Download OTC_Protocol_Template_Pediatrics

Ovarian Tissue Freezing Prior to Chemotherapy or Radiation Therapy 1
Ovarian Tissue Freezing For Fertility Preservation In Girls Facing A Fertility
Threatening Medical Diagnosis Or Treatment Regimen: A Study By The National
Physicians Cooperative of the Oncofertility Consortium At [INSTITUTION]
PRINCIPAL INVESTIGATOR:
[INSERT PI NAME]
CO-INVESTIGATORS:
[INSERT CO-INVESTIGATORS]
FUNDING AGENCY: National Institute of Health P50HD076188
National Physicians Cooperative of the Oncofertility Consortium at Northwestern
University
Version: 5/5/2017
Ovarian Tissue Freezing Prior to Chemotherapy or Radiation Therapy 2
TABLE OF CONTENTS
1.0
Background
2.0
Specific Aims
3.0
Significance
4.0
Primary Studies
5.0
Patient Eligibility
6.0
Methods
6.1
Informed Consent and Survey
6.2
Collection of Ovarian Tissue
6.3
Methods of Ovarian Cryopreservation
6.4
Ovarian Tissue Storage
6.5
Clinical Data to be Collected
6.6
Efficacy of Cryopreservation
6.7
Feasibility of 3-Dimensional System for In Vitro Maturation of Immature
Ovarian Follicles
7.0
Statistical Considerations and Data Management
8.0
Ethical Issues
9.0
Reimbursements and Research Costs
References
Version: 5/5/2017
Ovarian Tissue Freezing Prior to Chemotherapy or Radiation Therapy 3
1.0 INTRODUCTION – BACKGROUND AND RATIONALE
Although malignancy remains a critical health concern, significant medical advances in
cancer detection and treatment have improved survival rates for patients. As patients
live longer, the early and late consequences of cancer management are beginning to
assume greater importance for survivors, their families and providers. For instance,
when considering the long term sequelae of cancer therapy, infertility surfaces as a
primary concern, particularly, among female survivors (Zeltzer, 1993). Unlike other late
effects of cancer treatment, such as complications in cardiovascular or liver function,
female infertility has biological and psychosocial implications that cannot be narrowly
defined, nor easily addressed given the number of ethical and legal questions
surrounding fertility preservation (Patrizio et al., 2005). The clinical implications can
range from acute ovarian failure, to induced premature menopause and ultimately may
result in difficulty with getting pregnant (Nieman et al, 2006). Particularly at risk are
females who are of or approaching reproductive age at the time of cancer diagnosis,
those who have received abdominopelvic radiation or high doses of alkylating agents
(particularly cyclophosphamide and procarbazine) and those diagnosed with Hodgkin
lymphoma (Chemaitilly et al., 2006; Sklar et al., 2006; Larsen et al., 2003; Chiarelli et
al., 1999; Mattison, 1990). These concerns are not limited to cancer patients; treatment
for other medical conditions such as rheumatoid arthritis and lupus may also result in
infertility.
Limited options are currently available for girls and young women suffering from cancer
to ensure future reproductive capacity. Patients who will receive radiation to the ovaries
may opt to have their ovaries shielded from the ovary or transposed to a part of the
body where it will be protected from the harmful effects of radiation (oophoropexy). This,
however, will not protect the ovaries from the gonadotoxic effects of chemotherapy.
(Meirow & Nugent, 2001; Sonmezer & Oktay, 2004; Wallace et al., 2005)
The most successful options for fertility preservation in women facing cancer is embryo
banking (formerly called emergency IVF) or egg banking prior to chemotherapy. The
pregnancy rate with this technique averages 30-40% (Lobo, 2005) but in patients under
35 years old is often higher. This success rate is inferred from embryo cryopreservation
programs in the general IVF population, with only 1 report specifically addressing
pregnancy outcomes in cancer survivors who opted to freeze embryos prior to cancer
treatment (Ginsburg et al, 2001). However, there is no reason to believe that embryo or
oocyte viability following freezing would be any different in cancer patients. While often
successful, this option requires time for ovarian stimulation, oocyte retrieval, and in-vitro
embryo development, which delays cancer treatment 2-3 weeks (Kim et al, 2006). This
process can be even longer if ovarian hyperstimulation syndrome (OHSS) occurs, as is
expected in 5% of all IVF cases and can be severe in 1%. Younger patients with
presumably normal ovarian function prior to cancer treatment are particularly
susceptible to developing OHSS, and ovarian stimulation regimens are individually
tailored and adjusted during the cycle to avoid such an outcome. Embryo banking is
ideally used when there is a partner involved, but can be performed in single, young
cancer patients who are willing to use donor sperm. One concern with using donor
Version: 5/5/2017
Ovarian Tissue Freezing Prior to Chemotherapy or Radiation Therapy 4
sperm pertains to the uncertain acceptance of the cryopreserved embryos should the
patient find a new partner in the future. In addition, embryo banking cannot be offered to
patients diagnosed with cancer before puberty because mature oocytes cannot be
collected. While embryo banking is the preferred way to preserve fertility for young
adults, a great deal of emotional and physical stress is associated with the procedure.
The challenges and stress of issues surrounding fertility preservation motivate the
desire to find alternatives for young cancer patients.
Mature oocytes can be retrieved following hormonal stimulation that is identical to that
used for in vitro fertilization and cryopreservation (Chen et al, 1986). Until recently, post
thaw recovery of cryopreserved oocytes with subsequent fertilization and embryo transfer
lead to disappointing results (Tucker et al, 1996; Porcu et al, 1999; Marina and Marina,
2003). However, improvements in both freezing and thawing techniques for human
oocytes necessitated by the ban in Italy on freezing embryos (only eggs can be frozen) are
currently leading to post thaw recovery of oocytes that approaches 80% with pregnancy
rates that are the same as for fresh eggs following in vitro fertilization (on an egg per egg
basis) Coticchio et al., (2006); Paynter et al., (2005); Fabbri R, et al., (2001); Bianchi et
al., (2007); Borini A et al., (2004); Borini et al., (2006)). Like in vitro fertilization with
freezing of embryos, the patient must undergo hormonal therapies to stimulate the growth
of multiple follicles, followed by a surgical oocyte retrieval: a process that may take up to 3
weeks and can put the patient at risk of hyperstimulation syndrome. Therefore, the use of
emergency IVF with the freezing of embryos or oocytes may delay the start of cancer
therapies and may not be feasible for patients who have not yet reached puberty.
Fertility preservation options for female patients who cannot delay treatment or are too
young to undergo hormonal stimulation rely upon ovarian tissue cryopreservation with
subsequent transplantation and/or in vitro follicle maturation (IFM). Ovarian tissue
containing immature oocytes (primordial follicles, antral and pre-antral follicles) has been
successfully cryopreserved in several animal models including rodents, sheep, and nonhuman primates (Harp et al, 1995; Oktay et al, 1998; Gosden, et al, 1994; Baird et al,
1999). When thawed, this tissue can be grafted into a host with resumption of both
endocrine and reproductive function. In 2004, the first successful application of this
technology was reported in humans (Donnez, et al.,2004) using autologous orthotopic
transplantation of frozen thawed human ovarian tissue. In this study, the patient had return
of endocrine function within 3 months of the transplant and achieved a spontaneous
pregnancy. As of March 2010, there have been 15 spontaneous live births following
transplantation of frozen/thawed ovarian tissue and in one case production of 3 embryos
through in vitro fertilization that were transferred in a single procedure without a pregnancy
(Donnez et al.2004; Meirow et al.2005; Meirow et al.2007; Anderson et al.2007;
Demeestere et al, 2006; Schmidt et al, 2005; Oktay and Oktem; 2005). In addition, there
have been 10 pregnancies (all but one spontaneous) from direct ovarian tissue
transplantation (Silber et al.2005, 2007 a,b) in a series of identical twins. Oral
presentations at international meetings suggest that the number of live births following
transplant of frozen ovarian tissue is higher but only case reports have been published.
