Ovarian tissue cryopreservation: Low usage rates and high live‐birth rate after transplantation

Abstract Introduction The likelihood of survival after cancer treatment among young women with cancer has increased considerably, quality of life after treatment has drawn more attention. However, in young fertile women, fertility preservation is an important issue with regard to quality of life. One of the options of fertility preservation is ovarian tissue cryopreservation. The purpose of this follow‐up study is to present our clinical experiences and evaluate the long‐term follow up of ovarian cryopreservation to improve future patient selection. Material and methods From July 2002 to December 2015 at the Leiden University Hospital, the Netherlands, 69 young women underwent ovarian tissue cryopreservation when they were at risk of iatrogenic premature ovarian insufficiency. Follow‐up data with regard to ovarian function were obtained until October 2018, from medical records and questionnaires. Results Of the 69 women in whom ovarian tissue cryopreservation was performed, 12 died (15.9%), 57 were approached to participate, of which 6 were lost to follow up. The indications for ovarian tissue cryopreservation were malignant (81.1%) and benign (18.9%) diseases in which gonadotoxic treatment was scheduled. In total, twenty women (39.2%) are known to have premature ovarian insufficiency due to gonadotoxic treatment. Fifteen women conceived spontaneously, and delivered 25 babies. In this cohort, the usage rate of autotransplantation is 8.7% (7/69). In total, nine autotransplantations of cryopreserved ovarian tissue were performed in seven patients (of which 1 ovarian tissue cryopreservation was performed in another hospital) after which 6 babies were born to four women, giving a live‐birth rate of 57%. Conclusions Ovarian tissue cryopreservation followed by autotransplantation is an effective method to restore fertility (live‐birth rate of 57%). The usage rate of 8.7% (6/69) indicates that more knowledge about the risk of premature ovarian insufficiency after gonadotoxic treatment is needed to be able to offer ovarian tissue cryopreservation more selectively.


| INTRODUC TI ON
Improved cancer treatment has resulted in decreased overall cancer mortality rates. From 1991 to 2006, overall cancer death rates decreased by 12.3%. This allows. and necessitates. young women to consider quality-of-life issues such as fertility preservation.
Especially as the Dutch cancer center has indicated that in 2017, 4.8% of all cancers were diagnosed in women under the age of 40, the need for safe and effective fertility preservation is mandatory. 1,2 It is well known that chemotherapy regimens as well as radiotherapy may compromise future fertility. 3 The risk of premature ovarian insufficiency (POI) depends on various factors such as age, type and dose of cytotoxic therapy, and ovarian radiation dosage. As a result of the increasing emphasis on fertility issues of young survivors, ovarian tissue cryopreservation (OTC) has been developed as 1 of the options to preserve fertility in case of gonadotoxic treatment.
When the patient is free of the disease and is diagnosed with POI, cryopreserved ovarian tissue can be autotransplanted to restore fertility. Over the last 2 decades, OTC has developed from the first successful report of a live birth in sheep, to the resumption of follicular activity and menstrual cycles after orthotopic autotransplantation in humans described by Oktay and Karlikaya. Since then, this technique has resulted in more than 130 healthy infants. [4][5][6][7] Cryopreservation of ovarian tissue is proposed as a fertility preservation option for various indications in a growing number of centers around the world. OTC benefits not only those with oncological diseases, but also girls and women with benign diseases such as β-thalassemia and autoimmune diseases requiring bone marrow transplantation, and benign ovarian diseases that require oophorectomy (ie endometriosis) with risk of POI. Finally, OTC can be used in women with genetic disorders associated with POI. [8][9][10][11] Despite the large series of OTC that have been performed, the number of women in whom autotransplantation of cryopreserved ovarian tissue has been performed is low. Some research groups have described the safety and usefulness of ovarian cryopreservation to preserve fertility. 12

| Patients
From July 2002 to December 2015, 69 women and young girls underwent OTC to preserve fertility and ovarian function at Leiden University Medical Center, the Netherlands. OTC was offered to women who were at high risk of iatrogenic POI (>50%), 17-21 had minimal risk of ovarian involvement of their primary malignancy, 22 were <36 years of age, and had normal uterus and ovaries on pelvic ultrasound. Women who had been treated with gonadotoxic agents previously, who had an expected 5-year survival rate of <50%, who were already known to have POI, in whom surgery was contraindicated and/or who were infected by human immunodeficiency virus or hepatitis B/C viruses were not offered OTC. 23 All patients were informed about the experimental procedure of ovarian cryopreservation, with no guarantees of fertility restoration or live birth and the small risk of reintroducing malignant cells when the ovarian tissue has to be thawed and transplanted into the patient. The study was approved by the local ethics committee and the national societies of gynecology and embryologists.

