Follow-up of ovarian function post-chemotherapy following ovarian cryopreservation and transplantation

K.L.T. Schmidt1,2,4, C. Yding Andersen2, A. Loft1, A.G. Byskov2, E. Ernst3 and A. Nyboe Andersen1

1 The Fertility Clinic, section 4071 and 2 The Laboratory of Reproductive Biology, section 5712, Copenhagen University Hospital, Rigshospitalet, DK-2100 Copenhagen and 3 The Fertility Clinic, Skejby Sygehus, DK-8200 Aarhus N, Denmark

4 To whom correspondence should be addressed. E-mail: tryde{at}dadlnet.dk


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
BACKGROUND: The purpose of this study was to assess the ovarian function after treatment of a malignant disease in women who previously had cortical tissue from an entire ovary cryopreserved prior to chemotherapy, and to assess ovarian function after autotransplantation of cryopreserved ovarian tissue. All were treated with chemotherapeutic drugs with an estimated high risk of inducing ovarian failure. METHODS: Twenty-two women with breast cancer (n = 8), Hodgkin’s disease (n = 6), non-Hodgkin’s (n = 2), leukaemia (n = 5) or brain tumour (n = 1) underwent a clinical examination >18 months after cryopreservation. Three patients with premature ovarian failure had ovarian tissue autotransplanted orthotopically and heterotopically. Ovarian function was assessed by ultrasonography of the remaining ovary and hormone measurements. RESULTS: Nine of 22 women (41%) had sonographic and hormonal signs of ovarian failure with ovarian volumes <1.3 cm3, no antral follicles and high FSH levels (median 57.1 IU/l). Thirteen of the 22 women (59%) still menstruated and 10 had a seemingly normal ovarian function, with a median ovarian volume of 6.8 cm3, a median number of antral follicles of six, FSH <15 IU/l and normal estradiol levels. All three patients with autotransplanted ovarian tissue regained ovarian function as confirmed by return of menses, follicles on ultrasonography and normalized hormone levels. Two embryos were created from the crypreserved tissue after IVF. CONCLUSIONS: Treatment with bone-marrow transplantation and/or high doses of alkylating agents led to ovarian failure in all patients. Autotransplantation of ovarian tissue led to return of ovarian function.

Key words: autotransplantation/cancer survivors/cryopreservation/IVF/premature ovarian failure


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
One of the side-effects of chemo- and/or radiation therapy in young female cancer patients is the risk of loss of ovarian function and thus fertility. As the survival rates of many cancer forms have increased dramatically during the past decades (McVie, 1999Go), so has the number of long-term survivors. However, many of these experience premature ovarian failure (POF) owing to the irreversible loss of ovarian follicles caused by anti-neoplastic treatment. This unwanted side-effect of an otherwise efficient cancer treatment may now be alleviated by cryopreserving ovarian tissue and thus preserving the fertility of young female patients. Many clinics now offer this to patients at risk of POF (Donnez and Bassil, 1998Go; Meirow et al., 1999Go; Radford et al., 2001Go; Hreinsson et al., 2002Go; Poirot et al., 2002Go; Demeestere et al., 2003Go; Schmidt et al., 2003Go; Oktay et al., 2004Go).

Although cryopreservation of ovarian tissue is still not an established method to guarantee return of ovarian function, autotransplantation of cryopreserved ovarian tissue has recently produced some promising results. Orthotopic transplantation of cryopreserved/thawed cortical fragments has led to the development of functional follicles and endogenous hormone production in patients with chemotherapy-induced POF (Radford et al., 2001Go; Schmidt et al., 2004Go). IVF in patients with autotransplanted cryopreserved ovarian tissue has resulted in the aspiration of mature metaphase II oocytes (Oktay et al., 2004Go; Schmidt et al., 2004Go) and embryos have been created following ICSI (Oktay et al., 2004Go). Just recently, the first baby was born after autotransplantation of cryopreserved ovarian tissue in a woman cured of Hodgkin’s disease (HD) (Donnez and Dolmans, 2004Go).