Version: 5/5/2017
Ovarian Tissue Freezing Prior to Chemotherapy or Radiation Therapy 5
An important concern with transplanting ovarian tissue is the potential reintroduction of
cancerous cells into a patient in remission, as seen in rodent lymphoma. Ovarian tissue
transplant using this leukocyte rich tissue may be contraindicated in patients with certain
types of cancer (hematologic malignancies) (Shaw et al., 1996 a, b). Recent findings by
Meirow et al. (2008) suggest that testing of the stored tissue at the time of thaw may
avoid reimplantation of tissue with malignant cells. However, use of in vitro follicle
maturation using the thawed tissue with subsequent IVF may be a safer option.
An alternative to transplantation of the ovarian tissue is isolation of the immature
oocytes from the cryopreserved ovarian tissue and maturation in vitro with subsequent
in vitro fertilization (Oktay, et al.1997). Traditional in vitro maturation systems make use
of a 2-dimensional culture (Cortvrindt et al., 1996) that yields few oocytes that are
viable, mature or fertilizable. NU researchers (Xu et al., 2006) have demonstrated that
a 3-dimensional scaffold system using alginate (a polysaccharide material which gels in
the presence of calcium), which may more closely mimic the in vivo ovarian physiologic
conditions, produces a high follicle survival rate (93%) and oocyte maturation success
(73%) in mice. Embryos derived from these oocytes were successfully fertilized in vitro
and transferred to pseudopregnant female mice to produce live young. Both male and
female offspring were also fertile. Parallel studies are underway in non-human primates.
Ovarian follicles are contained in the cortex of the ovary. Large numbers of follicles can be
obtained from strips of the ovarian cortex thus eliminating the need for hormonal
stimulation of the ovary. Restoration of fertility and endocrine function made possible by
maintaining the structural integrity of follicle using in vitro follicle maturation (IFM) could
substantially improve the quality of life for women of reproductive age receiving cancer
therapies which may otherwise impair ovarian function. Patient age, marital status, time
available before treatment and specific diagnosis are just a few of the factors that affect
the choice of fertility preservation options in each individual patient.
Ovarian cryopreservation has been performed in children and adolescents in other
countries. A French group has published their experience with human ovarian
cryopreservation in children beginning in 1998. Approximately 78 ovaries have now
been cryopreserved, 63 from girls <18 years old (the youngest was 0.1 years old at the
time of ovarian cryopreservation). In a cohort of 47 prepubertal girls, they found
laparoscopic oophorectomy to be a safe and time efficient procedure with low postoperative pain and excellent cosmetic results. Additionally, they report a strong
correlation between age and follicular density with younger patients having a higher
concentration of primordial and primary follicles compared to older patients. Five
families refused ovarian cryopreservation because it was too new and uncertain (Poirot
et al, 2002; Martelli et al., 2006, Poirot et al., 2007.). Feigin et al. reported on a series of
23 patients (mean age=14 years; range=5-17.5 years) with similar findings in Israel
(Feigin, et. al, 2007). In addition, this group reported harvesting oocytes from the
removed ovary in 17 out of 19 patients and a 34% in vitro maturation rate (Revel et al.,
2008).
[INSTITUTION NAME] is part of the National Physicians Cooperative of the
Oncofertility Consortium which includes 50 fertility preservation centers
Version: 5/5/2017
Ovarian Tissue Freezing Prior to Chemotherapy or Radiation Therapy 6
nationwide including 12 serving pediatric and adolescent patients. Six centers in
the Consortium have been freezing ovarian tissues for patients for over 5 years (two of
them including children: Children’s Hospital of Milwaukee and University of Alabama).
Ten of the sites have approval to see patients and perform ovarian tissue
cryopreservation on patients under the age of 12:
- University of Alabama (8 years and older)
- University of Louisville (3 years and older)
- Infertility Center of St. Louis (2 years and older)
- University of North Carolina (7 years and older)
- Oregon Health Science University (Newborn and older)
- Children's Hospital of Pennsylvania (10 years and older)
- Children's Hospital of Pittsburgh (Newborn and older)
- Women and Infants Hospital of Rhode Island (Newborn and older)
- Wisconsin Gundersen (8 years and older)
- Medical College of Wisconsin (Newborn and older)
To date, 5 children under the age of 12 years have participated in ovarian tissue
cryopreservation and 3 patients in the age range of 13-14 years old have participated.
Limited data is available on the feasibility of performing ovarian tissue cryopreservation
(OTC) in patients who have received therapy that is considered as likely to result in
complete and permanent loss of ovarian function. Preliminary data from the Oncofertility
Consortium National Physician’s Cooperative on a small number of patients who have
undergone ovarian tissue cryopreservation after receiving potentially sterilizing
chemotherapy reveal presence of normal-appearing follicles, which seem comparable to
those patients who did not have prior treatment before OTC. Appendix 8 shows a
comparison of histopathology of ovarian tissue in 2 patients who underwent OTC after
chemotherapy considered as likely to result in complete and permanent loss of ovarian
function compared with those from 2 patients who either did not receive any therapy
prior to OTC or received chemotherapy that is considered as unlikely to cause
permanent and complete loss of ovarian function. In addition, studies done by Abir et
al. (2008) and Fabbri et al (2012) showed no significant difference in follicular density of
ovaries removed post-chemotherapy, although some damage in follicular quality such
as increased oocyte vacuolization, detachment of oocyte from granulosa cells, and
abnormal granulosa cell nuclei were noted after chemotherapy. Nevertheless, they
suggest that while performing ovarian tissue cryopreservation prior to any therapy would
be ideal, it may still be considered after treatment in younger patients (≤20 years old,
and possibly 20-30 years old). Thus, these patients will be allowed enrollment onto this
protocol but will be required to donate 20% of their tissue for research.
2.0 SPECIFIC AIMS
The primary objective of this study is to utilize tissue donated to a national repository (at
the time of ovarian tissue cryopreservation for fertility preservation) to establish
technologies that will enable long-term preservation of ovarian function, including the
production of viable oocytes, through cryopreservation of ovarian tissue prior to
Version: 5/5/2017
Ovarian Tissue Freezing Prior to Chemotherapy or Radiation Therapy 7
chemotherapy, radiation or treatment that is expected to reduce fertility. This study will
provide research tissue for a national research repository that can be used to:
 Optimize techniques for freezing and thawing of ovarian tissue for use in transplant
or in vitro follicle maturation (IFM).
 Investigate factors affecting successful maturation of immature follicles obtained
from ovarian tissue including the use of 3-dimensional biogel scaffolds, growth
factors, hormones and other culture conditions.
 Determine the efficacy of ovarian cryopreservation techniques.
 Provide long term follow up on patients who have ovarian tissue frozen for their
own use.
 A substantial portion of the patient’s tissue will be cryopreserved and reserved for
her own use.
3.0 SIGNIFICANCE
Fertility preservation is an important quality of life issue for cancer survivors. While cancer
treatments have been become more efficacious, some can be expected to reduce fertility.
This study will provide a pool of research ovarian tissue to use to develop and test
methods to expand the range of fertility preservation options available to female cancer
patients. At the same time, a substantial portion of the patient’s tissue will be
cryopreserved and reserved for her own use using methods that have been published and
shown to result in pregnancies in humans.
4.0 PRIMARY STUDIES
This project will be conducted under the auspices of the “NIH Oncofertility
Consortium: Fertility Preservation in Women with Cancer” with the intent to
preserve fertility in females with cancer before they undergo chemotherapy or
radiation therapy. The program has evolved from the fortuitous coincidence of
remarkable scientific discovery and a new process of funding created by the National
Institutes of Health to transform the nation’s medical research capabilities and speed
the movement of research discoveries from the bench to the bedside.
The investigators of the proposed study have extensive clinical or laboratory experience in
the utilization of Assisted Reproductive Technologies for infertile patients. The clinical
investigators are fully trained and highly skilled in all the surgical procedures and clinical
counseling needed in this study. The laboratory investigators of the study have extensive
training in the physiology of oocyte and embryo development and have all the laboratory
skills required for this study, including cell/tissue culture, culture and manipulation of
gametes and embryos in vitro and gamete/embryo cryopreservation. They also have
extensive knowledge of the federal regulations governing reproductive tissue banking,
tissue preparation and patient screening and testing for long term tissue banking.
