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Pregnancies and live births after 20 transplantations of cryopreserved ovarian tissue in a single center
Pregnancies and live births after 20 transplantations of cryopreserved ovarian tissue in a single center Ralf Dittrich, Ph.D., Janina Hackl, M.D., Laura Lotz, M.D., Inge Hoffmann, M.D., and Matthias W. Beckmann, M.D. Department of Gynecology and Obstetrics, Erlangen University Hospital, Friedrich-Alexander University of Erlangen-Nuremberg, Erlangen, Germany
Objective: To report the results of 20 orthotopic retransplantations of cryopreserved ovarian tissue after cancer treatment. Design: Retrospective analysis. Setting: Tertiary gynecology department. Patient(s): Twenty patients with malignant disease: 11 with hematological malignancies (55%), four with breast cancer (20%), three with anal cancer (15%), and two with ovarian cancer (10%); the mean age before oncological treatment was 30.5 years. Intervention(s): Ovarian tissue was removed from patients in various centers in Germany in 2005–2009. All patients received chemotherapy and/or radiotherapy. Afterward, 17 patients had complete premature ovarian insufficiency, while three still showed some ovarian activity. Overnight transportation of tissue before freezing was necessary in eight cases. Cryopreservation followed slow freezing protocols in all cases. Retransplantation was performed at Erlangen University Hospital 3.75 years after extraction, on average. Thawed tissue was transplanted into a peritoneal pouch in the broad ligament region, below the tube, in 16 cases. Fragments were sutured both onto the remaining ovary and into a peritoneal pouch in four cases. Main Outcome Measure(s): Restoration of ovarian activity, pregnancy, birth. Result(s): Ovarian activity resumed in all patients except one. Seven patients conceived, with one miscarriage and four ongoing pregnancies. Four patients delivered healthy babies. One pregnancy and live birth after oocyte donation need to be considered separately. Conclusion(s): These data clearly demonstrate that preserving fertility by cryopreserving ovarian tissue is a successful and safe clinical option that can be considered for selected cancer patients. (Fertil SterilÒ 2015;103:462–8. Use your smartphone Ó2015 by American Society for Reproductive Medicine.) to scan this QR code Key Words: Fertility preservation, cryopreserved ovarian tissue, transplantation, live birth, and connect to the pregnancy Discuss: You can discuss this article with its authors and with other ASRM members at http:// fertstertforum.com/dittrichr-transplantations-ovarian-tissue/
M
odern treatments for oncological diseases have significantly increased survival rates in cancer patients, but chemotherapy and radiotherapy often lead to sterility due to destruction of the ovarian reserve. It is therefore important to advise patients who require gonadotoxic treatment on the options that are available for preserving their
fertility. The ability to have children of one's own is an important aspect of quality of life (1). A number of strategies have therefore been developed in recent years to enable these patients to have children using their own gametes (2). The individual patient's specific situation needs to be taken into account when fertility-preserving measures are being selected. Several
Received August 21, 2014; revised October 23, 2014; accepted October 28, 2014; published online December 6, 2014. R.D. has nothing to disclose. J.H. has nothing to disclose. L.L. has nothing to disclose. I.H. has nothing to disclose. M.W.B. has nothing to disclose. R.D. and J.H. should be considered similar in author order. €tsklinikum Erlangen, UniverReprint requests: Professor Ralf Dittrich, Ph.D., Frauenklinik, Universita €tsstrasse 21-23, D-91054 Erlangen, Germany (E-mail: [email protected]). sita Fertility and Sterility® Vol. 103, No. 2, February 2015 0015-0282/$36.00 Copyright ©2015 American Society for Reproductive Medicine, Published by Elsevier Inc. http://dx.doi.org/10.1016/j.fertnstert.2014.10.045 462
discussion forum for this article now.*
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options are currently available for preserving fertility, and cryopreservation of oocytes (3) and cryopreservation of embryos (4) are well-established procedures. The choice of which procedure is used depends on various parameters: the type and timing of gonadotoxic therapies, the type of cancer (with possible involvement of the ovaries), the patient's age, and her partner status. However, cryopreservation of ovarian tissue is a promising method for fertility preservation because it not only restores fertility after retransplantion but also is able to avoid hormone insufficiency (5). Ovarian tissue can be easily extracted laparoscopically, regardless of the current phase of the menstrual VOL. 103 NO. 2 / FEBRUARY 2015
Fertility and Sterility® cycle, without significantly delaying chemotherapy or radiotherapy. Patients who are younger than 35 years are particularly suitable, as the ovaries contain large numbers of oocytes in these patients and the chances of successful retransplantation are therefore greater. The ovarian tissue can be cryopreserved at specialized centers for reproductive medicine, and—depending on the oncological therapy required—can be retransplanted after a certain time if the patient has treatment-induced premature ovarian failure. Cryopreservation of ovarian tissue before oncological treatment is a promising method, since a large number of follicles survive the freeze-thaw procedure (6). Donnez et al. reported the first live birth after autotransplantation of human ovarian tissue in 2004 (7). In Germany, the first live birth after retransplantation of cryopreserved ovarian tissue was reported in 2012 (8). Using this technique, more than 25 live births have been reported worldwide to date (9). It can be expected that in the near future, more and more oncology patients who have survived cancer and have been cured of their disease will wish to undergo reimplantation of ovarian tissue. The purpose of the present review is to report on and discuss the techniques used and the results in 20 orthotopic retransplantations of cryopreserved tissue in a single center.
MATERIALS AND METHODS Patient Cohort Ovarian tissue was removed from 20 premenopausal women with various types of cancer; the patients' mean age was 30.5 years (range, 20–37 years). Three patients had anal cancer, 10 Hodgkin's lymphoma, one non-Hodgkin's lymphoma, four breast cancer, and two ovarian tumors (Table 1). At the time of retransplantation, their average age was 34.2 years. All patients received chemotherapy, including two patients with Hodgkin's lymphoma who received high-dose chemotherapy. The three patients with anal cancer received pelvic radiotherapy. Signs of residual hormone activity were present before retransplantation in only three of the patients. These patients did not wish to await a natural course and requested retransplantation. Values for antim€ ullerian hormone (AMH) levels after oncological treatment and before retransplantation were obtained in 12 of the women. All AMH levels were below detection limits. Tubal patency was tested in 14 women, and bilateral obstruction of the fallopian tubes was noted in four of them. The study design was approved by the Institutional Review Board (IRB) at Erlangen University Hospital, which had no concerns about it (IRB no. 522_14 Bc).