However, little is known about the degree of POF following anti-neoplastic treatment in women with cryopreserved ovarian tissue. Despite the fact that cryopreservation of ovarian tissue is only offered to women at high risk of POF, it is reasonable to assume that only a percentage of these women will actually develop ovarian failure after treatment, dependent on the age of the patient, and the type of drugs used and at what doses.

The purpose of this study was to investigate ovarian function in a group of women who had previously undergone unilateral ovariectomy with subsequent cryopreservation of the cortex prior to treatment for cancer, and to report the results of autotransplantation of some of this tissue in three of the women.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Patients
Since August 1999 cryopreservation of ovarian tissue has been performed at the University Hospital of Copenhagen in 102 girls and young women at risk of losing their fertility due to a disease requiring the use of gonadotoxic drugs for treatment. All patients had an entire ovary removed laparoscopically, the cortex was isolated, cut into fragments of 5 x 5 x 1 mm and cryopreserved as described previously (Schmidt et al., 2003Go). Other methods to preserve the fertility potential, such as cryopreservation of embryos or oocytes, were not offered to these patients.

The inclusion criteria for entering the present study were as follows: the cryopreservation procedure should have taken place >18 months before entering the study and the age of the patient at study inclusion should be >18 years. Twenty-five patients fulfilled these criteria, and of these 22 agreed to participate, two patients were too sick at study inclusion to participate and one patient did not want to participate. Another four patients had died from their disease, but would have fulfilled the criteria had they been alive at the time of the study.

The original study on cryopreservation of ovarian tissue has been approved by the Ethical Comitee for Copenhagen and Frederiksberg.

Clinical control of ovarian function
All 22 women were examined by the same physician (K.L.T.Schmidt). They were interviewed about their menstrual cycles, use of oral contraceptives or hormone replacement therapy (HRT), the occurrence of a pregnancy after cryopreservation of their ovary and the result of this pregnancy, and finally asked about the presence of any symptoms associated with menopause (hot flashes, dryness of vagina, etc.)

Hospital records were tracked to obtain information on the exact treatment given for the cancer disease.

To assess ovarian function, a transvaginal ultrasonography of the remaining ovary was performed and blood was drawn for analysis of FSH, LH, estradiol and inhibin A and B. The ultrasonographic examination included measurement of ovarian volume by measuring the length and height of the ovary in the sagittal section and the width of the ovary in the transverse section. The volume of the ovary was then calculated using the formula of an ellipse: d1 x d2 x d3 x 0.523. Additionally, the number of antral follicles was counted.

FSH and LH were measured using the AxSym® system (Abbott Laboratories, IL, USA), estradiol was measured using a Pantex (E2) 125I kit (Pantex, Santa Monica, CA, USA). An FSH level <15 IU/l was considered normal. Inhibin A and B were measured in duplicate in double antibody enzyme immunometric assays (Oxford Bio-Innovation Ltd, Oxford, UK). The detection limit was 7 pg/ml for inhibin A and 20 pg/ml for inhibin B. Patients were examined at random days of their menstrual cycle, since a fixed time of examination at day 2–5 of the menstrual cycle was not possible in this group of patients.

A total of three patients [two patients with HD, cases 9 and 10; and one patient with non-Hodgkin’s lymphoma (NHL), case 16] who were all menopausal at the time of follow-up decided to have some of their cryopreserved ovarian tissue reimplanted.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Diagnoses, treatment, age at diagnosis, current medication and menstrual patterns are given for each patient in Table I, as well as the results of the clinical examination.