5.0 PATIENT ELIGIBILITY
Version: 5/5/2017
Ovarian Tissue Freezing Prior to Chemotherapy or Radiation Therapy 8
According to American Society for Clinical Oncology (ASCO) Recommendations for
Fertility Preservation in Cancer Patients (Lee, et. Al, 2006; see Appendix 6) all patients
should be apprised in advance of the potential effects of their proposed treatment on
fertility, be offered a fertility preservation consult and access to fertility preservation
interventions. The consultation will be conducted by a reproductive or medical
endocrinologist at either [INSTITUTION] During the fertility preservation consultation,
patients and their parents will be presented with a range of options, including oocyte
banking, use of donor eggs or adoption later in life, having no fertility preservation
treatment and ovarian tissue freezing. The American Society for Reproductive Medicine
(ASRM) Ethics Committee has also stated that it is ethical for parents to give consent
for these procedures for their children (2005; see Appendix 7). While the range of
options available will be discussed, fertility preservation options vary according to the
age of the patient, time line to cancer treatment, marital status and other individual
cultural and/or personal wishes or constraints. The treatment algorithms are presented
in the ASCO guidelines (see Appendix 7).
Only patients and their parents who choose ovarian tissue cryopreservation as their
means of fertility preservation and who meet the following criteria will be approached to
enroll in this study to donate 20% of their tissue to the research repository.
5.1 Inclusion criteria:
5.1.1. Be females, ≤30 years old who are post-menarche.
5.1.2. Will undergo imminent surgery, chemotherapy or radiation therapy which
is viewed as likely to result in a permanent and complete loss of subsequent
ovarian function (Chemaitilly et al., 2006; Sklar et al., 2006; Larsen et al., 2003;
Chiarelli et al., 1999; Mattison, 1990; Fertile Hope, 2007). These include:
5.1.2.1. any health condition or malignancy that requires removal of all or
part of one or both ovaries,
5.1.2.2. whole abdomen or pelvic irradiation ≥10Gy in post-pubertal girls
or ≥15Gy in pre-pubertal girls
5.1.2.3. total body irradiation, and
5.1.2.4. alkylating-intensive chemotherapy
5.1.2.4.1. cyclophosphamide cumulative dose ≥7.5 g/m 2
5.1.2.4.2. any treatment regimen containing procarbazine
5.1.2.4.3. busulfan cumulative dose >600 mg/m2
5.1.2.4.4. alkylating chemotherapy conditioning prior to stem cell
transplantation
5.1.2.5. combination of any alkylating agent with total body irradiation or
whole abdomen or pelvic radiation
5.1.2.6. cranial radiation ≥30 Gy
5.1.2.7. summed alkylating agent dose score ≥3 (see Appendix 9; Green
et al., 2009)
5.1.2.8. cyclophosphamide equivalent dose (CED) ≥ 4,000 mg/m 2 (see
Appendix 10. Green et al., 2014)
5.1.3. Be of reasonably good health otherwise.
Version: 5/5/2017
Ovarian Tissue Freezing Prior to Chemotherapy or Radiation Therapy 9
5.1.4. A pre-pubertal adult patient with Turner syndrome is eligible for this study
if other eligibility criteria are met.
5.1.5. For patients undergoing elective removal of an ovary for fertility
preservation only, have two normal-appearing ovaries as determined at the time
of surgery.
5.1.6. Have conditions for which definitive chemotherapy or radiation therapy
can be deferred for approximately 3 days until the subject recovers from
laparoscopic oophorectomy.
5.2 Exclusion criteria:
5.2.1. All boys,
5.2.2. Patients not about to undergo chemotherapy and radiation therapy,
5.2.3. Girls undergoing these therapies who are viewed as likely to retain ovarian
function,
5.2.4. Pregnant girls
5.2.5. Girls with only one ovary
5.2.6. Anyone at high risk for complications from the surgery and/or anesthesia.
5.2.7. Anyone unable to provide consent or assent (i.e. significant psychiatric
problems/cognitive delay).
5.2.8. Girls who have a large mass in the ovary that is being removed (abnormal
ovary) will not be enrolled in the study. That is, ovarian tissue cryopreservation will
not be performed on portions of an ovary that contained a large mass as
experience in this study indicates that this tissue is not suitable for patient use in the
future (contains limited or no follicles).
5.2.9. Post-menarche with serum FSH levels above 20 mIU/ml and AMH levels
below 0.1 ng/mL (Gleicher et al., 2010; Themmen et al., 2005) when no
chemotherapy has been administered. FSH levels measured at the time of
consideration for fertility preservation.
5.3. Patients may have newly diagnosed or relapsed disease. Those who were not
enrolled at diagnosis are eligible even if they have received therapy that is viewed as
likely to result in complete and permanent loss of ovarian function. However, these
patients will be required to provide 20% of their tissue for research.
5.4. The patient and/or the patient’s legally authorized guardian must acknowledge in
writing that consent for specimen collection has been obtained, in accordance with
institutional policies approved by the U.S. Department of Health and Human Services.
6.0 METHODS
6.1 Informed Consent
All potential participants will be informed of the risks of the planned treatment
(surgery/chemotherapy/radiation) for subsequent infertility. Information about different
options for fertility preservation: oophoropexy (ovarian transposition), ovarian radiation
Version: 5/5/2017
Ovarian Tissue Freezing Prior to Chemotherapy or Radiation Therapy 10
shielding, use of GnRH agonists/antagonists, embryo banking, oocyte banking and
ovarian tissue preparation, freezing and cryopreservation, will be provided. The
experimental nature of ovarian tissue cryopreservation will be emphasized. They will be
informed of the extent to which participating in this study might be of benefit to them. In
addition, they will be counseled prior to study entry for the unknown risk of possible
genetic damage/fetal defects from cryopreservation and subsequent in vitro handling of
the gametes and embryos.
6.2 Collection of Ovarian Tissue
If the patient chooses to participate and provides informed consent, she will have a
serum FSH and AMH drawn, (FSH on cycle day 3 where possible) as a marker of
ovarian reserve to use in counseling her on her options. (Only post-menarchal patients)
Patients in the following categories may participate in this study:
1. Patients who are having one or both ovaries removed for the treatment or
prevention of a disease.
2. Patients who are having surgery to remove one or both ovaries or parts of
ovaries for medical reasons where cryopreservation of normal ovarian cortex is
the only option for fertility preservation at the time but who cannot or will not
provide tissue to the research pool (except that ovarian cortex from the ovary that
contains a mass (regardless of whether it is benign or malignant) will not be
cryopreserved).These patients may participate if they are willing to consent to the
long term (annual) follow-up described in this study.
3. Patients having one ovary removed electively, solely for the purpose of fertility
preservation after considering the range of options available for fertility
preservation.
Patients in Categories 1-2 will have surgical removal of their ovarian tissue using the
methods determined by their surgeon based on their medical/surgical diagnosis or
treatment.
Patients undergoing elective removal of an ovary (Category 3 above) will undergo a
procedure called laparoscopy to remove the ovary. This surgery will be performed under
general anesthesia and one ovary will be removed in total through an instrument channel
employing standard techniques of operative laparoscopy. This surgical procedure is
performed solely for elective fertility preservation but can potentially be coordinated with
another procedure such as placement of a central line for future chemotherapy or
laparotomy for another purpose. Although only one ovary will be removed, both of the
patient's ovaries must appear normal for the procedure to be completed. The ovary to
be removed will be chosen at the time of surgery based on appearance and ease of
removal. After the surgery is complete, the patient will not have any further procedures
except for a routine post-operative visit. If indicated by the patient’s medical diagnosis, a
small piece of the ovarian tissue will be provided to pathology for routine histological
evaluation, and the remaining tissue will be processed for cryopreservation. If pathology
finds evidence of cancer in the ovarian tissue provided at surgery, they may request that
Version: 5/5/2017
Ovarian Tissue Freezing Prior to Chemotherapy or Radiation Therapy 11
all of the patient tissue (even tissue that has been frozen for patient use) be returned to
pathology for a more detailed examination. In this case, no tissue will be available for the
patient to use for fertility preservation purposes.
Despite a pre-operative treatment plan to remove and cryopreserve ovarian tissue for
fertility preservation, the surgeon or pathologist may determine intraoperatively that the
entire ovary or a significant portion of the cortical tissue is needed for diagnostic purposes.
Therefore, there may be no tissue available for cryopreservation for either patient use or
research use.