| Ovarian tissue cryopreservation
To harvest ovarian tissue, unilateral oophorectomy was performed under general anesthesia by laparoscopy or at open surgery depending on the individual situation.
In the operating room, the ovarian cortex was dissected and sliced into small pieces (10 × 5 × 1 mm), according to the description of Radford in 2001. The slices of ovarian cortex were transferred into vials and transported to the assisted reproductive technology (6/69) indicates that more knowledge about the risk of premature ovarian insufficiency after gonadotoxic treatment is needed to be able to offer ovarian tissue cryopreservation more selectively.

K E Y W O R D S
cryopreservation, fertility preservation, ovarian tissue, premature ovarian insufficiency, ovarian tissue transplantation

Key message
Ovarian tissue cryopreservation has a low usage rate of 8.7% but a high success rate after autotransplantation of 86% restoration of ovarian function and 57% live-birth rate. laboratory, where a slow freezing protocol was used to cryopreserve the slices. 24 From 2012, the whole ovary was transported to the assisted reproductive technology laboratory, where it was processed according to the Denmark technique and subsequently cryopreserved using a slow freezing protocol. 16

| Follow up
Follow up to determine survival, course of the disease, ovarian function, and fertility over time was obtained until October 2018 by consulting the referring doctor and by a review of medical records from the referring hospital and/or our own electronic patient dossier.
Additionally, a questionnaire was sent to all patients, addressing the following topics: current age, received gonadotoxic treatment, ovarian function, contraception method, diagnostics, hormone replacement therapy and pregnancies.

| Ovarian transplantation technique
If a patient requested autotransplantation of ovarian tissue, the treating oncologist/hematologist was contacted for approval. The tissue was thawed as described by Rosendahl et al, 16 followed by the operation as previously described by Andersen et al. 25 Whenever possible the tissue was transplanted under the cortex of the remaining ovary left in situ and in some cases, into peritoneal pockets on the anterior abdominal wall and the lateral pelvic wall.

| Statistics
POI was defined as 1 measurement of follicle-stimulating hormone >40 IU/L (with or without the measurement of estradiol) and/or when the woman did not menstruate after treatment. Collected data were analyzed using SPSS version 25 (IBM, Armonk, NY, USA) to perform descriptive statistics and to determine the 5-year survival rate with the Kaplan-Meijer method.

| Ethical approval
Ethical approval for this follow-up assessment was given by the  In 2 women, OTC was performed despite a moderate to high risk of ovarian metastases: 1 had rectal cancer, and 1 was diagnosed with abdominal disease of the esophageal cancer during the laparoscopy to harvest ovarian tissue. Six women had already been treated with low-risk gonadotoxic treatment, but these women were included because of normal ovarian function and the need for additional gonadotoxic treatment. In total, 3 minor complications occurred (4.3%): 1 woman (1.4%) had an injury to the bladder due to the laparoscopy that was diagnosed after surgery. An indwelling catheter was inserted for 14 days, which resulted in complete recovery. One laparoscopic procedure (1.4%) was converted to a minilaparotomy because of instrumental defects. In 1 woman (1.4%), the insertion of the uterus mobilizer resulted in a laceration of the vagina that had to be sutured. All 3 woman recovered completely.

| Gonadotoxic treatment
Follow up to determine survival, course of the disease, ovarian function, and fertility over time was obtained until October 2018.
Sixty-seven women have completed the gonadotoxic treatment, one woman is still under treatment and in 1 woman gonadotoxic treatment for breast cancer was scheduled but she rejected this treatment after OTC and hormonal treatment were administered after lumpectomy.
The different regimens of gonadotoxic treatment are shown in   approached to answer questionnaires of whom 6 were lost to follow up (8.7%).

| Survival
The cumulative 5-year survival rate of women who underwent OTC in our hospital is 80.5% (Figure 3). In total, 12 women died because of recurrence of disease, therapy resistance or after complications of hematopoietic stem cell transplantation.