The ovarian tissue was harvested laparoscopically, with no use of electrocoagulation or thermal cutting to avoid damaging the tissue. In the present group of 20 patients, an average of two thirds of one ovary was removed. The tissue samples from each patient were analyzed histologically to prevent transmission of the underlying disease, and the presence and density of primordial follicles were documented. No complications such as bleeding or infections were observed.
Transportation before Freezing The ovarian tissue was removed, processed for cryopreservation, and stored at the Department of Gynecology and Obstetrics at Erlangen University Hospital in six cases so that no transportation was necessary. In another six cases, the tissue was frozen outside Erlangen, but within 6 hours after tissue collection, and in eight cases overnight transportation of the removed tissue was required. After laparoscopic removal, the tissues were sent in a special insulated transportation box (Z100037054; delta T Ltd.) at 4 C by same-day courier service or express overnight transportation for cryopreservation to a specialized processing unit (in either Bonn or Erlangen) (10). If the tissues were stored in an external cryobank, the tissue pieces were sent in shipping containers cooled with liquid nitrogen at 196 C to the Reproductive Medicine Laboratory in Erlangen.
Cryopreservation Procedure Slow freezing methods were used in all cases. Between 6 C and 40 C, the cooling rate was 0.3 C/min. Dimethyl sulfoxide was used as the cryoprotectant in 11 cases; the cryopreservation method used in these cases has been described elsewhere in detail (11). Ethylene glycol was used as the cryoprotectant in six cases (12). In brief, pieces of ovarian tissue (3 3 1 mm) were equilibrated for 30 minutes in 1.5 mol/L ethylene glycol and 0.1 mol/L sucrose in phosphatebuffered saline in 2 mL standard cryovials (Simport T3092A) on a tilting table on ice and then loaded into the open freezing system (CTE-920, CTE). The following cooling program was used: 2 C/min to 1 C, 0.5 C/min to 5 C, 0.3 C/min to 9.3 C, 10 minutes of soaking, then 0.3 C/min to 40 C and 10 C/min to 140 C, at which temperature the samples were plunged into liquid nitrogen at 196 C. Propanediol was used as the cryoprotectant in the remaining three cases.
Storage Extraction of Ovarian Tissue In the present study, ovarian tissue was extracted from 20 cancer patients in Germany between 2005 and 2009. The tissue was removed in the Department of Gynecology and Obstetrics at Erlangen University Hospital in six of these patients. In the 14 other patients, the tissue was removed in other centers for reproductive medicine in various cities in Germany (D€ usseldorf, Dresden, Munich, Hamburg, W€ urzburg, Cologne, and Essen). VOL. 103 NO. 2 / FEBRUARY 2015
The tissue was stored in highly specialized cryobanks in Germany that have expertise in cryopreserving ovarian tissue. The cryobanks are members of the FertiPROTEKT network, a collaborative group of centers in Germany, Austria, and Switzerland.
Thawing Thawing was fast in a warm water bath (37 C). The tissue fragments were released from the protective cryopreservation 463
Patient characteristics and results of autotransplantation.
Disease
Age at tissue removal
Age at TX
1 2 3
Anal cancer Anal cancer Hodgkin's lymphoma
28 34 20
30 38 23
CTX, RT pelvis CTX, RT pelvis CTX
4
Hodgkin's lymphoma
26
31
CTX
5
Hodgkin's lymphoma
27
32
6
Breast cancer
35
38
7
Breast cancer
28
31
8 9
Anal cancer Hodgkin's lymphoma
33 35
36 37
High-dose CTX, RT mediastinum Surgery. CTX, RT breast Surgery, CTX, RT breast RT pelvis CTX
10 11
Hodgkin's lymphoma Breast cancer
33 37
39 42
12
34
38
13 14
Non-Hodgkin's lymphoma Hodgkin's lymphoma Hodgkin's lymphoma
21 28
27 30
15
Breast cancer
34
38
16 17 18
Hodgkin's lymphoma Ovarian cancer Hodgkin's lymphoma
27 30 31
31 35 37
19 20
Dysgerminoma Hodgkin's lymphoma
31 33
35 37
Patient no.
Treatment
CTX, RT Surgery, CTX, RT breast CTX CTX CTX, RT mediastinum Surgery, CTX, RT thorax, axilla
VOL. 103 NO. 2 / FEBRUARY 2015
High-dose CTX CTX, surgery CTX, RT, mediastinum Surgery CTX
AMH level before TX
Overnight transport
– – <0.1
No No No
–
No
0.18
Yes
Assisted reproductive techniques
First sign of E2 or follicle (months after TX)
Residual ovarian activity before TX
Pelvic wall Pelvic wall Pelvic wall & ovary Pelvic wall & ovary Pelvic wall
Yes Yes Yes
3 4 5
No No No
No
5
No
No
2
No
Yes
4
No
No
2
No
3 4 month
No No
4 month
No Yes
Irregular menses Regular menses Lost of follow up
Graft site
Outcome
Irregular menses Regular menses Regular menses Live birth after oocyte donation Live birth October 2011
No Pregnant
<0.1
No
–
No
<0.1 –
Yes Yes
Pelvic wall & ovary Pelvic wall & ovary Pelvic wall Pelvic wall
No No
Pelvic wall Pelvic wall
Yes No; induced ovulation No No
<0.1
Yes
Pelvic wall
No
4 month
No
<0.01 <0.08
Yes No
Pelvic wall Pelvic wall
No No
2 month 6 month
No No
Ongoing pregnancy
Pregnant Lost of follow up
<0.1
Yes
Pelvic wall
No
5 month
No
Pregnant
<0.1 0.4 0.18
Miscarriage December 2013 Second pregnancy
No No Yes
Pelvic wall Pelvic wall Pelvic wall
No No No
4 month
No Yes Yes
– –
No Yes
Pelvic wall Pelvic wall
No Yes
No 2 month
0.05 –
No No
No wish to become pregnant, 1 IVF Live birth August 2012 Second pregnancy Live birth December 2013
Live birth July 2014
Ongoing pregnancy
Note: The mean age at tissue removal was 30.5 years. The mean age at transplantation was 34.2 years. The baby take home rate was 20%. CTX ¼ chemotherapy; E2 ¼ 17b-estradiol; RT ¼ radiotherapy; TX ¼ transplantation. Dittrich. Cryopreserved ovarian tissue transplants. Fertil Steril 2015.