6.3. Cryopreservation:
Ovarian tissues will be cryopreserved using modifications of the techniques described by
Gosden et al. (1994) or will be vitrified using a modification of the techniques of Kuwayama
et.al, 2007. The ovary will be transported from the operating room to the laboratory for
cryopreservation.
As part of the study protocol, a small fraction of the ovarian tissue, not to exceed 20% of
the ovary (remaining after a portion has been provided to pathology, if indicated) will be
provided to the National Physicians Cooperative of the Oncofertility Consortium for
research purposes. The research tissue may be used fresh or may be frozen as described
above. The remainder (majority) of the ovarian tissue will be cryopreserved for the
patient’s own potential use in the future. Therefore, approximately 80% of the patient’s
tissue will be frozen for her own future use in fertility treatment; she may utilize that tissue
at any institution for any treatment modality she chooses. The 20% of the ovary
designated for research will go into a research pool and will be used for the research aims
described above. None of the research tissue will be used for experiments that
involve fertilization.
The ovarian cortex will be dissected from the medulla and cut into strips in culture/holding
media, washed to remove blood cells, and passed through a series of cryopreservation
solutions with increasing concentrations of cryoprotectants (including but not limited to
propanediol, glycerol or ethylene glycol, 0 - 15 %; sucrose, 0 - 0.3 M) (Freezing Media).
The tissues will be placed in cryovials containing freezing media and frozen using a
programmable freezer to cool tissues from 20o to -40o C using defined ramps or vitrified
directly or will be vitrified by direct plunge into liquid nitrogen. The vials will be placed in
liquid nitrogen for storage. The procedure for cryopreservation/vitrification may be
modified as improvements become available.
Quality Control procedures for the freezing and storage processes will be according to
FDA regulations for reproductive tissues (Federal Register 21 CFR Part 1271 and Part
1270), guidelines of the American Association of Tissue Banks and any other applicable
federal, state and local regulations. The designated Laboratory Director, Marybeth Gerrity,
Ph.D is responsible for the overall quality control of all of these clinical laboratory activities,
including ovarian tissue cryopreservation.
Version: 5/5/2017
Ovarian Tissue Freezing Prior to Chemotherapy or Radiation Therapy 12
6.4. Tissue storage and infectious disease testing:
6.4.1 Ovarian Tissue Storage: All cryopreserved tissues obtained, including both the
research portion (if frozen) and the patient portion, will be transferred to Reprotech, Ltd.
(RTL) in St. Paul, MN for storage and subsequent release. Reprotech, Ltd. is an FDA
compliant and American Association of Tissue Banks accredited long term storage facility
for reproductive tissues. Based on the extended periods of time that these tissues are
likely to be stored (patients may wait for five years from cancer treatment to be considered
cancer free and begin a family; some may wait longer based on age), Reprotech, Ltd.
provides maximum flexibility for the patients involved. At anytime while the tissue is in
storage, the patient may request that the tissue be shipped to another facility, discarded or
donated to research. The patient can store tissues as long as they wish and ship them to a
fertility treatment center of their choice at the time of use. The patient can determine how
her own tissue will be used as technology changes and based on her unique
circumstances. Reprotech, Ltd. does not perform fertility treatments and is not
affiliated with any fertility center so there is no potential conflict of interest. Patients
will execute a separate storage agreement with Reprotech, Ltd which defines the length of
storage, shipping requirements, infectious disease testing, screening and disposition of the
tissues in the event of their death.
Tissues designated for research and stored at Reprotech will be allocated to investigators
of NIH Oncofertility Consortium Grant by the Steering Committee of the Oncofertility
Consortium.
6.4.2 Infectious Disease Screening and Testing: Tissue banking and subsequent use
of ovarian tissue is currently regulated by the Food and Drug Administration
(FDA). In order to comply with current tissue banking regulations and to be prepared for
any future changes in regulations while these ovarian tissues are in storage, patients
will be tested and screened for a number of infectious diseases prior to banking ovarian
tissue. The testing will include but not be limited to testing for Hepatitis B and C and
HIV. The screening and testing that will be performed are the same as would be
performed on an anonymous reproductive tissue donor and include a physical
examination and questions regarding potential high risk behaviors. The testing that will
be performed will be testing that is mandated for donors of leukocyte rich tissues and
must be performed within 7 days of tissue procurement. In this way, the tissue could
potentially be used by the patient herself and it could also be suitable for use in another
individual (such as a gestational surrogate) in the future if indicated by the patient’s
medical diagnosis. (For example, use of in vitro maturation of follicles followed by IVF
with embryo transfer to a gestational carrier for a patient without a uterus). In addition,
a sample of the patient’s blood plasma will be stored with the ovarian tissue to
permit any future testing required under federal tissue banking regulations. In
spite of storing blood plasma, it is still possible that federal regulations may
change and therefore, it may not be possible to perform the appropriate testing to
permit heterologous use of the tissue in the future. Infectious disease testing is
performed in this study to permit patient use of her own tissue and not for the purposes
of research tissue or research study.
Version: 5/5/2017
Ovarian Tissue Freezing Prior to Chemotherapy or Radiation Therapy 13
6.5 Clinical Data to be Collected
6.5.1. Safety data
6.5.1.1. Adverse events after an elective laparoscopic oophorectomy (Category 3
Patients, see 6.2) will be identified using the Common Toxicity Criteria for Adverse
Events (CTCAE). A copy of the CTC version 3.0 can be downloaded from the CTEP
home page (http://ctep.info.nih.gov). All appropriate treatment areas should have
access to a copy of the CTCAE version 3.0. The severity of the event should then be
graded using the CTCAE criteria. Determination of whether the event was related to the
surgical procedure and whether the adverse event was expected or unexpected will be
made. Data on the time from surgery to start of definitive therapy (chemotherapy or
radiation) will also be collected.
6.5.2. Other data to be collected:
Name, address, telephone number
E-mail address
Personal and family medical history
Age
Information from physical examinations, including Tanner stage at time of
consent
Planned treatment associated with high risk of sterility
Diagnosis
Prior Cancer Treatment, if any
Treatment sequence associated with high risk of sterility
Method of ovarian collection
Time from surgery to start of definitive therapy (chemotherapy or radiation)
Outcome: Remission, Relapse or Death
Reproductive history before, during and after cancer diagnosis with long term
follow up
Choices regarding use of cryopreserved stored tissues for fertility restoration
6.5.3 Long Term Follow Up. Patients will be contacted by phone on an annual
basis by a member of the research team at [INSTITUTION]. The patient will be
asked questions about the use of their tissue and attitudes toward fertility
preservation and outcomes of any pregnancies that have occurred using the
attached phone script. Patients will be contacted annually until they use their
tissue or drop out of the study. A telephone script for long term study follow up is
attached (Appendix 5).
6.6 Efficacy of Cryopreservation
In 1986, Chen reported the first successful attempt at cryopreserving and thawing a
human oocyte resulting in a twin pregnancy after in vitro fertilization and embryo
transfer. Progress in the field moved slowly for the next decade after murine data
Version: 5/5/2017
Ovarian Tissue Freezing Prior to Chemotherapy or Radiation Therapy 14
suggested that cryopreservation showed higher levels of chromosomal anomalies when
compared with fresh eggs (Johnson and Pickering, 1987). However work in the early
1990s by Gook et al. demonstrated that cryopreservation was not as detrimental as
originally thought, leading to renewed research interest in human oocyte
cryopreservation (Gook et al, 1994).
By 2004, 100 human babies had been born from cryopreserved oocytes. These infants
were produced with great inefficiency (Stachecki and Cohen, 2004). For instance,
Marina and Marina (2003) reported a 4% live birth rate from 99 oocytes frozen. Results
with larger sample sizes were even less impressive. In fact, Porcu’s research group
(1999) reported only 16 pregnancies from 1502 thawed oocytes (slightly more than 1%).
Breakthroughs made by Italian researchers in optimizing freezing and thawing methods
greatly improved pregnancy rates with mature human oocytes (Coticchio et al., (2006);
Paynter et al., (2005); Fabbri R, et al., (2001); Bianchi et al., (2007); Borini A et al.,
(2004); Borini et al., (2006)) yielding pregnancy rates per thaw cycle of 19% and 22%
per patient. However, these techniques make use of oocytes retrieved after hormonal
stimulation utilized for in vitro fertilization which requires several weeks of treatment.