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Table I. Clinical data and post-treatment fertility in 22 patients with cryopreserved ovarian tissue

 

Patient characteristics
The diagnoses of the 22 patients were as follows: eight patients suffered from breast cancer, six from HD, two from NHL, five from leukaemia and one patient had a brain tumour (corpus pineale). Additionally, one patient with HD and three patients with metastazising breast cancer had died from their disease after cryopreservation of ovarian tissue. Apart from the patient with a brain tumour metastazising to the spinal cord, who received radiation therapy as the only treatment, all patients received chemotherapy. The different combinations of chemotherapy given all involved the use of at least one alkylating agent. Six patients received bone marrow transplantation (BMT) following conditioning with total body irradiation (TBI) and/or high-dose chemotherapy with alkylating agents, and one patient received autologous stem cell transplantation. Seven of the patients with breast cancer were on anti-estrogenic medication with tamoxifen. Seven patients took HRT; six of these had undergone BMT and one high-dose chemotherapy with alkylating agents due to relapse of HD. Age at diagnosis ranged from 17–36 years (median 26) and time of follow-up between 18–45 months (median 26) after cryopreservation.

Menstrual pattern and menopausal status
Thirteen patients still had spontaneous menstrual cycles; of these three had irregular cycles whereas the rest had regular menstrual cycles defined as 21–35 days between each cycle day 1. In nine patients menstruations ceased after treatment and seven of these were on HRT. The two patients who were not on HRT reported the presence of hot flashes and dryness of the vagina, symptoms that had also been present in the other seven patients before they started to take HRT. None of the patients with existing menstrual cycles reported any hot flashes.

Ultrasonographic findings
Two patients (cases 14 and 21) were virgins at the time of follow-up and thus a transvaginal ultrasonography was not performed. A transabdominal ultrasonography was tried, but failed to visualize the remaining ovary. In the rest of the patients ovarian volumes ranged from 0.3 to 13.3 cm3 and the number of antral follicles from zero to 12.

Hormone measurements
Blood was drawn at the same day as the ultrasonography in all but one patient (case 11), who preferred to have the blood tests done 2 months later (at which time she was in very early pregnancy, explaining the high estradiol level) coinciding with her clinical follow-up at the haematological department. Estradiol levels ranged from <0.04 (undetectable) to 3.75 nmol/l. FSH ranged from <0.2 to 95.9 IU/l and LH from <0.2 to 73.1. Inhibin B was undetectable (<20 pg/ml) in 15 patients, in three patients the inhibin data are missing and in the remaining four patients inhibin B ranged from 21 to 167 pg/ml. Inhibin A ranged from <7 to 89 pg/ml.

Fertility after cancer treatment
One patient with breast cancer (case 2) had given birth to a healthy baby prior to the clinical follow-up. Immediately after delivery she resumed anti-estrogenic medication. After the follow-up, against the advice of her oncologists, she became pregnant again and delivered a healthy baby by Caesarian section 9 months later. During the operation multiple metastases were found in the pelvis, spine and abdomen. High-dose chemotherapy treatment was initiated. Another patient with HD (case 11) became pregnant after the follow-up and delivered a healthy baby 9 months later. One patient with NHL (case 15) became pregnant after the follow-up, but had the pregnancy terminated for personal reasons.

Autotransplantation of ovarian tissue
Table II gives the results of the autotransplantation procedure. Patient 16 had six pieces of cryopreserved/thawed ovarian tissue autotransplanted to the remaining ovary and subsequently underwent IVF with down-regulation in the long protocol with Suprefact (buserelin; Hoechst, Copenhagen, Denmark) and stimulation with Menopur 450 IU/day (Menotropin; Ferring, Copenhagen, Denmark) for a total of 17 days, giving a total dose of 7650 IU, resulting in the aspiration of a mature metaphase II oocyte, as published earlier (Schmidt et al., 2004Go). However, a later attempt at IVF, this time with stimulation with Gonal-F® (Serono Nordic, Copenhagen, Denmark) 450 IU/l for 9 days, resulted in the development of a two-cell stage embryo 48 h after ICSI (Figure 1). This embryo was replaced, but the woman did not become pregnant. Today, 22 months after autotransplantation she still has functional ovarian tissue and an irregular menstrual cycle ranging from 20–35 days. However, for private reasons she has decided to pause with further IVF attempts.


