Live birth after autograft of ovarian tissue cryopreserved during childhood

Abstract

Ovarian insufficiency is a major long-term adverse event, following the administration of a myeloablative conditioning regimen, and occurring in >80% of children and adolescents receiving such treatment for malignant or non-malignant disease. Cryopreservation of ovarian tissue is currently offered to preserve the fertility of these young patients. At least 35 live births have been reported after transplantation of cryopreserved ovarian tissue in adult patients, but the procedure remains unproven for ovarian tissue harvested at a prepubertal or pubertal age. We report here the first live birth after autograft of cryopreserved ovarian tissue in a woman with primary ovarian failure after a myeloablative conditioning regimen as part of a hematopoietic stem cell transplantation performed for homozygous sickle-cell anemia at age 14 years. This first report of successful fertility restoration after the graft of ovarian tissue cryopreserved before menarche offers reassuring evidence for the feasibility of the procedure when performed during childhood.
© The Author 2015. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Full text

CASE REPORT Infertility
Live birth after autograft of ovarian
tissue cryopreserved during childhood
Isabelle Demeestere
1,2,
*
, Philippe Simon
3
, Laurence Dedeken
4
,
Federica Moffa
1,6
, Sophie Tse
´
pe
´
lidis
1,2,7
, Cecile Brachet
5
,
Anne Delbaere
1,2
, Fabienne Devreker
1,5
, and Alina Ferster
4
1
Research Laboratory on Human Reproduction, Faculty of Medicine, Universite
´
Libre de Bruxelles (ULB), Campus Erasme, CP636, 808 route de
Lennik, 1070 Brussels, Belgium
2
Fertility Clinic, Department of Obstetrics and Gynaecology, Erasme Hospital, Universite
´
Libre de Bruxelles,
1070 Brussels, Belgium
3
Gynecology Clinic, Department of Obstetrics and Gynaecology, Erasme Hospital, Universite
´
Libre de Bruxelles (ULB),
1070 Brussels, Belgium
4
Department of Hematology-Oncology, Ho
ˆ
pital Universitaire des Enfants Reine Fabiola (HUDERF), 1020 Brussels,
Belgium
5
Department of Endocrinology, Ho
ˆ
pital Universitaire des Enfants Reine Fabiola (HUDERF), 1020 Brussels, Belgium
6
Present address: Instituto Marque
´
s, Assisted Reproduction Service, Manuel Girona 33, MASIA, 08034 Barcelona, Spain
7
Present address: Nivelles Hospital, Rue Samiette, 1400 Nivelles, Belgium
*Correspondence address. Tel: +32-2-5556358; E-mail: idemeest@ulb.ac.be
Submitted on April 3, 2015; resubmitted on May 7, 2015; accepted on May 14, 2015
abstract: Ovarian insufficiency is a major long-term adverse event, following the administration of a myeloablative conditioning regimen,
and occurring in .80% of children and adolescents receiving such treatment for malignant or non-malignant disease. Cryopreservation of ovarian
tissue is currently offered to preserve the fertility of these young patients. At least 35 live births have been reported after transplantation of
cryopreserved ovarian tissue in adult patients, but the procedure remains unproven for ovarian tissue harvested at a prepubertal or pubertal
age. We report here the first live birth after autograft of cryopreserved ovarian tissue in a woman with primary ovarian failure after a myeloablative
conditioning regimen as part of a hematopoietic stem cell transplantation performed for homozygous sickle-cell anemia at age 14 years. This first
report of successful fertility restoration afterthe graft of ovarian tissue cryopreserved before menarche offers reassuringevidence for the feasibility
of the procedure when performed during childhood.
Key words: fertility preservation / childhood / ovarian tissue cryopreservation / transplantation / live birth
Introduction
Long-term survivors of hematological diseases diagnosed during
childhood are increasing in number, owing to improvements in treat-
ments and supportive care. Hematopoi etic stem cell transplanta-
tion (HSCT) is now offered as curative therapy for a wide v ariety of
malignant and non-malignant hematological disorders. However, the
risk of subfertility and premature ovarian insufficiency (POI) following
HSCT exceeds 80% in childhood cancer survivors, including teenagers
(Brougham and Wallace, 2005; Borgmann-Staudt et al., 2012). For
prepubertal female patients who face a high risk of treatment-induced
POI, the only option available to preserve fertility is the cryopreserva-
tion of ovarian tissue (Demeestere et al.,2009; Imbert et al., 2014;
Wallace et al., 2014). However, the successful outcome of this ap-
proach has been demonstrated only in pat ients who were adults at
thetimeoftheirdiagnosis(Stoop et al., 2014; Donnez et al., 2015).
As yet, the efficacy of the procedure in children has remained
unproven.
Case Report
We report the case of a woman aged 27 years, who was born in the
Republic of Congo and diagnosed with sickle-cell anemia at the age of
5, having experienced several episodes of fever and severe anemia.