Continuing hormonal activity
No Regular menses Irregular menses Still breast feeding
Regular menses Still breast feeding Regular menses No menses Pregnant
ORIGINAL ARTICLE: FERTILITY PRESERVATION
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TABLE 1
Fertility and Sterility® medium in reverse order, with the addition of 0.25 mol/L sucrose.
FIGURE 1
Time of Autotransplantation Retransplantation was performed on average 3.7 years (range, 1–5 years) years after extraction of the tissue. The patients' mean age at the time of first retransplantation was 34.2 years (range, 27–42 years).
Autotransplantation Techniques The thawed tissue was transplanted into a 1.5 cm deep pouch of peritoneum in the region of the broad ligament, below the fallopian tube, in 16 cases. Fragments of ovarian tissue 1–3 mm in size were introduced into this pouch, and the pocket was closed with a Vicryl suture if necessary, if the opening was gaping. Fragments were sutured both onto the remaining ovary and also into a peritoneal pouch in four cases. For transplantation onto the remaining ovary, a longitudinal incision of approximately 1 cm was made in the surface of the remaining ovary, and the tissue pieces were then threaded onto a suture and fixed into the incision. The amount of transplanted tissue was approximately 20% of the surface of one ovary. Four patients requested a second transplantation, and two even requested a third retransplantation when the initially transplanted tissue lost function.
Follow-up after Retransplantation The first assessment of E2 and FSH was carried out in each patient 2 months after retransplantation. If the FSH level had decreased and E2 had increased, monitoring of the cycle with hormone measurements and ultrasound controls was initiated. Initial signs of an increase in the E2 level or follicle growth were documented in 16 women an average of 3–6 months after retransplantation. An absence of hormone activity has so far been observed in only one patient.
A typical ‘‘neo-ovary’’ that developed from the transplanted tissue in the pelvic wall. Dittrich. Cryopreserved ovarian tissue transplants. Fertil Steril 2015.
Pregnancies Seven women have so far become pregnant. The ovarian tissue was transplanted into the pelvic wall in six of these cases only, and in one further case additionally into the remaining ovary. In five cases, the ovarian tissue was also transported overnight before freezing. Six of these pregnancies were spontaneous and one followed IVF. In the case of IVF, the tissue was transplanted into the pelvic wall. Four children have been born in the meantime; one pregnancy ended with a miscarriage in gestational week 10, and two are ongoing. Two patients became pregnant a second time after spontaneous conception (no. 5 and no. 15). One additional pregnancy and live birth needs to be considered separately, as it developed after oocyte donation. The average age of the patients who became pregnant was 29.7 years (median, 30; minimum, 21; maximum, 35) at the time of tissue collection and 33.8 (median, 35; minimum, 27; maximum, 37) at the time of transplantation.
RESULTS Hormone Activity in the Transplanted Tissue
Outcome Regarding the Site of Transplantation
No surgical complications occurred in any cases during transplantation of the ovarian tissue. In 19 cases (95%), hormone activity in the ovary was observed at least in the form of an increase in serum E2 levels. Three of these patients had residual activity in the ovary before transplantation. These patients cannot therefore be included in calculations of the restoration of ovarian function. One patient showed no activity after transplantation. Restoration of ovarian function was therefore achieved in 94% of the patients. The mean time to the first increase in E2 (in patients who had no residual hormone activity at the time of retransplantation) was 3.7 months (median, 4 months; range, 2–6 months). Figure 1 shows a typical ‘‘neo-ovary’’ that developed from the transplanted tissue in the pelvic wall. The average duration of hormone activity in the tissue after transplantation is 1.7 years to date, and activity is still present in 13 patients. The longest period of hormonal activity observed so far is 62 months.
The tissue was transplanted into the pelvic wall in all cases, and in four cases additionally into the remaining ovary. In these four cases, one patient became pregnant and delivered a child (25%) and has also become pregnant for a second time in the meantime. In the 16 patients in whom the tissue was transplanted into the pelvic wall alone, seven pregnancies occurred (two in one patient) and three babies have been delivered (19%).
VOL. 103 NO. 2 / FEBRUARY 2015
Follow-up of Patients at Risk for Possible Relapse of Cancer Due to Ovarian Tissue Transplantation The group of patients included three with cancer types involving a certain risk of relapse: one with non-Hodgkin's lymphoma, one with a dysgerminoma, and one with ovarian cancer (Table 2). In the patient with a dysgerminoma, the transplanted tissue showed no hormone activity and was removed 11 months after transplantation to ensure that no 465
ORIGINAL ARTICLE: FERTILITY PRESERVATION
TABLE 2 Tumor entities (with classification) involving a certain risk of relapse for the patients. Patient no. 12 17 19
Cancer entity
Tumor classification
Non-Hodgkin's lymphoma Serous adenocarcinoma in the ovary Ovarian dysgerminoma
Highly malignant B-cell lymphoma, Ann Arbor Iva pT3c pN0 M0 G1, International Federation of Gynecology and Obstetrics IIIc rpT1a NX L0 V0 Pn0 R0
Dittrich. Cryopreserved ovarian tissue transplants. Fertil Steril 2015.
relapse would occur. The patient with ovarian cancer became pregnant, and removal of the ovarian tissue 6 weeks after birth is being considered. In addition, one patient with breast cancer had a local relapse, which is not regarded as having been caused by the retransplantation. All of the patients were informed that there is a certain risk for minimal residual disease and provided written informed consent. All of the transplanted tissue fragments were analyzed histologically for the presence or absence of malignant cells.