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Table II. Clinical data and results of autotransplantation of cryopreserved/thawed ovarian tissue in three cancer survivors

 


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Figure 1. The oocyte immediately after aspiration (A), after denudation (B), the now fertilized oocyte with two pronuclei 24 h after ICSI (C) and a two-cell stage embryo 48 h after ICSI (D).

 

Patient 9 became amenorrhoic after receiving BMT due to relapse of HD. She had ovarian tissue cryopreserved in December 2001 prior to BMT. In May 2004, on the patient’s request, pieces of thawed ovarian tissue were autotransplanted. Four pieces were transplanted to the remaining ovary, four pieces corresponding to the site of the removed ovary in a subperitoneal pocket, and four pieces were placed laparoscopically from the inside in a small pocket created in the anterior abdominal wall approximately between the umbilicus and the pubic bone. After 19 weeks a transvaginal ultrasound examination demonstrated a follicle in the remaining ovary (Figure 2) and menstruation returned 22 weeks after autotransplantation. Table II gives the hormonal levels after the appearance of follicles on ultrasonography. After this she has had irregular menstruations with intervals from 4 to 8 weeks. Ten months post-operatively a follicle appeared corresponding to the abdominal wall (Figure 2). IVF was attempted when the follicle had reached a size of 20 mm, but the aspirate did not contain an oocyte or granulosa cells. A serum progesterone analysis a week later was 1.7 nmol/l, suggesting that ovulation had not occurred.



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Figure 2. A 15-mm diameter follicle in the remaining ovary 19 weeks post-operatively (A) and the endometrium at the same time (B). A follicle corresponding to the abdominal transplantation site 10 months post-operatively (C).

 

Patient 10 became amennorhoic during treatment with high-dose chemotherapy with alkylating agents due to relapse of HD in the beginning of 2002. Just prior to this she had ovarian tissue cryopreserved. In June 2004, on the patient’s request, 12 pieces of thawed ovarian tissue were autotransplanted in the exact same manner as described for patient 9. After 18 weeks follicles were present in the remaining ovary on ultrasonography and a spontaneous bleeding occurred after 25 weeks. In February 2005 she underwent IVF in the long protocol with Synarela® nasal spray (Pharmacia, Copenhagen, Denmark) and 450 IE/day of Gonal-F (Serono Nordic) for a total of 17 days, giving a total dose of 7650 IE. Three follicles were aspirated from the orthotopically replaced ovarian tissue in the remaining ovary, two oocytes were obtained [one metaphase II (MPII) and one germinal vesicle (GV)] and one developed into a four-cell stage embryo after 48 h of culture. The embryo was replaced, but pregnancy was not achieved.

In all three cases autotransplantation was performed after consultation with the patient’s haematologist.


    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The risk of loss of ovarian function is a well-known side-effect of anti-neoplastic treatment. Several studies have demonstrated that certain types of chemotherapy as well as abdominal radiation therapy may harm the gonads of both males and females (Wallace et al., 1989Go; Reichmann and Green, 1994Go; Bines et al., 1996Go; Blumenfeld et al., 1999Go; Bath et al., 2002Go; Presti et al., 2004Go). The alkylating agents, such as cyclophosphamide or procarbazine, are particularly gonadotoxic with a higher risk of inducing POF than other chemotherapeutics (Bokemeyer et al., 1994Go; Meirow and Nugent, 2001Go). Today, many young patients faced with a potentially gonadotoxic treatment are offered the option to have ovarian tissue or, alternatively, embryos or oocytes, cryopreserved. To our knowledge this is the first study to assess the ovarian function post-treatment in women with cryopreserved ovarian tissue due to a malignant disease.

Twenty-two patients were included in our study. Of these, nine showed sonographic and biochemical signs of POF with very small ovarian volumes (range 0.3–1.3 cm3), no antral follicles, elevated FSH levels >20 IU/l (median 57.1 IU/l), elevated LH levels >30 IU/l (median 42.6 IU/l) and undetectable inhibin B (<20 pg/ml) and A (<7 pg/ml). All of the nine patients had ceased menstruating either during the course of their treatment or soon after the end of treatment, although five of seven patients currently using HRT were menstruating on this treatment.