Cryopreservation of human ovarian tissue containing oocytes poses additional
challenges. Oocytes have larger cell volumes, different freezing characteristics and
varying membrane permeability to cryoprotectants compared to ovarian tissue.
Developing a method for tissue cryopreservation that permits recovery of a
maximum number of mature follicles is one aim of this study. Tissue donated to
the research pool will be frozen using varying cryopreservation methods and
thawed to determine the efficacy of the freeze/thaw techniques. Follicles may be
isolated from the thawed tissue and matured in vitro. The number of viable follicles
isolated and their performance in culture will be recorded for each tissue specimen as
indices of the efficacy of the cryopreservation process. Maturation of at least 50% of the
oocytes/follicles recovered from the cryopreserved tissue is expected based on the
literature. Data gathered from this research will assist in identifying and overcoming some
of the challenges to successful freezing and thawing of tissue with subsequent in vitro
maturation of follicles or oocytes.
6.7 Feasibility of 3-Dimensional System for In Vitro Maturation of Immature Ovarian
Follicles
Tissue provided to the research pool at the time of surgery (no more than 20% of total
tissue obtained from each patient) will be thawed as described by Gosden et al.(1994).
Preantral and antral follicles (if available) will be isolated from the tissues and placed in
culture. Factors that affect in vitro maturation of human oocytes will be investigated using
the research tissue including but not limited to:
(1) Growth factors such IGF-1, IGF-2, EGF, GDF-9 and activin have been implicated in
promoting follicular growth and oocyte maturation. These growth factors and others will be
tested individually and in combination.
Version: 5/5/2017
Ovarian Tissue Freezing Prior to Chemotherapy or Radiation Therapy 15
(2) Hormones such as recombinant human FSH and LH will be included in the culture
medium at different concentrations.
(3) Co-culture of oocytes with certain somatic cells or cell lines is beneficial for in vitro
maturation.
(4) Three-dimensional culture environment. Under conventional culture conditions
using plastic culture dishes, most types of cells will attach to the bottom of culture dish and
spread. When follicular cells attach and spread on culture dishes, the 3-D structure of a
follicle is lost and cellular contact between oocytes and surrounding follicular cells is
disrupted, which may be detrimental to oocyte growth and maturation. In this study,
preantral follicles will be encapsulated in biogel materials, such as agar or alginate,
to prevent attachment and spreading of the follicular cells and maintain the three
dimensional structure of the follicle. Agar has been used successfully to culture
hamster and human antral follicles (Roy and Greenwald, 1989; Roy and Treacy, 1993),
but it has a relatively high gel temperature (45 degree C), which may damage the cells
during encapsulation. Alginate is a polysaccharide material which gels in the
presence of calcium and cause less damage to the cells during the encapsulation
process. Gels from both materials permit ready diffusion of growth factors, hormones and
other factors in and out of the follicle. The 3-D culture conditions needed to maximize in
follicle maturation will be examined. Endpoints studied will include hormone production,
follicle size and growth rate, oocyte maturity, structural changes using light and electron
microscopy and gene expression. None of the endpoints will involve fertilization of the
oocytes.
7.0 STATISTICAL CONSIDERATIONS AND DATA MANAGEMENT
7.1 Accrual
Up to [INSERT NUMBER] patients per year are estimated to be enrolled at
[INSTITUTION].
7.2 Data Collection and Access
Participation in this research is confidential. All research tissues will be de-identified;
participants will be identified by number, not name. No information by which the patient
can be identified will be released or published in connection with this study. Only the PI
and co-investigators will have access to files matching the patient with tissue specimen
numbers. All data will be stored in a confidential database including the Cancer
Central Clinical Database (C3D) provided by NIH/NCI for the Oncofertility
Consortium. Data will be permanently stored by the PI and co-investigators. The
researchers using the research portion of the tissue will not have access to any
identifying information regarding that tissue.
8.0 RISKS
Version: 5/5/2017
Ovarian Tissue Freezing Prior to Chemotherapy or Radiation Therapy 16
The patient's participation in this study may involve the following risks. Most of these
are related to the general risks of surgery.

FOR PATIENTS UNDERGOING ELECTIVE LAPAROSCOPIC OOPHRECTOMY
that was required for fertility preservation only (Category 3 patients, see 6.2):
1. Risks of laparoscopy include infection, damage to the patient's
internal organs or bleeding problems as a result of insertion or
manipulation of the laparoscopic instruments. The chance of the
patient requiring hospitalization for complication(s) is about 4%
(Kauff et al., 2002). The patient's chance of dying as a result of
such complication(s) about 2 in 1,000 (Chi et al., 2004).
2. Risks of general anesthesia: The patient's risk of death from
anesthesia is less than 1 in 100,000 in children older than 3 years
and less than 1 in 10,000 in children less than 3 years (Arbous et
al., 2001; Gibbs et al., 2006; Kawashima et al., 2003; Mackay et al.,
2002; Gouhua et al., 2009).

REMOVAL OF THE OVARY: There is a theoretical risk that the patient may
experience a loss of fertility due to the removal of an ovary and/or may
experience an early menopause caused by the loss of hormones produced by
ovaries. In addition, it is possible that the surgery itself could cause scar tissue or
damage to the remaining ovarian tissue, so that chances for pregnancy could be
reduced. The patient may regain spontaneous ovarian function. If so, the surgery
to remove tissue would then have been unnecessary for the patient. These
circumstances are very unlikely and much less likely than the patient’s chance of
losing ovarian function as a consequence of her medical, surgical or radiation
treatment of her cancer or medical condition. She may also be at risk for the
psychological consequences, including emotional upset, of having one ovary
removed

CRYOPRESERVATION: Although care will be taken, damage to the removed
ovarian tissue may occur during any part of the cryopreservation (freezing),
shipping or storage process. The effects of cryopreservation and long term
storage on human ovarian tissues are not known and possible damage to the
tissues may occur. The risk of birth defect(s) and/or genetic damage to any child
who may be born following such a procedure is also unknown. However,
thousands of children have been born worldwide from frozen embryos and eggs
and there has been no report of increased risk of birth defects in these children.
The ovarian tissue removed may not yield usable eggs, or pregnancy may not
result when the eggs are ultimately used. Some patients may have particular
risks associated with their underlying disease. If a cancer or other disease
already affects the ovarian tissue, then it may never be possible to use the tissue
in the future. This may not be known until the patient wishes to use her ovarian
tissue. Tissue could be lost or made unusable due to equipment failure, or
unforeseeable natural disasters beyond the control of this program.
Version: 5/5/2017
Ovarian Tissue Freezing Prior to Chemotherapy or Radiation Therapy 17

INFECTIOUS DISEASE TESTING. Infectious disease testing may indicate that
the patient has an infectious disease of which she was previously unaware and
which may require treatment or may delay the start of her cancer or medical
treatment. Although infectious disease testing will be performed within 7 days of
removal of the ovary and additional plasma samples from the patient will be
stored with her ovarian tissue, a future change in federal regulations for testing
prior to use of this tissue may render the tissue unusable. Infectious disease
testing and screening performed around the time of surgery may be inadequate
to permit safe use of your tissue after the long term storage of the tissue. While a
sample of blood plasma will be stored to minimize this risk, the tests required in
the future may require a sample other than plasma, the plasma sample may be
inadequate or it may be lost or damaged during cryopreservation or storage.
Infectious disease testing is only required for the patient to make use of her own
tissue and is not required for use of the research tissue or the research study.
There may be no direct benefit to the patient from participating in this research study,
but, as described above, there is a possibility that the portion of the tissue stored for her
own use may eventually be used successfully to initiate a pregnancy. Participation in
this research study may indirectly help other patients, who could benefit from ovarian
tissue storage by helping to advance understanding of how to successfully freeze and
thaw ovarian tissue in a manner that permits future use.
The use of ovarian transplant, in vitro maturation or other new technology that cannot
be envisioned now may allow the patient to use some of her cryopreserved and stored
ovarian tissue in the future. However this possibility cannot be assured and the patient
will be thoroughly counseled about the experimental nature of this protocol.
