Reproductive BioMedicine Online (2014) 29, 722–728

w w w. s c i e n c e d i r e c t . c o m w w w. r b m o n l i n e . c o m

ARTICLE

Obstetric outcome after oocyte vitrification and warming for fertility preservation in women with cancer Maria Martinez a, Susana Rabadan a, Javier Domingo b, Ana Cobo c, Antonio Pellicer c, Juan A Garcia-Velasco a,* a

IVI-Madrid, Rey Juan Carlos University, Av. del Talgo 68, 28023 Madrid, Spain; b IVI-Las Palmas, Av. Juan Carlos I, 17 35019, Las Palmas de Gran Canaria, Spain; c IVI-Valencia, Plaza de la Policía Local, 3 46025 Valencia, Spain * Corresponding author.

E-mail address: [email protected] (JA Garcia-Velasco). Maria Martinez studied medicine and specialized in obstetrics and gynaecology in Madrid, Spain. She received her initial training at Assisted Reproduction in IVI Madrid and at IVI Valencia, and has been working at IVI Madrid since 2007. She specializes in fertility preservation in oncology patients.

Abstract Obstetric outcome of first pregnancies achieved after vitrification and warming oocytes from women being treated for cancer

was evaluated. Of a total of 493 women who consulted for fertility preservation, 357 had their oocytes cryopreserved after being diagnosed with cancer, and 11 returned after being cured for assisted reproduction treatments (eight had breast cancer, one Hodgkin lymphoma, one endometrial adenocarcinoma, and one thyroid cancer). The oocyte survival rate was 92.3%, the fertilization rate was 76.6%, and the mean number of embryos transferred was 1.8 ± 0.7. Beta-human chorionic gonadotropin was detected in seven out of the 11 embryo transfers carried out. Four ongoing pregnancies were achieved and delivered at term with normal fetal weight and no major or minor malformations. Women diagnosed with cancer who have their eggs cryopreserved before anti-cancer treatment have good assisted reproductive technology performance and good perinatal outcomes. Cryopreservation of oocytes seems to be a good alternative for fertility preservation in these women. © 2014 Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved. KEYWORDS: fertility preservation, oocyte, perinatal outcome, vitrification

http://dx.doi.org/10.1016/j.rbmo.2014.09.002 1472-6483/© 2014 Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved.

Obstetric outcome after oocyte vitrification and warming

Introduction Cancer is a disease with a high prevalence and a tremendous affect on our society. Until recently, it was considered a long-term incurable disease. In the past decades, survival rates of many different oncological diseases have drastically improved. Patients and physicians now focus on surviving the disease, and also on quality of life after survival, such as fertility. The incidence of breast cancer in the USA is increasing in women aged less than 40 years, currently representing 7% of cases; however, survival rates are greater than 70% (Del Mastro et al., 2011; Linet et al., 1999; Rodríguez-Wallberg and Oktay, 2010; Sonmezer and Oktay, 2006; Surveillance, Epidemiology and End Results Program, 2013, 2006. USA: Division of Cancer Control and Population Sciences, National Cancer Institute, 2013). Around 250,000 cancer survivors are women between the ages of 20 and 39 years (Linet et al., 1999; National Cancer Institute, 2006), of which 42% will develop premature ovarian failure (Eskander et al., 2011; Larsen et al., 2003). Therefore, the effect of radiotherapy and chemotherapy on the gonads and the uterus is crucial to understanding the future fertility potential of these patients. Fertility preservation is a new area in reproductive medicine. Cryopreservation of oocytes or ovarian tissue gives oncological patients at high risk of becoming infertile after their treatment the possibility of becoming pregnant with their own gametes. Different fertility preservation strategies have been described, ranging from surgical ovarian transposition to ovarian quiescence with gonadotrophin-releasing hormone (GnRH) agonists, in-vitro oocyte maturation, freezing of ovarian tissue, or oocyte and embryo vitrification (Donnez and Dolmans, 2013). Among the different alternatives, cryopreservation of eggs has been extremely successful, with excellent survival rates and similar rates of fertilization, implantation and pregnancy as fresh oocytes in many different IVF indications (Cobo et al., 2008, 2010; Garcia-Velasco et al., 2013; Rienzi et al., 2012). Oocyte cryopreservation is significantly simpler and does not require a laparoscopic approach under general anaesthesia. Another advantage is that oocyte cryopreservation can be applied in women without a partner, and allows avoidance of ethical and legal complications, which are often related to embryo cryopreservation. Since the American Society of Clinical Oncology (Loren et al., 2013) and the Practice Committees of the American Society for Reproductive Medicine (2013) decided that this procedure is not experimental – unlike freezing ovarian tissue (Loren et al., 2013) – many different centers throughout the world are offering oocyte vitrification as an option for fertility preservation in women with cancer. In this study, the first results are presented of patients who had oocytes vitrified for oncological reasons, and who, after being treated for their condition and realizing that their fertility was compromised, returned to have their oocytes warmed to try to achieve a pregnancy.