Thirteen of the patients were still menstruating at the time of the clinical follow-up (median time of follow-up 26 months, range 19–45). Ten reported regular menstrual cycles of between 21 and 35 days in cycle length, whereas three had irregular menstrual cycles. Two of these also had elevated FSH levels (55 and 27.7 IU/l) and few antral follicles on ultrasonography, suggesting a diminished ovarian reserve. One of the patients with a regular cycle had an elevated FSH level of 18.7 IU/l, also suggesting a diminished ovarian reserve, but in the remaining 10 patients transvaginal ultrasonography of the remaining ovary as well as hormone measurements suggested a normal ovarian function >18 months after treatment of a malignant disease.

When looking at the different diagnoses in this cohort of patients and their relation to POF it is clear to see that all of the patients with leukaemia (n = 5) experienced POF after treatment, as did three out of six patients with HD and one patient with NHL. Not surprisingly, six of these patients had received BMT, one patient had received autologous stem cell transplantation, one patient high-dose chemotherapy with alkylating agents and only one patient had received first-line combination chemotherapy for NHL.

The risk of developing POF following BMT is well known. One study found that only five of 63 cancer patients undergoing BMT kept their ovarian function post-BMT (Meirow, 2000Go). Sanders and co-workers evaluated the ovarian function in 187 women between 13 and 49 years of age and found that BMT following conditioning with chemotherapy only induced POF in 11/16 women >26 years of age and in 0/27 women <26 years of age, whilst conditioning with both chemotherapy and TBI induced POF in 135/144 women (Sanders et al., 1988Go). Spinelli and co-workers concluded in their study that ovarian failure is common following TBI and that gonadal recovery was dependent on age and menarchal status at the time of BMT (Spinelli et al., 1994Go). In our study all patients receiving BMT experienced POF, a finding very much in accordance with previous studies.

Surprisingly, we found that all of the women with breast cancer (n = 8) were still menstruating, even though all but one took anti-estrogenic medication (tamoxifen). These women had all been treated with either CEF or CMF, both protocols including cyclophosphamide, an alkylating agent, in doses between 3 and 8 g in total. Five of these women had a seemingly normal ovarian function, whereas three did show some impairment of ovarian function (cases 3, 4 and 7). In a review of the literature Bines and colleagues found that, on average, chemotherapy-related amenorrhea in premenopausal breast cancer survivors was seen in 68% of the patients, dependent on age, and type of drug used and cumulative dose (Bines et al., 1996Go). An earlier study found that 17 of 18 patients treated with cyclophosphamide only for breast cancer developed POF and the dose at which amenorrhea occurred was 5.2 g in women in their forties, 9.3 g in women in their thirties and 20.4 g in women in their twenties (Koyama et al., 1977Go). The women with breast cancer in our study were young (mean age 29.1 years) and they all received a total dose of cyclophosphamide of <10 g, which could explain why we did not see POF in any of these patients, although three of them did show some impairment of the ovarian function. Additionally, five of the women did have undetectable inhibin B levels, suggesting some degree of reduction in the follicle number.

The three patients in this study who only received first-line chemotherapy with adriamycine, bleomycin, vincristine and decarbazine (ABVD) for HD had a seemingly normal ovarian function 19–26 months after cryopreservation. This supports the findings of previous studies which concluded that treatment with ABVD for HD seems to be less gonadotoxic than treatment with mechlorethamine, oncovin, prednisone and procarbazine (MOPP) (Santoro et al., 1987Go; Clark et al., 1995Go; Brusamolino et al., 2000Go).