The participant has the alternative to choose not to participate in this study. Instead she
may receive cancer treatment, chemotherapy or radiation therapy, without undergoing
surgery for the removal of one of her ovaries. If she is already undergoing surgery to
remove all or part of her ovary (ovaries) she can choose not to have the tissue
cryopreserved. Participants undergoing radiation therapy may have the option of having
ovarian tissue transplanted to another part of their body and shielded from radiation.
The patient may also choose to delay her treatment and undergo hormonal stimulation
with harvest of oocytes with or without in vitro fertilization and freezing of embryos.
There are no other alternative methods for preserving ovarian tissue available to the
participant at this time based upon current scientific technology. If the subject becomes
infertile from chemotherapy or radiation therapy, and desires to have a child, she may
opt to adopt a child or use donor eggs for in vitro fertilization.
9.0 REIMBURSEMENTS AND RESEARCH COST
9.1. Reimbursement
No direct reimbursement will be made to patients or their families.
Version: 5/5/2017
Ovarian Tissue Freezing Prior to Chemotherapy or Radiation Therapy 18
9.2. Research costs
Patients in the following categories may participate in this study:
1. Patients who are having one or both ovaries removed for the treatment or
prevention of a disease.
2. Patients who are having surgery to remove one or both ovaries or parts of
ovaries for medical reasons where cryopreservation of normal ovarian cortex is
the only option for fertility preservation at the time but who cannot or will not
provide tissue to the research pool (except that ovarian cortex from the ovary that
contains a mass (regardless of whether it is benign or malignant) will not be
cryopreserved).These patients are willing to participate in the long term follow-up
described in this study.
3. Patients having one ovary removed electively, solely for the purpose of fertility
preservation after considering all of the options available for fertility preservation.
9.2.1 Hospital, Surgical and Physician Costs:
All costs will be billed to the patient or patient’s insurance. All non-covered
services are the patient’s or their family’s responsibility, but may be provided free
of charge if the family cannot afford to pay for the procedure and they qualify for
financial assistance. Estimated costs are in Appendix 2. Pathology services,
where indicated by the standard of care, are patient or family responsibility. The
patient will be charged for the freezing of her ovarian tissue (approximately
$695).
9.2.2 Patient Tissue Storage

The cost of shipping the patient’s ovarian tissue to Reprotech, Ltd (approximately
$215) and the yearly storage of the tissue at Reprotech, Ltd (approximately
$275) will be the patient’s responsibility. Reprotech, Ltd has a financial
assistance program in place to reduce this annual fee for those who can
demonstrate financial need based on income.

9.2.3 Infectious Disease Testing

Infectious disease testing is required for storage and use of the patient’s own
tissue and is not part of the research study. Infectious disease testing will be
billed to the patient or patient’s insurance. Non-covered services are the patient’s
responsibility.
9.3. Provisions for dealing with research-related injury
In the event of injury or illness resulting from the research procedures, medical
treatment for injuries or illness is available through [INSTITUTION].
Version: 5/5/2017
Ovarian Tissue Freezing Prior to Chemotherapy or Radiation Therapy 19
REFERENCES:
 Abir R, Ben-Haroush A, Felz C, Okon E, Raanani H, Orvieto R, Nitke S and B
Fisch (2008). Selection of patients before and after anticancer treatment for
ovarian cryopreservation. Human Reproduction; 23(4): 869-877.
 American Society for Reproductive Medicine Ethics Committee . Fertility
Preservation and Reproduction in Patients Facing Gonadotoxic Therapies: A
Committee Opinion. (2013). Fertil Steril; 100: 1224-31.
 Anderson et al. (2007) Personal communication to J. Donnez.
 Arbous MS, Grobbee DE, van Kleef JS et al. (2001) Mortality associated with
anaesthesia: A qualitative analysis to identify risk factors. Anaesthesia; 56: 11411153.
 Baird DT, Webb R, Campbell BK, Harkness LM, Gosden RG. (1999) Long-term
ovarian function in sheep after ovariectomy and transplantation of autografts
stored at -196 C. Endocrinology; 140: 462-71.
 Borini A, Bonu M, Coticchio G, Bianchi V, Cattoli M and C Flamigni (2004).
Pregnancies and births after oocyte cryopreservation. Fertil Steril; 601-605.
 Borini A, Sciajno R, Bianchi V, Sereni E, Flamigni C and G Coticchio (2006).
Clinical outcome of oocyte cryopreservation after slow cooling with a protocol
utilizing a high sucrose concentration. Human Reproduction; 21: 512-517.
 Bianchi V, Coticchio G, Distratis V, DiGuisto N, Flamigni C and A Borini (2007).
Differential sucrose concentration during dehydration (0.2 mol/l) and rehydration
(0.3 mol/l) increases the implantation rate of frozen human oocytes. RBM
OnLine; 14: 64-71.
 Chemaitilly W, Mertens A, Mitby P, Whitton J, Stovall J, Yasui Y, Robison L,
Sklar C. (2006) Acute ovarian failure in the Childhood Cancer Survivor Study.
The Journal of Clinical Endocrinology & Metabolism; 91: 1723-1728.
 Chi DS, Abu-Rustum NR, Sonoda Y, Awtrey C, Hummer A, Venkatraman E,
Franklin CC, Hamilton F, Gemignani ML, and RR Barakat. (2004) Ten year
experience with laparoscopy on a gynecologic oncology service: Analysis of risk
factors for complications and conversion to laparotomy. American Journal of
Obstetrics and Gynecology; 191: 1138-1145.
 Chiarelli AM, Marrett LD, Darlington G. (1999) Early menopause and infertility in
females after treatment for childhood cancer diagnosed in 1964-1988 in Ontario,
Canada. American Journal of Epidemiology; 150: 245-254.
 Chen, C. (1986) Pregnancy after human oocyte cryopreservation. Lancet; 1: 884886.
 Chian RC, Gilbert L, Huang JYJ, Demirtas E, Holzer H, Benjamin A, Buckett WM,
Tulandi T and SL Tan. Live Birth after Vitrification of In Vitro Matured Oocytes.
Fertility and Sterility 2008 (early view)-a
 Chian RC, Huang JYJ, Gilbert L, Son WY, Holzer H, Cui SJ, Buckett WM,
Tulandi T and SL Tan. Obstetric Outcomes After Vitrification of In Vitro and In
Vivo Matured Oocytes. Fertility and Sterility 2008 (early view)-b
 Cortvrindt R, Smitz J, Van Steirteghem AC. (1996) In-vitro maturation, fertilization
and embryo development of immature oocytes from early preantral follicles from
prepubertal mice in a simplified culture system. Human Reproduction; 11: 2656.
Version: 5/5/2017
Ovarian Tissue Freezing Prior to Chemotherapy or Radiation Therapy 20
















Coticchio G, DeSantis L, Rossi G, Borini A, Albertini D, Scaravelli G, Alecci C,
Bianchi V, Nottola S and S Cecconi (2006). Sucrose Concentration influences
the rate of human oocytes with normal spindle and chromosome configurations
after slow-cooling cryopreservation. Human Reproduction; 21: 1771-1776.
Demeestere I, Simon P, Buxant F, Robin V, Fernandez S, Centner J, Delbaere A
and Y Englert (2006). Ovarian function and spontaneous pregnancy after
combined heterotopic and orthotopic cryopreserved ovarian tissue
transplantation in a patient previously treated with bone marrow transplantation:
Case Report. Human Reproduction; 21: 2010-2014.
Donnez J, Dolmans MM, Demylle D, et al. (2004) Live birth after orthotopic
transplantation of cryopreserved ovarian tissue. Lancet; 364:1405-1410.
Fabbri R, Porcu E, Marsella T, Rocchetta G, Venturoli S and C Flamigni (2001).
Human oocyte cryopreservation: new perspectives regarding oocyte survival.
Human Reproduction; 16: 411-416.
Fabbri R, Vicenti R, Macciocca M, Pasquinelli G, Lima M, Parazza I, Magnani V
and S Venturoli (2012). Cryopreservation of Ovarian Tissue in Pediatric Patients.
Obstetrics and Gynecology international; Epub 2012 Feb 13..
FDA Tissue Banking Regulations. Human Cells, Tissues, and Cellular and
Tissue Based Products. Federal Register 21 CFR 1271.