Materials and methods Between May 2007 and November 2012, 493 women were selected for inclusion in our fertility preservation programme

723 for women with cancer. The mean age of patients was 31.9 years (range 15–43 years). All had recently been diagnosed with cancer and were about to start oncological treatment with chemotherapy, radiotherapy, or both. In all cases, written informed consent was obtained from the patient as well as authorization from the clinical oncologist to proceed with ovarian stimulation. If there was disagreement with the oncologist, the procedure was not carried out. This study was exempt from Institutional Review Board as existing information had been recorded in such a manner that participants could not be identified directly or through identifiers to investigators. The most frequent indication for fertility preservation in our programme was breast cancer (67%) followed by Hodgkin lymphoma (11%). Most patients opted for oocyte vitrification to preserve their fertility (357/493 [72.4%]). A total of 375 controlled ovarian stimulation cycles were carried out. The remaining either had their ovarian tissue frozen or decided not to have either their oocytes or ovarian tissue cryopreserved.

Ovarian stimulation protocol In non-hormone-dependent cancers, an ovarian stimulation protocol was started with 150–225 IU/day of recombinant FSH (Gonal F; Merck-Serono) on day 2 or 3 of a spontaneous cycle under GnRH antagonist protocol (Domingo et al., 2012). In ‘hormone-dependent’ cancer patients (i.e. all breast, ovarian, and endometrial cancers irrespective of the hormone receptor status), aromatase inhibitors were used for ovarian stimulation. Letrozole 5 mg/day (Femara 2.5 mg; Novartis, Spain) was administered orally starting on the second or third day of a spontaneous cycle until the day of triggering and then until the first day of menstruation after oocyte retrieval. After 2 days of letrozole administration, 150–225 IU/day of recombinant FSH (Gonal F; Merck-Serono) was added. A GnRH antagonist 0.25 mg/day (Cetrotide; Merck-Serono) was administered when the leading follicle reached 14 mm, and final oocyte maturation was triggered with 0.2 mg of the GnRH agonist, triptorelin (Decapeptyl 0.1; Ipsen-Pharma, Spain) as soon as two follicles were 20 mm or greater. No complications or moderate or severe side-effects were noted.

Oocyte vitrification and warming The Cryotop method was used for oocyte vitrification as described by Kuwayama et al. (2005) with minimal modifications. Oocytes were denuded by enzymatic means 2 h after ovum retrieval. They were then equilibrated at room temperature for 15 min in 7.5% (v/v) ethylene glycol plus 7.5% dimethylsulphoxide in tissue culture medium (TCM) 199 plus 20% synthetic serum substitute (SSS) (Kitazato, Tokyo, Japan). After 12 min, the oocytes were checked for rehydration. If complete, the oocytes were subjected to the vitrification step. Oocytes were then placed in vitrification solution containing 15% ethylene glycol plus 15% dimethylsulphoxide plus 0.5 M sucrose. After 1 min in this solution, the oocytes were placed on a Cryotop strip and immediately submerged in liquid nitrogen. No more than four oocytes per Cryotop were loaded. When the patient was ready to attempt to conceive, all

724

M Martinez et al. Table 1

Cycle outcome.