DISCUSSION After autotransplantation of cryopreserved ovarian tissue, 19 of the 20 patients showed ovarian function and four live births of healthy children occurred. These data demonstrate that cryopreservation of ovarian tissue and subsequent retransplantation is a successful option for preserving fertility in patients with malignancies; the results are consistent with those of other studies reporting a high level of success with regard to restoration of ovarian function and live birth. Donnez et al. reported in 2011 on 13 live births worldwide after retransplantation of cryopreserved ovarian tissue (13), and in 2013 on 24 live births after 60 retransplantations worldwide (9). Andersen et al. reported restoration of ovarian function in six patients, with live births of two healthy
babies (14). The first live birth in Germany was reported in 2011 (8). Table 3 summarizes all of the live births after retransplantation of cryopreserved ovarian tissue reported in the international literature to date (Pubmed search, 7–10, 13–29). To the best of our knowledge, 24 live births have so far been described; the pregnancy rate after autografting of cryopreserved tissue at orthotopic sites is estimated at approximately 30% (28), which was also observed in the present group of patients. This shows that cryopreservation of ovarian tissue and subsequent retransplantation is a promising method that enables women affected by cancer to have children after recovering from the disease. However, several questions still remain regarding cryopreservation and transplantation of ovarian tissue that have not yet been fully answered (30). Two different techniques of orthotopic autotransplantation have proved to be successful (7). The tissue can be either transplanted into or onto the remaining ovary or into a peritoneal pocket in the pelvic peritoneum of the ovarian fossa; both techniques have been successful (9). In this study, the ovarian tissues were retransplanted orthotopically into peritoneal pockets in the pelvic wall in all cases and additionally in four cases into the remaining ovary. In the present authors' view, transplantation into a peritoneal pocket provides a better blood supply than transplantation to a heterotopic site or onto the remaining ovary, and it is therefore associated with better follicle development and better functionality of the tissue (31). It also makes natural conception possible. Figure 1 shows a neo-ovary with growing follicles after transplantation into the ovarian fossa. In the present group of 20 patients, the tissue was frozen at the center in which surgery was carried out in 12 cases. In the other eight cases, the tissue was transported to a center specializing in cryopreservation. In contrast to the practice in Denmark (14), the tissue was transported overnight before being frozen in these eight cases. However, five of these eight patients became pregnant. This demonstrates that overnight transportation of ovarian tissue before freezing is possible. The first live birth in Germany originated from tissue that had been transported overnight before freezing (10). In that
TABLE 3 Published reports of live births following cryopreservation and retransplantation of ovarian tissue. Research group Donnez et al. Meirow et al. Andersen et al. Demeestere et al. Silber et al. Roux et al. S anchez-Serrano et al. Revel et al. García Rada et al. Revelli et al. Devi and Kumar Dittrich et al. Kawamura et al. Ernst et al. Callejo et al. Yang et al.
Cryopreservation procedure
Live births
Year
References
Slow freezing Slow freezing Slow freezing Slow freezing Slow freezing Slow freezing Slow freezing, oocyte vitrification Slow freezing Slow freezing Slow freezing
6 1 2 1 1 1 1 1 1 1 1 1 1 1 1 3
2004–2013 2008 2008 2010 2010 2010 2010 2011 2012 2013 2005 2012 2013 2010 2013 2011
(7, 9, 13, 15) (16) (14) (17) (18) (19) (20) (21) (22) (23) (24) (10) (25) (26) (27) (29)
Slow freezing Vitrification Slow freezing Slow freezing Slow freezing
Dittrich. Cryopreserved ovarian tissue transplants. Fertil Steril 2015.
466
VOL. 103 NO. 2 / FEBRUARY 2015
Fertility and Sterility® case, the ovarian tissue was kept cool for >20 hours in a special transport medium, with a special cooling device, before it was cryopreserved. Although it is still a matter of debate whether transporting the tissue is acceptable or may lead to additional loss of follicles, this result shows that sufficient follicles survive the procedure for a successful pregnancy to result. A research group in Denmark has reported three live births after successful transportation of ovarian tissue for 4–5 hours before freezing (32). The present data show that overnight transportation of ovarian tissue can be offered as an opportunity for women to preserve fertility even when they do not have direct access to a tissue-processing unit. With this method, it is possible to provide fertility preservation for a larger number of women who may be at risk of induced ovarian failure and might otherwise have no access to ovarian cryopreservation. It is beyond dispute that frozen tissues—such as oocytes, sperm, or embryos—can be transported using special shipping containers. Transplantation of the tissue back into the patient can be therefore be carried out in specialized centers to which the tissue has been transported. It remains to be confirmed whether such specialized centers are better than centers in which only one or two ovarian tissue retransplantations per year are carried out. Hematological malignancies are the most common indications for cryopreserving ovarian tissue, and this was also observed in the present cohort, with 50% of the patients having Hodgkin's lymphoma (33). An important concern that still remains is the safety of ovarian tissue transplantation in patients who have cancer, in view of the potential risk that malignant cells present in the frozen tissue may lead to recurrence of the primary disease after transplantation. An option for cancer patients with a low risk of reimplantation of tumor cells might be to remove the transplanted tissue after a successful pregnancy or after a defined time limit. In the present cohort, it was decided to remove the transplanted tissue for safety reasons in the two patients who had had ovarian tumors. Another option for these patients might be maturation of follicles in vitro without transplantation. Total in vitro maturation of oocytes from cryopreserved ovarian tissue in humans has not yet been possible (34). However, it is quite conceivable that the technique may be successful in humans within the next few years (35). Immature oocytes can also be obtained during the processing of biopsied ovarian tissue. However—with the exception of figures from a few research groups—data on this procedure are still very limited (36). Another theoretical option is ovarian tissue xenotransplantation. Transplantation of frozen-thawed ovarian tissue into an animal host, with subsequent maturation and collection of oocytes, eliminates the possibility of cancer transmission and relapse, as the cancer cells are not able to penetrate the zona pellucida. However, fertilization and embryo development of human oocytes developed in animal hosts have not so far been investigated, owing to ethical issues (37). Another option for solving this problem might be artificial formation of ovaries with isolated follicles, as described by Luyckx et al. (38). Some additional remarks may be appropriate with regard to the present group of patients. Three of the women had anal VOL. 103 NO. 2 / FEBRUARY 2015
cancer and were therefore undergoing radiotherapy of the pelvis before retransplantation. The radiation dose was guidelines based and therefore over 50 Gy—implying an estimated organ dosage to the uterus of more than 30 Gy. None of the four women concerned conceived, despite having regular cycles over an extended period. One explanation for the lack of conception might be damage to the myometrium, with impaired function in the musculature and atrophic endometrium and impaired vasculature (39, 40). Although the data are limited, it may be suggested that women who wish to have children but who have been exposed to radiation are less likely to conceive. Another special feature of the present study is that patient counseling, tissue harvesting, cryopreservation, and retransplantation of the tissue and follow-up were all carried out in centers belonging to the FertiPROTEKT network (www.fertiprotekt.com). FertiPROTEKT—a collaboration among highly specialized cryobanks and reproductive medicine specialists—was founded in Germany in 2006 and offers a nationwide cryopreservation service (41). A further goal of the network is to carry out quality-control procedures. The network currently includes more than 100 centers in Germany, Switzerland, and Austria.
