. Infertility services are offered that include a board-certified infertility specialist, a urologist specializing in male infertility and a full-service Andrology lab.
Cleveland Clinic also offers infertility services at main campus, Beachwood Family Health & Surgery Center, Solon Family Health Center, Strongsville Family Health & Surgery Center and the Twinsburg Family Health & Surgery Center.
To schedule an appointment for infertility services, call 216.444.6601.
Now On Bookshelves
Cleveland Clinic Gynecologists Collaborate on New Text
Basic, Advanced, and Robotic Laparoscopic Surgery: Female Pelvic Surgery Video Atlas Series
Tommaso Falcone, MD, FRCSC, FACOG
Vice Chair, Office of Professional Staff Affairs
Professor and Chair, Ob/Gyn & Women’s Health Institute
Jeffrey M. Goldberg, MD
Section Head, Reproductive Endocrinology and Infertility
Ob/Gyn & Women’s Health Institute
This book and accompanying videos demonstrate the most important aspects of laparoscopy including anatomy, instrumentation and principles of laparoscopic suturing.
Hours of video footage, detailed discussion and illustrations provide a clear description of the most effective laparoscopic procedures, how to manage complications and more.
Treatment Guide Available
Cleveland Clinic has created a comprehensive guide to the treatment of infertility, which is available online.
This free downloadable guide includes:
- The causes of infertility
- When couples should seek help
- Available treatment options
- A directory of Cleveland Clinic physicians and locations providing care
If your patients are interested in comprehensive information on infertility, they can access it at clevelandclinic.org/infertilityguide.