The methods employed in the present study to assess ovarian function included hormone level analyses and ultrasonography of the remaining ovary. Concentrations of FSH, LH, estradiol and inhibins are good predictors of ovarian reserve (Ahmed-Ebbiary et al., 1994Go; Groome et al., 1996Go; Welt et al., 1999Go; van Zonneveld et al., 2001Go), as are measurements of ovarian volumes (Lass and Brinsden, 1999Go; Pavlik et al., 2000Go; Flaws et al., 2001Go) and antral follicle counts (Scheffer et al., 1999Go; Flaws et al., 2001Go). HRT is known to decrease endogenous serum gonadotropins significantly (Casson et al., 1997Go), but nevertheless, all but one of the patients on HRT in this study still had FSH levels >15 IU/l.

In most previous studies on ovarian function after treatment of a malignant disease the women have two intact ovaries. In our study, however, all women only have a single ovary because of cryopreservation of the other. Little is known about fertility and age at menopause in women with a single ovary. In a review, Lass concluded that women with a single ovary did not in general have a reduced fertility potential to conceive, either naturally or via IVF treatment (Lass, 1999Go), while other studies have shown a trend towards younger age at menopause and higher FSH levels in women with only one ovary (Cramer et al., 1995Go; Cooper and Thorp, 1999Go). Demeestere and colleagues reported experience with 19 patients undergoing cryopreservation of an entire ovary (n = 16), both ovaries (n = 1) or ovarian biopsies (n = 2) and found that nine patients had POF after treatment (two of these had undergone bilateral oophorectomies), five patients had intact ovarian function, two patients had died and in three patients the ovarian function was unknown, although it is not mentioned how the ovarian function was assessed (Demeestere et al., 2003Go).

In the present study 10 of 22 patients had a seemingly normal ovarian function, despite the fact that they only had a single ovary and had undergone treatment with alkylating agents, whereas three of 22 patients showed some impairment of ovarian function. What is uncertain though, is the time at which these patients will enter menopause. Studies on female childhood cancer survivors have shown that although these patients may be cycling regularly they have significantly smaller ovarian volumes (P < 0.001) and lower antral follicle counts (P < 0.001) as compared with healthy age-matched controls, suggesting some impairment of ovarian function (Larsen et al., 2003Go). Hopefully, further long-term follow-up studies on patients like those in the present study will help to clarify this question.

Three of the women with POF following treatment had some of their cryopreserved ovarian tissue autotransplanted in an effort to restore ovarian function. One patient had the tissue transplanted orthotopically and two patients both orthotopically and heterotopically. In all patients ovarian function returned between 8 and 19 weeks post-operatively and it is still functioning today after 12–24 months. Other groups have described the return of ovarian function in women with orthotopically transplanted cryopreserved ovarian tissue (Oktay and Karlikaya, 2000Go; Radford et al., 2001Go; Donnez and Dolmans, 2004Go) as well as heterotopically transplanted cryopreserved ovarian tissue (Callejo et al., 2001Go; Oktay et al., 2004Go). Because of a pregnancy wish, IVF was performed in all three women after return of their ovarian function, resulting in the aspiration of a total of three MPII oocytes and one GV oocyte, and the development of two embryos (a two-cell and a four-cell). Only once previously has the development of an embryo resulting from cryopreserved, autotransplanted ovarian tissue been reported in the literature, when Oktay and co-workers reported on the development of a four-cell embryo after ICSI in a breast cancer survivor (Oktay et al., 2004Go).

Collectively, these studies confirm that autotransplantation of cryopreserved ovarian tissue can lead to resumption of follicular growth, endogenous hormone production and possibly pregnancies, either spontaneously occurring or after IVF. Additionally, the results of this study showed that patients treated with BMT, autologous stem cell transplantation and/or high doses of alkylating agents experienced POF in all cases, whereas patients treated with first-line combination chemotherapy in ‘standard’ protocols in this study had some degree of ovarian function at the time of follow-up. But of course, the number of patients and the study length is too small to draw any conclusions on the risk of developing POF after standard treatments. Certainly, tools better at predicting patients at high risk of losing their fertility due to a given treatment are needed and hopefully, further long-term studies on higher numbers of patients and at longer follow-up periods will help us in that direction.


    References
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
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Submitted on April 2, 2005; resubmitted on July 6, 2005; accepted on July 15, 2005.





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