Age, years (mean ± SD) (range) Number of metaphase II oocytes vitrified per patient (mean ± SD) Oocyte survival, n/N (%) Two pronuclei fertilization, n/N (%) Number of embryos transferred (mean ± SD) Number of good embryos obtained (transferred + cryopreserved) (mean ± SD) Implantation rate, n/N (%) Clinical pregnancy rate, n/N (%) Ongoing pregnancy rate, n/N (%) Multiple pregnancy rate Congenital birth defects Pregnancy complications

vitrified oocytes were warmed. For warming, the Cryotop was removed from the liquid nitrogen and warmed in 1.0 M sucrose in TCM 199 plus 20% SSS at 37°C. Any manipulation of the oocytes was avoided at this point. After 1 min, the oocytes were placed in 0.5 M sucrose in medium 199 plus 20% SSS at room temperature for 3 min, with no additional manipulations. Finally, one 5-min wash followed by one 1-min wash was carried out with TCM 199 plus 20% SSS at room temperature before incubating the oocytes in routine culture media for 2 h before intracytoplasmic sperm injection (ICSI).

Embryo transfer Embryos were selected for transfer strictly according to their morphological appearance. Comparative genomic hybridization array pre-genetic screening was offered to women of advanced maternal age (>38 years), although only two patients opted for it. Surplus embryos suitable for additional cryopreservation were vitrified after the Cryotop protocol for embryos (Rienzi et al., 2012). A hormonal replacement therapy protocol was used to prepare the endometrium in five out of 12 cycles. Women with ovarian function but irregular cycles were first down-regulated in the luteal phase with a singledose of GnRH-agonist depot (Decapeptyl 3.75 mg; Ipsen Pharma; Procrin depot 3.75; Abbott; Gonapeptyl 3.75 mg; Ferring Pharmaceuticals) and received 6 mg of oral oestradiol valerate (Progynova; Schering Spain) after menses. About 10–15 days after starting oestradiol valerate, serum oestradiol levels and endometrial thickness were determined. If the oestradiol levels were over 150 pg/ml and the endometrial thickness was 7 mm or greater and a triple layer endometrial pattern was confirmed, administration of micronized progesterone (800 mg/day, vaginally) (Progeffik; Effik Laboratories, Spain) was started. Embryo transfer was scheduled according to the developmental stage of the embryo. Vaginal progesterone was administered starting from the day of fertilization until 12 weeks of gestation, or was discontinued if a pregnancy was not achieved. If patients had regular menstrual cycles and did not concieve for 6 months after trying, natural cycles were opted for. This occurred in seven out of 12 cycles, and follicular growth was monitored. When the leading follicle reached a mean diameter of 18 mm, 250 µg recombinant human chorionic gonadotropin (rhCG) (Ovitrelle, Merck-Serono) was administered to trigger ovulation. Micronized vaginal progesterone was

35.6 ± 3.4 (30–41) 5.9 ± 2.2 60/65 (92.3) 46/60 (76.7) 1.8 ± 0.7 (total 22) 2.2 ± 1.1 7/22 (31.8) 6/11 (54.5) 4/11 (36.4) 0 0 0

initiated on the day of embryo transfer (400 mg/day, vaginally). All embryo transfers were routinely carried out under transabdominal ultrasound guidance. Clinical pregnancy was defined as an intrauterine sac visualized on ultrasound at 6 weeks’ gestation, and ongoing pregnancies were defined as the presence of at least one developing embryo of over 12 weeks’ gestation. Early pregnancy loss was defined as positive pregnancy test, but an embryo failed to develop beyond 12 weeks; this included spontaneous abortions as well as biochemical pregnancies and ectopic pregnancies. The elapsed time between having the oocytes vitrified to embryo transfer was variable, ranging from 6 months in the endometrial carcinoma patient to 5 years in the breast cancer patients, with a mean time of 2.5 years.