Having emigrated to Belgium at the age of 11, her initial medical
work-up revealed abnormal blood flow velocities on a transcranial
Doppler echography. Subsequent treatment with hydroxyurea was
initiated. Due to her significant disease severity, curative therapy with
HSCT, made possible with the availability of a matched sibling donor,
was required. The option of ovarian tissue cryopreservation to preserve
fertility was offered and explained to both parents and child prior to the
initiation of the conditioning regimen. The parents signed the written
consent form. She had reportedly started puberty (breast development)
at around 10 years of age and her hormonal profile showed follicle-
stimulating hormone [FSH], luteinizing hormone [LH] and estradiol
levels of 6.3, 2.9 IU/l and 28 pg/ml, respectively, in October 2000. At
the time of the ovarian tissue cryopreservation procedure, she was
& The Author 2015. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved.
For Permissions, please email: journals.permissions@oup.com
Human Reproduction, Vol.30, No.9 pp. 2107–2109, 2015
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aged 13 years and 11 months (height 158 cm and weight 37 kg), and had
never experienced menstruation. A right oophorectomy was carried out
by laparoscopy in June 2001, and 62 fragments of ovarian tissue (2 ×
3 × 1– 2 mm) were cryopreserved, as previously described (Demees-
tere et al., 2006). The conditioning regimen consisted of oral busulfan
(16 mg/kg), cyclophosphamide (200 mg/kg) and anti-thymocyte globu-
lin (ATG 12 mg/kg), and graft-versus-host disease (GVHD) prophylaxis
with cyclosporin and methotrexate. Following HSCT, the patient devel-
oped grade II acute GVHD, and also limited chronic GVHD that required
the continuation of immunosuppressive drugs for 18 months. All hema-
tological values, as well as cerebral velocities, were normalized after
HSCT, and full donor chimerism was obtained. As expected, following
myeloablative conditioning, the patient developed primary ovarian
failure, with elevated gonadotropins. Menarche was induced at the age
of 15.5 years (Tanner stage A2P3M3) with the use of a third-generation
estrogen–progesterone preparation.
Ten years later, the patient was counseled for her wish to become
pregnant. After the interruption of hormonal supplementation, POI
was confirmed by the presence of permanent amenorrhea and high go-
nadotropin levels (FSH 59 IU/l, LH 32 IU/l). The patient underwent
ovarian tissue transplantation, in order to restore her fertility. Prior to
transplantation, two fragments of ovarian tissue were thawed for follicu-
lar density assessment. Two and six follicles per millimeter square were
observed in the fragments. Transplantation was performed by atwo-step
laparoscopy procedure (Demeestere et al., 2006), using a da Vinci surgi-
cal robotic system in May 2011. Four thawed ovarian fragments were
grafted on the residual left ovary, six were grafted in the right peritoneal
bursa, and five were grafted subcutaneously using a trocar incision.
Following the grafting procedure, the FSH level progressively
decreased, whereas both inhibin B and estradiol serum levels increased
(Fig. 1). Four months later, the hormonal levels reached the premeno-
pausal range (FSH 5 IU/l; LH 6 U/l; estradiol E2 166 pg/ml), and
ovarian activity wasobserved in all transplanted sites on pelvic ultrasound
imaging. Menstruation was first occurred 5 months post-transplantation
of ovarian tissue and was followed by regular menstrual cycles thereafter.
The patient had recourse to assisted reproductive technologies for
male infertility, although all such treatments were discontinued due
to relationship issues. Although she did not conceive during the first
2 years post-transplantation, she reported regular menstruation
throughout this period. Basal FSH levels remained within the normal
range throughout most of the menstrual cycles (Fig. 1), while anti-
Mu
¨
llerian hormone (AMH) levels remained undetectable. After more
than two years post-transplantation,the patient had a spontaneous preg-
nancy with a new partner and spontaneously delivered a healthy boy in
November 2014 (birthweight 3140 g, Apgar score of 9 and 10 at 1 and
5 min, respectively).
Ethical approval
The procedures were approved by ethical committees of the Erasme
Hospital and of the Ho
ˆ
pital Universitaire des Enfants Reine Fabiola
(Brussels, Belgium).
Discussion
This case reports the first live birth after transplantation of ovarian tissue
harvested before menarche. To our knowledge, only two cases of
cryopreserved ovarian tissue autograft, with the aim of inducing
puberty, have been reported in children (Poirot et al., 2012; Ernst
et al., 2013); in each case, ovarian function was restored for at least 19
months and puberty was successfully induced. While the use of an ex-
perimental and invasive procedure for this clinical indication raises a
number of concerns (Anderson et al., 2013), the authors showed, for
the first time, the possibility of restoring endocrine function from
Figure 1 FSH and estradiol levels before and after autograft of cryopreserved ovarian tissue. The autograft procedure is represented at time 0 months.
The arrow indicates the first menstruation.