CONCLUSIONS Although retransplantation of cryopreserved ovarian tissue is still regarded as experimental, the technique has now achieved an innovative status. All patients requiring gonadotoxic treatment must receive information about the risk of infertility and about methods available for protecting fertility. Goals for the future should include optimizing the outcomes of transplantation, with improved tissue quality, reduced follicle loss, shorter transportation times, and longer periods of functionality in cryopreserved ovarian tissue.
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Objective: To report the results of 20 orthotopic retransplantations of cryopreserved ovarian tissue after cancer treatment. Design: Retrospective analysis. Setting: Tertiary gynecology department. Patient(s): Twenty patients with malignant disease: 11 with hematological malignancies (55%), four with breast cancer (20%), three with anal cancer (15%), and two with ovarian cancer (10%); the mean age before oncological treatment was 30.5 years. Intervention(s): Ovarian tissue was removed from patients in various centers in Germany in 2005–2009. All patients received chemotherapy and/or radiotherapy. Afterward, 17 patients had complete premature ovarian insufficiency, while three still showed some ovarian activity. Overnight transportation of tissue before freezing was necessary in eight cases. Cryopreservation followed slow freezing protocols in all cases. Retransplantation was performed at Erlangen University Hospital 3.75 years after extraction, on average. Thawed tissue was transplanted into a peritoneal pouch in the broad ligament region, below the tube, in 16 cases. Fragments were sutured both onto the remaining ovary and into a peritoneal pouch in four cases. Main Outcome Measure(s): Restoration of ovarian activity, pregnancy, birth. Result(s): Ovarian activity resumed in all patients except one. Seven patients conceived, with one miscarriage and four ongoing pregnancies. Four patients delivered healthy babies. One pregnancy and live birth after oocyte donation need to be considered separately. Conclusion(s): These data clearly demonstrate that preserving fertility by cryopreserving ovarian tissue is a successful and safe clinical option that can be considered for selected cancer patients. (Fertil SterilÒ 2015;103:462–8. Use your smartphone Ó2015 by American Society for Reproductive Medicine.) to scan this QR code Key Words: Fertility preservation, cryopreserved ovarian tissue, transplantation, live birth, and connect to the pregnancy Discuss: You can discuss this article with its authors and with other ASRM members at http:// fertstertforum.com/dittrichr-transplantations-ovarian-tissue/
M
odern treatments for oncological diseases have significantly increased survival rates in cancer patients, but chemotherapy and radiotherapy often lead to sterility due to destruction of the ovarian reserve. It is therefore important to advise patients who require gonadotoxic treatment on the options that are available for preserving their
fertility. The ability to have children of one's own is an important aspect of quality of life (1). A number of strategies have therefore been developed in recent years to enable these patients to have children using their own gametes (2). The individual patient's specific situation needs to be taken into account when fertility-preserving measures are being selected. Several
Received August 21, 2014; revised October 23, 2014; accepted October 28, 2014; published online December 6, 2014. R.D. has nothing to disclose. J.H. has nothing to disclose. L.L. has nothing to disclose. I.H. has nothing to disclose. M.W.B. has nothing to disclose. R.D. and J.H. should be considered similar in author order. €tsklinikum Erlangen, UniverReprint requests: Professor Ralf Dittrich, Ph.D., Frauenklinik, Universita €tsstrasse 21-23, D-91054 Erlangen, Germany (E-mail: [email protected]). sita Fertility and Sterility® Vol. 103, No. 2, February 2015 0015-0282/$36.00 Copyright ©2015 American Society for Reproductive Medicine, Published by Elsevier Inc. http://dx.doi.org/10.1016/j.fertnstert.2014.10.045 462
discussion forum for this article now.*
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options are currently available for preserving fertility, and cryopreservation of oocytes (3) and cryopreservation of embryos (4) are well-established procedures. The choice of which procedure is used depends on various parameters: the type and timing of gonadotoxic therapies, the type of cancer (with possible involvement of the ovaries), the patient's age, and her partner status. However, cryopreservation of ovarian tissue is a promising method for fertility preservation because it not only restores fertility after retransplantion but also is able to avoid hormone insufficiency (5). Ovarian tissue can be easily extracted laparoscopically, regardless of the current phase of the menstrual VOL. 103 NO. 2 / FEBRUARY 2015
Fertility and Sterility® cycle, without significantly delaying chemotherapy or radiotherapy. Patients who are younger than 35 years are particularly suitable, as the ovaries contain large numbers of oocytes in these patients and the chances of successful retransplantation are therefore greater. The ovarian tissue can be cryopreserved at specialized centers for reproductive medicine, and—depending on the oncological therapy required—can be retransplanted after a certain time if the patient has treatment-induced premature ovarian failure. Cryopreservation of ovarian tissue before oncological treatment is a promising method, since a large number of follicles survive the freeze-thaw procedure (6). Donnez et al. reported the first live birth after autotransplantation of human ovarian tissue in 2004 (7). In Germany, the first live birth after retransplantation of cryopreserved ovarian tissue was reported in 2012 (8). Using this technique, more than 25 live births have been reported worldwide to date (9). It can be expected that in the near future, more and more oncology patients who have survived cancer and have been cured of their disease will wish to undergo reimplantation of ovarian tissue. The purpose of the present review is to report on and discuss the techniques used and the results in 20 orthotopic retransplantations of cryopreserved tissue in a single center.
MATERIALS AND METHODS Patient Cohort Ovarian tissue was removed from 20 premenopausal women with various types of cancer; the patients' mean age was 30.5 years (range, 20–37 years). Three patients had anal cancer, 10 Hodgkin's lymphoma, one non-Hodgkin's lymphoma, four breast cancer, and two ovarian tumors (Table 1). At the time of retransplantation, their average age was 34.2 years. All patients received chemotherapy, including two patients with Hodgkin's lymphoma who received high-dose chemotherapy. The three patients with anal cancer received pelvic radiotherapy. Signs of residual hormone activity were present before retransplantation in only three of the patients. These patients did not wish to await a natural course and requested retransplantation. Values for antim€ ullerian hormone (AMH) levels after oncological treatment and before retransplantation were obtained in 12 of the women. All AMH levels were below detection limits. Tubal patency was tested in 14 women, and bilateral obstruction of the fallopian tubes was noted in four of them. The study design was approved by the Institutional Review Board (IRB) at Erlangen University Hospital, which had no concerns about it (IRB no. 522_14 Bc).
The ovarian tissue was harvested laparoscopically, with no use of electrocoagulation or thermal cutting to avoid damaging the tissue. In the present group of 20 patients, an average of two thirds of one ovary was removed. The tissue samples from each patient were analyzed histologically to prevent transmission of the underlying disease, and the presence and density of primordial follicles were documented. No complications such as bleeding or infections were observed.
