Preservation of Ovarian Tissue and Chemo-Radiotherapy

At the present time, freezing of ovarian tissue is considered experimental and so thawing and
future use of the tissue to initiate a pregnancy must be performed as part of a research
program. With the patient's permission, a portion of her ovarian tissue will be frozen for
her own use and a portion will be donated to a research pool.

Twenty subjects/year will be participating in this study.

Participation in this study will last until the patient's ovarian tissue is all used, or the
patient decides to drop out of the study, or for the rest of her life. Freezing of ovarian
tissue can be done only for research. Thawing and future use of the tissue to start a
pregnancy must be done as part of a research program. After the age of 18, the patient will
decide how and at what institution she wishes to use her own tissue in the future, for the
purposes of becoming pregnant. The number of visits will be decided by the surgical procedure
performed. The Investigators will contact the patient by phone, email, or mail until she has
turned 18 years old, used her tissue or dropped out of the study (whichever comes first) to
follow the patient's medical and fertility status over time and to ask questions about any
future use of her frozen tissue and the possible results of the treatment. Depending on the
patient's age at the time of enrollment, follow-up might be up to 30 years.

PURPOSE Primary objective: To assess resumption/initiation of menses after surgical ovarian
preservation (ovarian tissue explant followed by freezing and autologous transplantation).

Secondary objective: To assess pre- and post-treatment production of various hormones, i.e.,
anti-mullerian hormone (AMH), insulin-like growth factor-1 (IGF-1), inhibin-B, follicle
stimulating hormone (FSH), luteinizing hormone (LH), and estradiol, by serum and histological
measurements.

Tertiary objective: To assess long-term sexual development, fertility competence, and
lifespan of ovarian preservation via surgical autotransplant.

RATIONALE Chemotherapy treatment with alkylating agents and/or total body irradiation will
prevent normal pubertal development of uterus, ovaries, and breasts because of primary
ovarian insufficiency (POI). Explant and autologous transplantation of ovarian tissue will
prevent ovarian insufficiency and allow normal sexual development through puberty and
fertility competence.

RESEARCH DESIGN Prospective cohort study.

STUDY PROCEDURES Subject Recruitment and Screening- Subjects will be recruited by referral
from the St. Jude Children's Research Hospital (SJCRH) and Methodist-Le Bonheur physicians to
the Reproductive Medicine clinics held by Dr. Laura Detti. Patients will undergo an informed
consent process in accordance with St. Jude Children's Research Hospital (SJCRH) and
Methodist-Le Bonheur Institutional Review Boards.

Screening Visit- Complete medical history and physical exam; vital signs, height, and weight.
Psychological Consult on impact of therapy.

Laboratory testing- Lab results for Estradiol, follicle stimulating hormone (FSH),
luteinizing hormone (LH), Anti-Mullerian hormone (AMH), Inhibin B, will be obtained from the
patients' clinical charts. In addition, Electrocardiogram (ECG) (12-lead); Bone age scan (if
pre-pubertal), Pelvic ultrasound (transabdominal or transvaginal, depending on the patient's
age) will be obtained from the patients' clinical charts. If a pelvic ultrasound has not been
obtained prior to surgery, the procedure will be performed under anesthesia prior to the
patient undergoing surgery for ovarian tissue harvesting).

Surgical Procedures- Unilateral oophorectomy will be performed by laparoscopy prior to
undergoing chemo-radiation treatment as specified above. Subsequent ovarian cortex
autotransplant will be performed by laparoscopy after the patients have been declared cured
by their physicians.

Laparoscopic procedures- Upon general anesthesia is induced, attention will be turned to the
patient's abdomen where 3-4 0.5 cm skin incisions will be performed (one in the umbilical
fold and 2 lateral incisions; possibly, a 4th suprapubic incision). Disposable, bladed,
trocars will be inserted in the abdominal cavity. The ovaries will be identified and a
unilateral oophorectomy will be performed by retroperitoneal clamping/dissection of the
infundibulo-pelvic and utero-ovarian ligaments.

A bipolar electrode will be used to assure accurate hemostasis and irrigation will be
performed as needed. Upon retrieval of the trocars from the abdomen, the skin incisions will
be reapproximated with 4-0 vycril sutures.

The second laparoscopic procedure will be done to perform autologous ovarian tissue
transplant by suturing the thawed cortex fragments to the recipient ovary using 7-0 Prolene
(Ethicon, Johnson and Johnson, USA), using the technique described by Silber et al. (Silber
et al., 2005; Silber and Gosden, 2007) applied during laparotomy for transplantation.

As the transplanted tissue exhausts its function after the 1st autologous transplant, other
laparoscopic procedures might become necessary over the years to transplant the rest of the
cryopreserved tissue until no more tissue is available in the cryobank storage.

Freezing Procedure- The process of vitrification will be started in the Operating Room or in
the lab within 120 minutes from collection of the sample and will be completed in the
University of Tennessee Center for Reproductive Medicine laboratory. The tissue will be
subdivided into numerous vials for individual storage/thawing. This will allow selective
thawing for autotransplant over multiple procedures, if necessary.

Embryology laboratory procedures- The ovarian tissue that is stored for the patients will be
used for autologous transplant. However, other techniques that are performed in the
embryology laboratory may be available to the patients in the near future and might represent
the best option for fertility preservation in patients with blood-borne cancers (i.e.:
leukemia), in whom the risk of re-seeding cancer cells with tissue transplant is high.

Follow-Up Evaluation- Study treatment will include a post-operative visit and following
scheduled follow-up visits for the patients' initial disease during which a complete physical
exam and psychological evaluation will be conducted, and laboratory testing will be obtained
for follicle stimulating hormone (FSH), luteinizing hormone (LH), anti-mullerian hormone
(AMH), and Inhibin B.

Visits will be scheduled one month after explant and every 6-12 months until autotransplant
of tissue and afterwards.

Laboratory testing, Electrocardiogram (ECG) (12-lead), Bone age, Pelvic ultrasound may be
repeated yearly, as part of the patient's scheduled follow-ups. Bone age assessment will stop
when a bone age of 18 years is achieved.

Patients who are going to develop ovarian insufficiency prior to or after puberty will
undergo estrogen/progesterone therapy as per our institutional protocols.

Follow-up will continue until the patient stays enrolled in the study or until she reaches 40
years of age, or she goes in menopause and does not have, or does not desire, further tissue
for transplantation.

Inclusion Criteria:

1. Pre- and post-pubertal patients who are diagnosed with conditions that necessitate
chemo- or radiotherapy and wish to preserve their ovarian function.

Exclusion criteria:

1. Patients with acute or chronic conditions that would preclude surgery. These include,
but are not limited to, hyper- or hypo-coagulation disorders, lung conditions
precluding mechanical ventilation, brain death.

2. Patients for whom the primary oncologist and/or pathologist discourage entering the
protocol because of specific condition characteristics. These include, but are not
limited to, cancers with high risk of ovarian metastasis such as leukemia and
non-Hodgkin lymphoma.

3. Patients with known positive bone marrow for leukemia or solid tumor.

4. Patients whose tumor is in an organ that communicates with the peritoneum and
peritoneal sampling at time of primary excision shows evidence of disease.

5. Patients older than 40 years of age.

6. Males.