2108 Demeestere et al.
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ovarian tissue harvested before puberty (Poirot et al., 2012; Ernst et al.,
2013). The prepubertal ovary contains many follicles at an early stage of
development, which is theoretically favorable for restoring ovarian func-
tion after grafting. However, the developmental competence of these
oocytes remains poorly investigated. Immature oocytes collected from
prepubertal ovarian tissue have been shown to mature in vitro, but to a
lesser degree than those retrieved from adult tissue (Revel et al., 2009;
Fasano et al., 2011). A recent studyreported that ovarian tissue frompre-
pubertal girls contains a larger proportion of abnormal follicles, compared
with adult ovarian tissue, and in vitro activation of quiescent follicles also
occurs differently, leading to limited follicular development (Anderson
etal.,2014). Thesedatahighlighttheneed tofurtherinvestigatethe viability
of ovarian tissuetransplantationfor restoring fertilitywhenthe cryopreser-
vation procedure occurs before the patient starts puberty.
While cryopreservation of ovarian tissue is a common procedure to
preserve fertility in children, this report offers, for the first time, evidence
for the long-term survival of autografts of ovarian tissue cryopreserved
before menarche and the efficiency of the procedure to restore fertility.
Acknowledgement
We thank J. Dechene for her contribution to the fertility preservation
program.
Authors’ roles
I.D. was responsible for the fertility preservation program and per-
formed the cryopreservation procedure. I.D. and L.D. contributed to
the data collection and analysis and wrote the manuscript. A.F. contrib-
uted to the data collection and analysis, performed the HSCT, referred
the patient for fertility preservationand revised the manuscript. C.B. con-
tributed to the data collection and manuscript revision. P.S. performed
the ovarian tissue transplantation procedure. F.M. and S.T. managed
the patient during the transplantation and the follow up. A.D. and F.D.
contributed to the development of the fertility preservation pro-
gram, managed the patient for assisted reproductive treatments and
contributed to the revision of the manuscript.
Funding
This program was supported by the Fondation Belge contre le Cancer
and the Fonds de la Recherche Scientifique FNRS-Te
´
le
´
vie. I.D. is a
research associate at the FNRS.
Conflict of interest
None declared.
References
Anderson RA, Hindmarsh PC, Wallace WH. Induction of puberty by
autograft of cryopreserved ovarian tissue in a patient previously treated
for Ewing sarcoma. Eur J Cancer 2013;49:2960 –2961.
Anderson RA, McLaughlin M, Wallace WH, Albertini DF, Telfer EE. The
immature human ovary shows loss of abnormal follicles and increasing
follicle developmental competence through childhood and adolescence.
Hum Reprod 2014;29:97 –106.
Borgmann-Staudt A, Rendtorff R, Reinmuth S, Hohmann C, Keil T,
Schuster FR, Holter W, Ehlert K, Keslova P, Lawitschka A et al. Fertility
after allogeneic haematopoietic stem cell transplantation in childhood
and adolescence. Bone Marrow Transplant 2012;47:271– 276.
Brougham MF, Wallace WH. Subfertility in childrenand young people treated
for solid and haematological malignancies. Br J Haematol 2005;131:
143–155.
Demeestere I, Simon P, Buxant F, Robin V, Fernandez SA, Centner J,
Delbaere A, Englert Y. 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. Hum Reprod 2006;21:2010 –2014.
Demeestere I, Simon P, Emiliani S, Delbaere A, Englert Y. Orthotopic and
heterotopic ovarian tissue transplantation. Hum Reprod Update 2009;
15:649–665.
Donnez J, Dolmans MM, Pellicer A, Diaz-Garcia C, Ernst E, Macklon KT,
Andersen CY. Fertility preservation for age-related fertility decline.
Lancet 2015;385:506 –507.
Ernst E, Kjaersgaard M, Birkebaek NH, Clausen N, Andersen CY. Case
report: stimulation of puberty in a girl with chemo- and radiation therapy
induced ovarian failure by transplantation of a small part of her frozen/
thawed ovarian tissue. Eur J Cancer 2013;49:911–914.
Fasano G, Moffa F, Dechene J, Englert Y, Demeestere I. Vitrification of in vitro
matured oocytes collected from antral follicles at the time of ovarian tissue
cryopreservation. Reprod Biol Endocrinol 2011;9:150.
Imbert R, Moffa F, Tsepelidis S, Simon P, Delbaere A, Devreker F, Dechene J,
Ferster A, Veys I, Fastrez M et al. Safety and usefulness of cryopreservation
of ovarian tissue to preserve fertility: a 12-year retrospective analysis. Hum
Reprod 2014;29:1931–1940.
Poirot C, Abirached F, Prades M, Coussieu C, Bernaudin F, Piver P. Induction
of puberty by autograft of cryopreserved ovarian tissue. Lancet 2012;
379:588.
Revel A, Revel-Vilk S, Aizenman E, Porat-Katz A, Safran A, Ben-Meir A,
Weintraub M, Shapira M, Achache H, Laufer N. At what age can human
oocytes be obtained? Fertil Steril 2009;92:458–463.
Stoop D, Cobo A, Silber S. Fertility preservation for age-related fertility
decline. Lancet 2014;384:1311 – 1319.
Wallace WH, Smith AG, Kelsey TW, Edgar AE, Anderson RA. Fertility
preservation for girls and young women with cancer: population-based
validation of criteria for ovarian tissue cryopreservation. Lancet Oncol
2014;15:1129–1136.
Graft of ovarian tissue harvested during childhood 2109
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