Fertile Hope. Cancer and Fertility. New York, NY. 2007 (Brochure)
Feigin E, Avir R, Fisch B, Kravarusic D, Steinberg R, Nitke S, Avrahami G, VenHaroush A, Freud E. Laparoscopic ovarian tissue preservation in young paitents
at risk for ovarian failure as a result of chemotherapy/irradiation for primary
malignancy. Journal of Pediatric Surgery 2007; 42: 862-864.
Gibbs N, Borton C. (2006) Safety of anesthesia in Australia: A review of
Anaesthesia related mortality, 2000-2002. Report of the committee convened
under the auspics of the Australian and New Zealand College of Anesthetists.
Melbourne, Australian and New Zealand College of Anesthetists.
Ginsburg E.S., Yanushpolsky E.H.Jackson K.V., (2001) In vitro fertilization for
cancer patients and survivors. Fertil Steril, 2001. 75(4): p. 705-710.
Gleicher N, Weghofer A, and DH Barad. (2010) Anti-Mullerian hormone (AMH)
defines, independent of age, low versus good livebirth chances in women with
severely diminished ovarian reserve. Fertility and Sterility, 2010. 94(7): 28242827.
Gook D., et al. (1994) Fertilization of human oocytes following cryopreservation:
Normal karyotypes and absence of stray chromosomes. Human Reproduction; 9:
684-691.
Gosden RG. (2005) Prospects for oocyte banking and in vitro maturation. Journal
of the National Cancer Institute Monograph; 34: 60-63.
Gosden RG, Baird D, Wade J and R. Webb (1994). Restoration of fertility to
oophorectomized sheep by ovarian autografts stored at -196 C. Human
Reproduction 9: 597-603.
Gouhua L, Warner M, Land BH, et al. (2009) Epidemiology of anesthesia-related
mortality in the United States, 1999-2005. Anesthesiology, 110: 759-765.
Green D., Kawashima T, Stovall M, Leisenring W, Sklar CA, Mertens AC,
Donaldson SS, Byrne J, and LL Robison. Fertility of Female Survivors of
Version: 5/5/2017
Ovarian Tissue Freezing Prior to Chemotherapy or Radiation Therapy 21














Childhood Cancer: A Report From the Childhood Cancer Survivor Study. Journal
of Clinical Oncology, 27(16): 2677-2685.
Green D, Nolan VG, Goodman PJ, Whitton JA, Srivastava D, Leisenring WM,
Neglia JP, Sklar CA, Kaste SK, Hudson MM, Diller LR, Stovall M, Donaldson SS,
and LL Robison. (2014) The Cyclophosphamide Equivalent Dose as an
Approach for Quantifying Alkylating Agent Exposure: A Report From the
Childhood Cancer Survivor Study. Pediatr Blood Cancer ;61:53–67
Johnson M and S Pickering. (1987) The effect of dimethylsulphoxide on the
microtubular system of the mouse oocyte. Development; 100: 313-324.
Harp R, Leibach J, Black J, Keldahl C. and A Karow (1994).Cryopreservation of
Murine Ovarian Tissue. Cryobiology; 31: 336-34.
Kauff ND, Satagopan JM, Robson ME, Scheuer L, Hensley M, Hudis CA, Ellis NA,
Boyd J, Borgen PI, Barakat RR, Norton L and K Offit (2002). Risk-Reducing
Salpingo-oophorectomy in Women with a BRCA1 or BRCA2 mutation. New
England Journal of Medicine; 346 (21): 1609-1615.
Kim S.S., Fertility preservation in female cancer patients: current developments
and future directions. Fertil Steril, 2006. 85(1): p. 1-11 (2006).
Kola I, Kirby C, Shaw J, Davey A, and Trounson A. (1988) Vitrification of mouse
oocytes results in aneuploid zygotes and malformed fetuses. Teratology; 38:
467-74.
Larsen EC, Muller J, Schmiegelow K, Rechnitzer C, Andersen AN. (2003)
Reduced ovarian function in long-term survivors of radiation- and chemotherapytreated childhood cancer. The Journal of Clinical Endocrinology & Metabolism;
88: 5307-5314.
Kawashima Y, Takahashi S, Suzuki M, et al. (2003) Anesthesia-related mortality
and morbidity over a 5-year period in 2,363,038 patients in Japan. Acta
Anaesthesiology Scandinavica, 47: 809-817.
Kuwayama M, Highly efficient vitrification for cryopreservation of human
oocytes and embryos: The Cryotop method. Theriogenology 67:73-80, 2007.
Marina F and S Marina. (2003) Comments on oocyte cryopreservation.
Reproductive BioMedicine Online; 6: 401-402.
Lee S, Shover L, Partridge A, Patrizio P, Wallace H, Hagerty K, Beck L, Brennan
L and K Oktay. American Society of Clinical Oncology Recommendations on
Fertility Preservation in Cancer Patients. Journal of Clinical Oncology 24: 29172931, 2006.
Lobo RA. Potential Options for Preservation of Fertility in Women. New Engl J
Med 2005; 353:64-73.
Mackay, P. (2002) Safety of Anaesthesia in Australia: A review of anaesthesia
related mortality, 1997-1999. Report of the committee convened under the
auspices of the Australian and New Zealand College of Anaesthetists.
Melbourne, Australian and New Zealand College of Anaesthetists.
Martelli H, Poirot C, Montupet P, Helardot P, Sarnacki S, Brugieres L. Ovarian
Tissue Harvesting for Cryopreservation in Prepubertal Girls. SIOP XXXVII
Annual Conference Abstracts P.O2.008. Pediatric Blood and Cancer 2006;
47:570-571.
Version: 5/5/2017
Ovarian Tissue Freezing Prior to Chemotherapy or Radiation Therapy 22















Mattison DR, Plowchalk DR, Meadows MJ, al-Juburi AZ, Gandy J, & Malek A.
(1990) Reproductive toxicity: Male and female reproductive systems as targets
for chemical injury. Medical Clinics of North America; 74: 391-411.
Meirow D, Hardan I, Dor J, Fridman E, Elizur S, Ra’anani H, et al., Searching for
evidence of disease and malignant cell contamination in ovarian tissue stored
from hematologic cancer patients. Human Reproduction 2008; 23(5): 100711013.
Meirow D, Nugent D. (2001) The effects of radiotherapy and chemotherapy on
female reproduction. Human Reproduction Update; 7(6): 535-43.
Meirow D, Levron J, Eldar-Geva T, et al. (2005) Pregnancy after transplantation
of cryopreserved ovarian tissue in a patient with ovarian failure after
chemotherapy. New England Journal of Medicine; 353: 318-321.
Meirow D, Levron J, Eldar-Geva, T., Hardan I, Fridman E, Yemini Z and Dor J
(2007). Monitoring the ovaries after autotransplantation of the cryopreserved
ovarian tissue: endocrine studies, in vitro fertilization cycles, and live birth. Fertil
Steril; 87: 7-15.
Nieman CL, Kazer R, Brannigan RE, Zoloth LS, Chase-Lansdale L, Kinahan K,
Dilley KJ, Roberts D, Shea LD, Woodruff TK. (2006) Cancer survivors and
infertility: A review of a new problem and novel answers. Journal of Supportive
Oncology; 4: 171-178.
Oktay K, Nugent D, Newton H, Salha O, Chatterjee P, Gosden R. (1997) Isolation
and characterization of primordial follicles from fresh and cryopreserved human
ovarian tissue. Fertility and Sterility 6: 481-486.
Oktay K, Newton H, Mullan J, Gosden RG. (1998) Development of human
primordial follicles to antral stages in SCID/hpg mice stimulated with follicle
stimulating hormone. Human Reproduction; 13(5):1133-8.
Oktay K, Buyuk E, Veeck L, et al. (2004) Embryo development after heterotopic
transplantation of cryopreserved ovarian tissue. Lancet; 363: 837-840.
Oktay, K. and O. Oktem. Sustained endocrine function and spontaneous
pregnancies after subcutaneous transplantation of cryopreserved ovarian tissue
in stem cell transplant recipients. Fertil Steril 2005; 84(Suppl 1): S68.
Patrizio P, Butts S, Caplan A. (2005) Ovarian tissue preservation and future
fertility: Emerging technologies and ethical considerations. Journal of the
National Cancer Institute Monograph; 34: 107-110.