F I G U R E 3
Five-year survival analysis use hormonal contraception, and one is under treatment ( Table 2).
In Figure 4, Fifteen women (29.4%) conceived spontaneously after OTC without autotransplantation, which resulted in 25 deliveries. One woman died due to recurrence of the myxoid liposarcoma after she had delivered a healthy baby. Of these 15 women, nine had breast cancer (which accounts for 42.9% of the breast cancer patients) ( Table 2).

| Fertility after ovarian autotransplantation
In our follow-up cohort, six women underwent 8 autotransplantations of cryopreserved ovarian tissue (Table 3) One woman did not benefit from the transplantation because ovarian function was not restored. In five women the transplants are still active at closure of follow up of this study, one woman underwent ovariectomy after delivery because of BRCA1 mutation. A total of seven pregnancies occurred in four of these women, which resulted in 6 term deliveries and one miscarriage. Two pregnancies were accomplished after in vitro fertilization: in one woman one oocyte was retrieved in a spontaneous cycle, which was fertilized and resulted in one live birth. In the other woman, eight oocytes were retrieved in a stimulated cycle, of which one was fertilized. However this pregnancy resulted in a missed abortion.

| D ISCUSS I ON
We showed that ovarian cryopreservation with subsequent autotransplantation is a safe and successful technique with six live births in four out of seven patients (57%). Additionally, our previous data showed no ovarian metastases in ovarian tissue. 26 The number of international reports and publications is increasing and OTC has been described in large series. 13,14 The usage rate of cryopreserved ovarian tissue transplantation (1.9%-4%) and success rates with regard to live-birth rate (31%-37%) have been described. [12][13][14]16,[27][28][29] In this series of women who underwent OTC, the autotransplantation rate was 8.7% (6/69), the restoration rate of ovarian function was   Thereafter, six other pregnancies occurred which resulted in a total of five live births and one missed abortion. Pacheco and Oktay reported a meta-analysis with success rates of 57.5% life-birth rate, 37.7% ongoing pregnancy rate and an endocrine restoration rate of 63.9%. 40 However, the meta-analysis did not rule out underreporting of failed cases by others. Although our study describes small numbers of patients, OTC with subsequent transplantation is a successful technique with an ongoing pregnancy rate of 57%.
When looking at our results (and we have to realize that the usage rates at this moment might be an underestimation as OTC), not all women actively wish for a child and a large number of women in our cohort are still at risk of developing premature failure later in life: resumption of cyclic menses after high gonadotoxic treatment does not guarantee normal fertility. 41 These women are future candidates for autotransplantation (resp. 8-41 patients). In addition, the risk of POI needs to be elaborated. Our data may help women in the future and their physicians to decide about the options to preserve fertility in case of scheduled gonadotoxic treatment.

| CON CLUS ION
We show that autotransplantation of cryopreserved ovarian tissue is an effective method (restoration of ovarian function 86%) and has a live-birth rate of 57% (4/7). However, given our low risk of POI  after gonadotoxic treatment (29.4% normal ovarian function), the usage rate was only 8.7% (6/69). Especially in breast cancer, and perhaps in osteosarcoma, the risk of POI after gonadotoxic treatment was significantly lower than estimated. One can argue that fertility preservation is not necessary in these patients because of the low risk of POI and high chance of (spontaneous) pregnancy. Our data may help future patients and physicians in their discussions and decisions about the need and possibilities to preserve fertility. This will lead to an increase in the efficiency and applicability of care.
Finally we would make a plea for an international collaboration to expedite the knowledge on need, safety, and effectiveness of OTC and autotransplantation.

ACK N OWLED G M ENTS
The authors thank Esther Jenninga, who was a dedicated medical doctor and researcher and was one of the first researchers in the LUMC, in the field of fertility preservation. Unfortunately, she passed away and could not finish what she started.

CO N FLI C T O F I NTE R E S T
The authors have stated explicitly that there are no conflicts of interest in connection with this article.