Transportation before Freezing The ovarian tissue was removed, processed for cryopreservation, and stored at the Department of Gynecology and Obstetrics at Erlangen University Hospital in six cases so that no transportation was necessary. In another six cases, the tissue was frozen outside Erlangen, but within 6 hours after tissue collection, and in eight cases overnight transportation of the removed tissue was required. After laparoscopic removal, the tissues were sent in a special insulated transportation box (Z100037054; delta T Ltd.) at 4 C by same-day courier service or express overnight transportation for cryopreservation to a specialized processing unit (in either Bonn or Erlangen) (10). If the tissues were stored in an external cryobank, the tissue pieces were sent in shipping containers cooled with liquid nitrogen at 196 C to the Reproductive Medicine Laboratory in Erlangen.
Cryopreservation Procedure Slow freezing methods were used in all cases. Between 6 C and 40 C, the cooling rate was 0.3 C/min. Dimethyl sulfoxide was used as the cryoprotectant in 11 cases; the cryopreservation method used in these cases has been described elsewhere in detail (11). Ethylene glycol was used as the cryoprotectant in six cases (12). In brief, pieces of ovarian tissue (3 3 1 mm) were equilibrated for 30 minutes in 1.5 mol/L ethylene glycol and 0.1 mol/L sucrose in phosphatebuffered saline in 2 mL standard cryovials (Simport T3092A) on a tilting table on ice and then loaded into the open freezing system (CTE-920, CTE). The following cooling program was used: 2 C/min to 1 C, 0.5 C/min to 5 C, 0.3 C/min to 9.3 C, 10 minutes of soaking, then 0.3 C/min to 40 C and 10 C/min to 140 C, at which temperature the samples were plunged into liquid nitrogen at 196 C. Propanediol was used as the cryoprotectant in the remaining three cases.
Storage Extraction of Ovarian Tissue In the present study, ovarian tissue was extracted from 20 cancer patients in Germany between 2005 and 2009. The tissue was removed in the Department of Gynecology and Obstetrics at Erlangen University Hospital in six of these patients. In the 14 other patients, the tissue was removed in other centers for reproductive medicine in various cities in Germany (D€ usseldorf, Dresden, Munich, Hamburg, W€ urzburg, Cologne, and Essen). VOL. 103 NO. 2 / FEBRUARY 2015
The tissue was stored in highly specialized cryobanks in Germany that have expertise in cryopreserving ovarian tissue. The cryobanks are members of the FertiPROTEKT network, a collaborative group of centers in Germany, Austria, and Switzerland.
Thawing Thawing was fast in a warm water bath (37 C). The tissue fragments were released from the protective cryopreservation 463
Patient characteristics and results of autotransplantation.
Disease
Age at tissue removal
Age at TX
1 2 3
Anal cancer Anal cancer Hodgkin's lymphoma
28 34 20
30 38 23
CTX, RT pelvis CTX, RT pelvis CTX
4
Hodgkin's lymphoma
26
31
CTX
5
Hodgkin's lymphoma
27
32
6
Breast cancer
35
38
7
Breast cancer
28
31
8 9
Anal cancer Hodgkin's lymphoma
33 35
36 37
High-dose CTX, RT mediastinum Surgery. CTX, RT breast Surgery, CTX, RT breast RT pelvis CTX
10 11
Hodgkin's lymphoma Breast cancer
33 37
39 42
12
34
38
13 14
Non-Hodgkin's lymphoma Hodgkin's lymphoma Hodgkin's lymphoma
21 28
27 30
15
Breast cancer
34
38
16 17 18
Hodgkin's lymphoma Ovarian cancer Hodgkin's lymphoma
27 30 31
31 35 37
19 20
Dysgerminoma Hodgkin's lymphoma
31 33
35 37
Patient no.
Treatment
CTX, RT Surgery, CTX, RT breast CTX CTX CTX, RT mediastinum Surgery, CTX, RT thorax, axilla
VOL. 103 NO. 2 / FEBRUARY 2015
High-dose CTX CTX, surgery CTX, RT, mediastinum Surgery CTX
AMH level before TX
Overnight transport
– – <0.1
No No No
–
No
0.18
Yes
Assisted reproductive techniques
First sign of E2 or follicle (months after TX)
Residual ovarian activity before TX
Pelvic wall Pelvic wall Pelvic wall & ovary Pelvic wall & ovary Pelvic wall
Yes Yes Yes
3 4 5
No No No
No
5
No
No
2
No
Yes
4
No
No
2
No
3 4 month
No No
4 month
No Yes
Irregular menses Regular menses Lost of follow up
Graft site
Outcome
Irregular menses Regular menses Regular menses Live birth after oocyte donation Live birth October 2011
No Pregnant
<0.1
No
–
No
<0.1 –
Yes Yes
Pelvic wall & ovary Pelvic wall & ovary Pelvic wall Pelvic wall
No No
Pelvic wall Pelvic wall
Yes No; induced ovulation No No
<0.1
Yes
Pelvic wall
No
4 month
No
<0.01 <0.08
Yes No
Pelvic wall Pelvic wall
No No
2 month 6 month
No No
Ongoing pregnancy
Pregnant Lost of follow up
<0.1
Yes
Pelvic wall
No
5 month
No
Pregnant
<0.1 0.4 0.18
Miscarriage December 2013 Second pregnancy
No No Yes
Pelvic wall Pelvic wall Pelvic wall
No No No
4 month
No Yes Yes
– –
No Yes
Pelvic wall Pelvic wall
No Yes
No 2 month
0.05 –
No No
No wish to become pregnant, 1 IVF Live birth August 2012 Second pregnancy Live birth December 2013
Live birth July 2014
Ongoing pregnancy
Note: The mean age at tissue removal was 30.5 years. The mean age at transplantation was 34.2 years. The baby take home rate was 20%. CTX ¼ chemotherapy; E2 ¼ 17b-estradiol; RT ¼ radiotherapy; TX ¼ transplantation. Dittrich. Cryopreserved ovarian tissue transplants. Fertil Steril 2015.
Continuing hormonal activity
No Regular menses Irregular menses Still breast feeding
Regular menses Still breast feeding Regular menses No menses Pregnant
ORIGINAL ARTICLE: FERTILITY PRESERVATION
464
TABLE 1
Fertility and Sterility® medium in reverse order, with the addition of 0.25 mol/L sucrose.
