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CASE REPORTS
Successful pregnancies after transplantation of ovarian tissue retrieved and cryopreserved at time of childhood acute lymphoblastic leukemia – a case report
Fertility preservation (FP) has gained recognition as an integral part of cancer-treatment in young patients and the interest in developing methods that can also be offered to children is increasing.1 Ovarian tissue cryop- reservation (OTC) is the only option for FP for prepuber- tal girls, and also a frequently offered method to young female adolescents.2 Retrieval of the tissue is usually per- formed through laparoscopy and can be planned without significant delay and with low postoperative risks, as reported in large series.2
The outcome for pediatric patients suffering from acute leukemia has improved dramatically over the last decades and OTC aimed at FP is becoming an established routine procedure in many cancer centers. However, fundamen- tal questions remain, especially with regard to the poten- tial of reseeding of malignant cells, as well as the future functionality of OTC if the patients have already received several chemotherapy rounds. At present, only a few cases of ovarian tissue transplantation (OTT) in survivors of adult acute leukemia have been reported,3-5 but no cases of OTC at time of childhood leukemia have been reported.
The case hereby described refers to a female pediatric patient, 14 years of age, diagnosed in 2001 with Philadelphia chromosome positive, high risk, acute lym- phoblastic leukemia (Ph+ ALL). Induction treatment according to the protocol of the Nordic Society Pediatric Hematology and Oncology, NOPHO ALL 2000 high risk protocol (Vincristine, Doxorubicine, Cytarabine and Prednisolone plus Methotrexate), was initiated immedi- ately upon diagnosis. After consolidation the patient achieved complete remission and she was planned for allogenic stem cell transplantation (HSCT), as stipulated by the protocol. Bone marrow flow-cytometry analysis showed cells with the leukemia-associated immunophe- notype below 0,1% at that time point. Due to the high gonadotoxicity of the conditioning regime (3 Gy x 4 total body irradiation combined with high dose cyclophos- phamide) the patient was referred for FP counselling (Figure 1). OTC was considered appropriate due to the confirmed remission status. An ovarian biopsy of half the right ovary was obtained by laparoscopy and cryopre- served in pieces of about 2 mm x 4 mm size, using a propanediol-based slow-freezing protocol.6 Following HSCT-conditioning the patient developed permanent ovarian failure (Figure 1).
In 2016, 15 years after remission and OTC, the patient consulted the fertility clinic expressing a wish to retrans- plant her ovarian tissue. The serum gonadotropin levels after interruption of hormonal substitution were in the postmenopausal range (Figure 1). The ovaries were small and appeared inactive on ultrasound scans.
Although the ovarian tissue had been harvested after several courses of chemotherapy and during confirmed remission, an additional molecular analysis to further evaluate the safety of the ovarian tissue to be transplant- ed was discussed with the patient in 2017. No molecular investigations had been performed at the time of leukemia-diagnosis; however, frozen blood cells taken at diagnosis in 2001 were available at the Karolinska Institute’s biobank and were used to establish that the patient’s leukemic cells harbored the BCR-ABL minor fusion transcript. Subsequently, the presence of the BCR-
ABL transcript was investigated in the patients’ cryopre- served ovarian tissue. Eight ovarian tissue pieces each approximately 1-2 mm x 4 mm, comprising approximate- ly 15% of the cryopreserved tissue (the rest was eventu- ally transplanted), were randomly selected and processed in parallel using the EZ1 RNATM tissue mini kit (Qiagen, Sweden) in 2017. In order to maximize sensitivity, we used all the RNA extracted from the ovarian tissue and reverse transcribed it using the SuperscriptTM VILOTM kit (ThermoFischer, Sweden). The presence of the BCR- ABL transcript was investigated in a total of 80 independ- ent polymerase chain reaction (PCR) reactions.7 GUS transcript was used to measure RNA integrity and quan- tity. No BCR-ABL transcript was detected in any of the real-time PCR reactions and we could estimate a detec- tion level corresponding to 1/105 malignant to non- malignant cells. As our investigations showed a reassur- ingly low likelihood for leukemic cell contamination, this supported the decision to proceed to transplantation of the ovarian tissue with reproductive purposes.
Twenty-seven ovarian tissue pieces were thawed and transplanted in November 2017 through a modified laparoscopic technique that allowed the ovary to be exte- riorized facilitating the insertion of the ovarian tissue transplants in ovarian subcortical pockets (Figure 2).
The patient suspended hormonal substitution therapy the day before surgery but gonadotropins still indicated postmenopausal levels 1 month post surgery. Ovarian engraftment was followed-up through monitoring of clinical signs of estrogen secretion, climacteric symp- toms, and serum levels of gonadotropins, estrogen and anti-Mullerian hormone (AMH). The gonadotropins returned to premenopausal levels 85 days after transplan- tation, increasing serum estradiol levels were demon- strated, and climacteric symptoms improved (Figure 1). Antral follicles were visualized on ultrasound and con- trolled ovarian stimulation with gonadotropins (COS) using an antagonist protocol aiming at in vitro fertilization (IVF) was initiated. Four attempts of IVF failed due to poor response, with only one oocyte retrieved that was not fertilized. A second transplantation was therefore performed in November 2018 using the remaining ovari- an tissue that included 19 thawed pieces; of those seven pieces had been previously thawed in 2017 but not used for the molecular analysis and re-cryopreserved. Spontaneous menstruation occurred 86 days following transplantation. On ultrasound scans the size of the uterus and the ovaries was significantly enlarged from baseline estimations, but the serum AMH levels were low, 0.15 mg/L, indicative of a much reduced ovarian reserve. A new attempt at IVF was initiated when gonadotropins returned to premenopausal levels and this treatment resulted in one oocyte retrieved and fertilized by standard IVF technique. Embryo morphological devel- opment was normal and transferred was at 4-cell stage. Pregnancy was established and the patient delivered a healthy baby boy at 35+5 gestational weeks in November 2019. No complications associated with pre- maturity occurred. Breast feeding was possible, but suc- cessful only for 3 months and thereafter suspended. Menstrual cycles resumed 3 months after delivery and continued for 9 months until the woman achieved a nat- ural conception confirmed incidentally in November 2020 at the time of consultation to our fertility center. On ultrasonography, the pregnancy was confirmed as ongo- ing at gestational week 8. At the time of this report the pregnancy is progressing into week 35th.
Although OTC have been reported in women and girls with leukemia in large series of fertility preservation,2,8
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