Paynter S, Borini A, Bianchi V, DeSantis L, Flamigni C and G Coticchio(2005).
Volume changes of mature human oocytes on exposure to cryoprotectant
solutions used in slow cooling procedures. Human Reproduction; 20: 1194-1199.
Peters, H & McNatty, KP. (1980) The Ovary. University of California Press, LA,
California.
Poirot C, Vacher-Lavenu MC, Helardot P, Guibert J, Brugieres L, Jouannet P.
Human Ovarian Tissue Cryopreservation: Indications and Feasibility. Human
Reproduction 2002: 17:1447-1452
Poirot C, Martelli H, Genestie C, Golmard JL, Valteau-Couanet D, Helardot P,
Pacquement H, Sauvat F, Tabone MD, Philippe-Chomette P, Esperou H,
Baruchel A. Feasibility of Ovarian Tissue Cryopreservation for Prepubertal
Females With Cancer. Pediatric Blood and Cancer 2007.
Version: 5/5/2017
Ovarian Tissue Freezing Prior to Chemotherapy or Radiation Therapy 23

















Porcu E et al. (1999) Cycles of human oocyte cryopreservation and
intracytoplasmic sperm injection: Results of 112 cycles. Fertility and Sterility; 72:
S2.
Revel A, Revel-Vilk S, Aizenman E, Porat-Katz A, Safran A, Ben-meir A,
Weintraub M, Shapira M, Achache H, and N Laufer. At what age can human
oocytes be obtained? Fertility and Sterility. 2008 (in press).
Roy, SK & Greenwald, GS. (1989) Hormonal requirements for the growth and
differentiation of hamster preantral follicles in long-term culture. Journal of
Reproduction and Fertility; 87:103-14.
Roy, SK & Treacy, BJ. (1993) Isolation and long-term culture of human preantral
follicles. Fertility and Sterility; 59: 783-90.
Schmidt K, Yding Andersen C, Loft A, Byskov A, Ernst E and A Nyboe Andersen
(2005). Follow up of ovarian function post-chemotherapy following ovarian
cryopreservation and transplantation. Human Reproduction; 20: 3539-3546.
Seli E and Tangir J. (2005) Fertility preservation options for female patients with
malignancies. Current Opinion in Obstetrics and Gynecology; 17: 299.
Shaw JM, Bowles J, Koopman P, et al. (1996) Fresh and cryopreserved ovarian
tissue samples from donors with lymphoma transmit the cancer to graft
recipients. Human Reproduction; 54: 197-207.
Shaw JM, Bowles J, Koopman P, Wood EC, Trounson AO. Fresh and
cryopreserved ovarian tissue samples from donors with lymphoma transmit the
cancer to graft recipients. Hum Reprod. 1996 Aug;11(8):1668-73.
Silber S, Lenahan K, Levine, D, Pineda J, Gorman K, Friez M, Crawford E and R
Gosden (2005). NEJM; 353: 58-63.
Silber S and R. Gosden (2007a). Ovarian Transplantation in a series of
monozygotic twins discordant for ovarian failure. NEJM 356: 13.
Silber S and R. Gosden (2007b). EMD Serono Biosymposia Presentation
Boston, Sept 2007.
Sklar C, Mertens AC, Mitby P, Whitton J, Stovall M, Kasper C, Mulder J, Green
D, Nicholson HS, Yasui Y, Robison LL. (2006) Premature menopause in
survivors of childhood cancer: A report from the Childhood Cancer Survivor
Study. Journal of the National Cancer Institute; 98: 890-896.
Sonmezer M, Oktay K. (2004) Fertility preservation in female patients. Human
Reproduction Update; 10(3): 251-66.
Stachecki J and Cohen J. (2004) Cryopreservation and assisted human
conception: An overview of oocyte cryopreservation. Reproductive BioMedicine
Online; 9(2): 152-163
Themmen APN. (2005) Anti-Mullerian Hormone: Its Role in Follicular Growth
Initiation and Survival and as an Ovarian Reserve Marker. 34: 18-21
Wallace WH, Thomson AB, Saran F, Kelsey TW. (2005) Predicting age of
ovarian failure after radiation to a field that includes the ovary. International
Journal of Radiation Oncology, Biology, Physics; 62(3): 738-44
Xu M, Kreeger PK, Shea LD, Woodruff TK. (2006) Tissue engineered follicles
produce live, fertile offspring. Tissue Engineering (accepted for publication)
Version: 5/5/2017
Ovarian Tissue Freezing Prior to Chemotherapy or Radiation Therapy 24

Zeltzer LK. (1993) Cancer in adolescents and young adults: Psychosocial
aspects in long-term survivors. Cancer; 71S: 3463-3468
Version: 5/5/2017
Ovarian Tissue Freezing Prior to Chemotherapy or Radiation Therapy 25
Appendix 1. Reprotech Registration and Agreement Forms
Version: 5/5/2017
Ovarian Tissue Freezing Prior to Chemotherapy or Radiation Therapy 26
Appendix 2. Estimated Costs associated with Ovarian Cryopreservation (per patient)
Consultation with surgeon (CPT code 99241-45: range $100-$800)
Outpatient Laparoscopic Oophorectomy (OR use, surgeon and anesthesiologist):
$18,000
Surgeon’s fee: $3,000 (CPT code 58661)
Anesthesia fee: $2,000 (CPT code 58662).
Hospital fee (OR time, equipment fees): $13,000
Infectious Disease testing: $240
Cost of Freezing the ovary: $695
Shipping of tissue to Reprotech: $215
Yearly storage: $275; Discount based on financial need to $75/year.
Costs may change based on changes on the Charge Master.
Version: 5/5/2017
Ovarian Tissue Freezing Prior to Chemotherapy or Radiation Therapy 27
Appendix 3. Requisition Form for Infectious Disease Testing
Version: 5/5/2017
Ovarian Tissue Freezing Prior to Chemotherapy or Radiation Therapy 28
Appendix 4. Application for Financial Assistance with Reprotech
Version: 5/5/2017
Ovarian Tissue Freezing Prior to Chemotherapy or Radiation Therapy 29
Appendix 5. Study Follow-up Telephone Script and Questionnaire
Version: 5/5/2017
Ovarian Tissue Freezing Prior to Chemotherapy or Radiation Therapy 30
Appendix 6. American Society for Clinical Oncology (ASCO) Recommendations for
Fertility Preservation in Cancer Patients
Version: 5/5/2017
Ovarian Tissue Freezing Prior to Chemotherapy or Radiation Therapy 31
Appendix 7. American Society for Reproductive Medicine Ethics Committee Position
Paper.
Version: 5/5/2017
Ovarian Tissue Freezing Prior to Chemotherapy or Radiation Therapy 32
Appendix 8. Ovarian Tissue Prior to and After Receiving Chemotherapy Considered as
Likely to Result in Complete and Permanent Loss of Ovarian Function
Version: 5/5/2017
Ovarian Tissue Freezing Prior to Chemotherapy or Radiation Therapy 33
Appendix 9. Alkylating Agent Dose Scoring
Add the tertile score (1, 2, or 3) for each alkylating agent given (or to be given) to a
patient
Source: Green et al., JCO, 2009. 27(16): 2677-2685.
Version: 5/5/2017
Ovarian Tissue Freezing Prior to Chemotherapy or Radiation Therapy 34
Appendix 10. Cyclophosphamide Equivalent Dose (CED) Calculation
CED (mg/m2) = 1.0 (cumulative cyclophosphamide dose (mg/m2))
+ 0.244 (cumulative ifosfamide dose (mg/m2))
+ 0.857 (cumulative procarbazine dose (mg/m2))
+ 14.286 (cumulative chlorambucil dose (mg/m2))
+ 15.0 (cumulative BCNU dose (mg/m2))
+ 16.0 (cumulative CCNU dose (mg/m2))
+ 40 (cumulative melphalan dose (mg/m2))
+ 50 (cumulative Thio-TEPA dose (mg/m2))
+ 100 (cumulative nitrogen mustard dose (mg/m2))
+ 8.823 (cumulative busulfan dose (mg/m2)).
Source: Green et al., PBC. 2014. 61:53–67
Version: 5/5/2017