FIGURE 1
Time of Autotransplantation Retransplantation was performed on average 3.7 years (range, 1–5 years) years after extraction of the tissue. The patients' mean age at the time of first retransplantation was 34.2 years (range, 27–42 years).
Autotransplantation Techniques The thawed tissue was transplanted into a 1.5 cm deep pouch of peritoneum in the region of the broad ligament, below the fallopian tube, in 16 cases. Fragments of ovarian tissue 1–3 mm in size were introduced into this pouch, and the pocket was closed with a Vicryl suture if necessary, if the opening was gaping. Fragments were sutured both onto the remaining ovary and also into a peritoneal pouch in four cases. For transplantation onto the remaining ovary, a longitudinal incision of approximately 1 cm was made in the surface of the remaining ovary, and the tissue pieces were then threaded onto a suture and fixed into the incision. The amount of transplanted tissue was approximately 20% of the surface of one ovary. Four patients requested a second transplantation, and two even requested a third retransplantation when the initially transplanted tissue lost function.
Follow-up after Retransplantation The first assessment of E2 and FSH was carried out in each patient 2 months after retransplantation. If the FSH level had decreased and E2 had increased, monitoring of the cycle with hormone measurements and ultrasound controls was initiated. Initial signs of an increase in the E2 level or follicle growth were documented in 16 women an average of 3–6 months after retransplantation. An absence of hormone activity has so far been observed in only one patient.
A typical ‘‘neo-ovary’’ that developed from the transplanted tissue in the pelvic wall. Dittrich. Cryopreserved ovarian tissue transplants. Fertil Steril 2015.
Pregnancies Seven women have so far become pregnant. The ovarian tissue was transplanted into the pelvic wall in six of these cases only, and in one further case additionally into the remaining ovary. In five cases, the ovarian tissue was also transported overnight before freezing. Six of these pregnancies were spontaneous and one followed IVF. In the case of IVF, the tissue was transplanted into the pelvic wall. Four children have been born in the meantime; one pregnancy ended with a miscarriage in gestational week 10, and two are ongoing. Two patients became pregnant a second time after spontaneous conception (no. 5 and no. 15). One additional pregnancy and live birth needs to be considered separately, as it developed after oocyte donation. The average age of the patients who became pregnant was 29.7 years (median, 30; minimum, 21; maximum, 35) at the time of tissue collection and 33.8 (median, 35; minimum, 27; maximum, 37) at the time of transplantation.
RESULTS Hormone Activity in the Transplanted Tissue
Outcome Regarding the Site of Transplantation
No surgical complications occurred in any cases during transplantation of the ovarian tissue. In 19 cases (95%), hormone activity in the ovary was observed at least in the form of an increase in serum E2 levels. Three of these patients had residual activity in the ovary before transplantation. These patients cannot therefore be included in calculations of the restoration of ovarian function. One patient showed no activity after transplantation. Restoration of ovarian function was therefore achieved in 94% of the patients. The mean time to the first increase in E2 (in patients who had no residual hormone activity at the time of retransplantation) was 3.7 months (median, 4 months; range, 2–6 months). Figure 1 shows a typical ‘‘neo-ovary’’ that developed from the transplanted tissue in the pelvic wall. The average duration of hormone activity in the tissue after transplantation is 1.7 years to date, and activity is still present in 13 patients. The longest period of hormonal activity observed so far is 62 months.
The tissue was transplanted into the pelvic wall in all cases, and in four cases additionally into the remaining ovary. In these four cases, one patient became pregnant and delivered a child (25%) and has also become pregnant for a second time in the meantime. In the 16 patients in whom the tissue was transplanted into the pelvic wall alone, seven pregnancies occurred (two in one patient) and three babies have been delivered (19%).
VOL. 103 NO. 2 / FEBRUARY 2015
Follow-up of Patients at Risk for Possible Relapse of Cancer Due to Ovarian Tissue Transplantation The group of patients included three with cancer types involving a certain risk of relapse: one with non-Hodgkin's lymphoma, one with a dysgerminoma, and one with ovarian cancer (Table 2). In the patient with a dysgerminoma, the transplanted tissue showed no hormone activity and was removed 11 months after transplantation to ensure that no 465
ORIGINAL ARTICLE: FERTILITY PRESERVATION
TABLE 2 Tumor entities (with classification) involving a certain risk of relapse for the patients. Patient no. 12 17 19
Cancer entity
Tumor classification
Non-Hodgkin's lymphoma Serous adenocarcinoma in the ovary Ovarian dysgerminoma
Highly malignant B-cell lymphoma, Ann Arbor Iva pT3c pN0 M0 G1, International Federation of Gynecology and Obstetrics IIIc rpT1a NX L0 V0 Pn0 R0
Dittrich. Cryopreserved ovarian tissue transplants. Fertil Steril 2015.
relapse would occur. The patient with ovarian cancer became pregnant, and removal of the ovarian tissue 6 weeks after birth is being considered. In addition, one patient with breast cancer had a local relapse, which is not regarded as having been caused by the retransplantation. All of the patients were informed that there is a certain risk for minimal residual disease and provided written informed consent. All of the transplanted tissue fragments were analyzed histologically for the presence or absence of malignant cells.