Results A total of 11 patients returned to have their oocytes warmed, ICSI carried out and have embryo transfer after surviving their disease and with the agreement of their oncologists. The different indications of the patients were thyroid cancer (n = 1), endometrial adenocarcinoma (n = 1, but two embryo transfers), Hodgkin lymphoma (n = 1), and breast cancer (n = 8). One of the breast cancer patients did not receive an embryo transfer because all embryos were found to be aneuploid after preimplantation genetic screening with comparative genomic hybridization-array for advanced maternal age. Therefore, a total of 11 embryo transfers were carried out. As shown in Table 1, the mean age of patients was 35.6 ± 3.4 (range, 30–41) years, average duration of the ovarian stimulation was 8.6 ± 3.2 days, with no serious side effects or complications observed; mean number of metaphase II oocytes retrieved was 5.9 ± 2.2 and the survival rate of the vitrified-warmed eggs was 92.3%. The fertilization rate was 76.7% and 46 embryos were obtained. The mean number of embryos per patient transferred was 1.8 ± 0.7. Only two patients had cryopreserved embryos after their fresh cycle, and only one patient (41 years of age) did not reach embryo transfer as all embryos were aneuploid after pre-genetic diganosis and comparative genomic hybridization array analysis. A total of 22 embryos were transferred. According to the Alpha/ European Society of Human Reproduction and Embryology classification (2011), 31.8% (n = 7) were grade A embryos, 18.2%

Obstetric outcome after oocyte vitrification and warming (n = 4) were grade B, 36.4% (n = 8) were grade C, and 13.6% (n = 3) were grade D. Out of the 11 embryo transfers, seven (63.6%) had positive beta human chorionic gonadotrophin (HCG) (Table 2). One was a biochemical pregnancy (14.3%), and two of the six clinical pregnancies (33.3%) were clinical spontaneous abortions, as a gestational sac was observed on ultrasound, but no heartbeat was detected. Of the four remaining ongoing pregnancies, the patient with Hodgkin lymphoma had a vaginal delivery and live birth at term (40 weeks). The fetal weight was 3440 g. One breast cancer survivor underwent caesarean section and a healthy male infant was born at 40 weeks with a fetal weight of 2850 g. Another breast cancer patient had a vaginal delivery and a healthy baby girl was born with a fetal weight of 3220 g at 40 weeks gestation. Finally, a breast cancer survivor delivered a healthy baby boy at 38 weeks through caesarean section because of pregnancy-induced hypertension. His fetal weight was 2950 g. None of these four pregnancies had additional obstetric complications (i.e. preeclampsia or abnormal bleeding). Also, neonatal intensive care unit was not required as all babies were born at term and had adequate weight for their gestational age.

Discussion Fertility preservation offers women with cancer the potential of becoming pregnant with embryos from their own oocytes. Until recently, the gonadotoxic effect of oncological treatments induced infertility in most patients, making these women opt for oocyte donation, adoption or having a childless family. The excellent results obtained after cryopreservation of oocytes has drastically changed this scenario. We have shown the first series of live births after fertility preservation in women with cancer, with success rates that are similar to those of women who have had their eggs vitrified for several other indications, such as cryo-banked oocytes in an ovum donation programme (Cobo et al., 2010), where the ongoing pregnancy rate was 43.7 and 41.7% in the vitrification and fresh groups. Because of the limited sample of this series, more studies are needed to confirm the efficiency and the safety of oocyte vitrification in this group of patients. Although freezing ovarian tissue is still considered experimental (Loren et al., 2013), cryopreservation of oocytes was no longer considered experimental in 2013 (Loren et al., 2013; The Practice Committees of the American Society for Reproductive Medicine, 2013). The technique of ultra-rapid cryopreservation (i.e. vitirification) has shown excellent survival rates (80–95%) (Cobo et al., 2013; Garcia-Velasco et al., 2013; Kim et al., 2011; Parmegiani et al., 2011; Rienzi et al., 2012), and great fertilization and implantation rates (Alpha Scientist in Reproductive Medicine and ESHRE Special Interest Group in Embryology, 2011; Cobo et al., 2013; Garcia-Velasco et al., 2013; Rienzi et al., 2012). We, and others (Cobo and Diaz, 2011; Cobo et al., 2010; Parmegiani et al., 2011) have consistently shown that cryopreservation of oocytes through vitrification is an effective, simple, safe, and efficient technique for fertility preservation in women who are about to start oncological treatments with gonadotoxic impact. Compared with slow freezing, oocyte vitrification provides better results according to a recent meta-analysis