DISCUSSION After autotransplantation of cryopreserved ovarian tissue, 19 of the 20 patients showed ovarian function and four live births of healthy children occurred. These data demonstrate that cryopreservation of ovarian tissue and subsequent retransplantation is a successful option for preserving fertility in patients with malignancies; the results are consistent with those of other studies reporting a high level of success with regard to restoration of ovarian function and live birth. Donnez et al. reported in 2011 on 13 live births worldwide after retransplantation of cryopreserved ovarian tissue (13), and in 2013 on 24 live births after 60 retransplantations worldwide (9). Andersen et al. reported restoration of ovarian function in six patients, with live births of two healthy
babies (14). The first live birth in Germany was reported in 2011 (8). Table 3 summarizes all of the live births after retransplantation of cryopreserved ovarian tissue reported in the international literature to date (Pubmed search, 7–10, 13–29). To the best of our knowledge, 24 live births have so far been described; the pregnancy rate after autografting of cryopreserved tissue at orthotopic sites is estimated at approximately 30% (28), which was also observed in the present group of patients. This shows that cryopreservation of ovarian tissue and subsequent retransplantation is a promising method that enables women affected by cancer to have children after recovering from the disease. However, several questions still remain regarding cryopreservation and transplantation of ovarian tissue that have not yet been fully answered (30). Two different techniques of orthotopic autotransplantation have proved to be successful (7). The tissue can be either transplanted into or onto the remaining ovary or into a peritoneal pocket in the pelvic peritoneum of the ovarian fossa; both techniques have been successful (9). In this study, the ovarian tissues were retransplanted orthotopically into peritoneal pockets in the pelvic wall in all cases and additionally in four cases into the remaining ovary. In the present authors' view, transplantation into a peritoneal pocket provides a better blood supply than transplantation to a heterotopic site or onto the remaining ovary, and it is therefore associated with better follicle development and better functionality of the tissue (31). It also makes natural conception possible. Figure 1 shows a neo-ovary with growing follicles after transplantation into the ovarian fossa. In the present group of 20 patients, the tissue was frozen at the center in which surgery was carried out in 12 cases. In the other eight cases, the tissue was transported to a center specializing in cryopreservation. In contrast to the practice in Denmark (14), the tissue was transported overnight before being frozen in these eight cases. However, five of these eight patients became pregnant. This demonstrates that overnight transportation of ovarian tissue before freezing is possible. The first live birth in Germany originated from tissue that had been transported overnight before freezing (10). In that
TABLE 3 Published reports of live births following cryopreservation and retransplantation of ovarian tissue. Research group Donnez et al. Meirow et al. Andersen et al. Demeestere et al. Silber et al. Roux et al. S anchez-Serrano et al. Revel et al. García Rada et al. Revelli et al. Devi and Kumar Dittrich et al. Kawamura et al. Ernst et al. Callejo et al. Yang et al.
Cryopreservation procedure
Live births
Year
References
Slow freezing Slow freezing Slow freezing Slow freezing Slow freezing Slow freezing Slow freezing, oocyte vitrification Slow freezing Slow freezing Slow freezing
6 1 2 1 1 1 1 1 1 1 1 1 1 1 1 3
2004–2013 2008 2008 2010 2010 2010 2010 2011 2012 2013 2005 2012 2013 2010 2013 2011
(7, 9, 13, 15) (16) (14) (17) (18) (19) (20) (21) (22) (23) (24) (10) (25) (26) (27) (29)
Slow freezing Vitrification Slow freezing Slow freezing Slow freezing
Dittrich. Cryopreserved ovarian tissue transplants. Fertil Steril 2015.
466
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Fertility and Sterility® case, the ovarian tissue was kept cool for >20 hours in a special transport medium, with a special cooling device, before it was cryopreserved. Although it is still a matter of debate whether transporting the tissue is acceptable or may lead to additional loss of follicles, this result shows that sufficient follicles survive the procedure for a successful pregnancy to result. A research group in Denmark has reported three live births after successful transportation of ovarian tissue for 4–5 hours before freezing (32). The present data show that overnight transportation of ovarian tissue can be offered as an opportunity for women to preserve fertility even when they do not have direct access to a tissue-processing unit. With this method, it is possible to provide fertility preservation for a larger number of women who may be at risk of induced ovarian failure and might otherwise have no access to ovarian cryopreservation. It is beyond dispute that frozen tissues—such as oocytes, sperm, or embryos—can be transported using special shipping containers. Transplantation of the tissue back into the patient can be therefore be carried out in specialized centers to which the tissue has been transported. It remains to be confirmed whether such specialized centers are better than centers in which only one or two ovarian tissue retransplantations per year are carried out. Hematological malignancies are the most common indications for cryopreserving ovarian tissue, and this was also observed in the present cohort, with 50% of the patients having Hodgkin's lymphoma (33). An important concern that still remains is the safety of ovarian tissue transplantation in patients who have cancer, in view of the potential risk that malignant cells present in the frozen tissue may lead to recurrence of the primary disease after transplantation. An option for cancer patients with a low risk of reimplantation of tumor cells might be to remove the transplanted tissue after a successful pregnancy or after a defined time limit. In the present cohort, it was decided to remove the transplanted tissue for safety reasons in the two patients who had had ovarian tumors. Another option for these patients might be maturation of follicles in vitro without transplantation. Total in vitro maturation of oocytes from cryopreserved ovarian tissue in humans has not yet been possible (34). However, it is quite conceivable that the technique may be successful in humans within the next few years (35). Immature oocytes can also be obtained during the processing of biopsied ovarian tissue. However—with the exception of figures from a few research groups—data on this procedure are still very limited (36). Another theoretical option is ovarian tissue xenotransplantation. Transplantation of frozen-thawed ovarian tissue into an animal host, with subsequent maturation and collection of oocytes, eliminates the possibility of cancer transmission and relapse, as the cancer cells are not able to penetrate the zona pellucida. However, fertilization and embryo development of human oocytes developed in animal hosts have not so far been investigated, owing to ethical issues (37). Another option for solving this problem might be artificial formation of ovaries with isolated follicles, as described by Luyckx et al. (38). Some additional remarks may be appropriate with regard to the present group of patients. Three of the women had anal VOL. 103 NO. 2 / FEBRUARY 2015
cancer and were therefore undergoing radiotherapy of the pelvis before retransplantation. The radiation dose was guidelines based and therefore over 50 Gy—implying an estimated organ dosage to the uterus of more than 30 Gy. None of the four women concerned conceived, despite having regular cycles over an extended period. One explanation for the lack of conception might be damage to the myometrium, with impaired function in the musculature and atrophic endometrium and impaired vasculature (39, 40). Although the data are limited, it may be suggested that women who wish to have children but who have been exposed to radiation are less likely to conceive. Another special feature of the present study is that patient counseling, tissue harvesting, cryopreservation, and retransplantation of the tissue and follow-up were all carried out in centers belonging to the FertiPROTEKT network (www.fertiprotekt.com). FertiPROTEKT—a collaboration among highly specialized cryobanks and reproductive medicine specialists—was founded in Germany in 2006 and offers a nationwide cryopreservation service (41). A further goal of the network is to carry out quality-control procedures. The network currently includes more than 100 centers in Germany, Switzerland, and Austria.
CONCLUSIONS Although retransplantation of cryopreserved ovarian tissue is still regarded as experimental, the technique has now achieved an innovative status. All patients requiring gonadotoxic treatment must receive information about the risk of infertility and about methods available for protecting fertility. Goals for the future should include optimizing the outcomes of transplantation, with improved tissue quality, reduced follicle loss, shorter transportation times, and longer periods of functionality in cryopreserved ovarian tissue.
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