725 (Cil et al., 2013). Although these results were obtained in agematched, infertile women, they could be extrapolated to special situations, such as women with cancer. Similarly, other investigators have shown better survival rates after vitrification compared with slow freezing (80–90% versus 60–85%) as well as higher pregnancy rates (20–40% versus 10–15%) (Cobo et al., 2010; Herrero et al., 2011; Smith et al., 2010). When analysing pregnancy rates after oocyte vitrification, initial reports suggested live birth rates of 21.6% per embryo transfer (Oktay et al., 2006). Today, however, the success rates mimic those obtained with fresh oocytes. For instance, Grifo and Noyes, 2010) reported a 57% live birth rate per embryo transfer. This published evidence has contributed to worldwide acceptance of vitrification as an attractive approach to preserving fertility. Data on the perinatal outcome of children born after fertilization of cryopreserved oocytes is still limited, although recent publications reinforce its safety (Chian et al., 2008; Cobo et al., 2001; Garcia-Velasco et al., 2013; Noyes et al., 2009). This is not the case, however, for cryopreserved embryos, as extensive data on perinatal outcome is available (Cobo et al., 2012; http://www.cdc.gov/art/ART2011). The freezing of oocytes is a more recent technology that was introduced in 1986 (Chen, 1986), and oocyte vitrification is even newer so data are scarce. When one considers that oocyte freezing and oocyte vitrification was only started in 1997 (Porcu et al., 1997) and 1999 (Kuleshova et al., 1999), respectively, and that women who underwent fertility needed to survive their disease and allow some time to establish the relevance of their oncological treatment on their fertility potential before returning for the cryopreserved eggs, there have not been many cases throughout the world. The first pregnancy described after oocyte slow freezing in a gestational carrier in a cancer patient was by Yang et al. (2007). The patient had been diagnosed with Hodgkin lymphoma and had her eggs frozen in 1997. After thawing the oocytes, fertilization, and embryo transfer in a surrogate, a child was born in 2005. Porcu et al. (2008) described one case of twins conceived with warmed oocytes and delivered by a cancer patient after oocyte cryopreservation and bilateral ovariectomy as a result of ovarian cancer. Additionally, Kim et al. (2011) described the second case in a patient with chronic myeloid leukaemia who had her eggs vitrified, and gave birth to a biological baby in 2011. In the present study, we describe the first series of initial pregnancies in our large fertility-preservation programme and the perinatal outcomes. Cancer can be considered as a systemic disease. In fact, there is a shortening of telomeres even before oncological treatments, suggesting cellular senescence may be induced by the disease itself. This has severe implications for ovarian reserve and ovarian response to ovarian stimulation. In fact, women with cancer have been described as having a poorer response to ovarian stimulation (Domingo et al., 2012). A recent study, however, showed that, in patients who cryopreserved oocytes either for oncological or for non-medical reasons, all delivered babies were healthy, at term, and with normal weight for gestational age, which is reassuring information to adequately counsel patients (Garcia-Velasco et al., 2013). Although women who undergo pelvic radiation have high-risk pregnancies (Critchley and Wallace, 2005; Larsen et al., 2003; Wallace and Thomson, 2003), pregnancy after chemotherapy has been shown to be

726

Table 2

Results of oocyte warming, embryo transfer, and obstetric outcome. Age 1a

Age 2b

Diagnosis

Treatment

Number

Ovarian

Number of

stimulation

metaphase

Survived

Sperm

Fertilization

Total

Embryo

Cryopresrved

rate (%)

embryos

transfer

embryos

HCG

Sac

II oocytes 1

35

37

Thyroid cancer

Surgery and

Embryonic

Live

Fetal

Gestational

Congenital

heart

birth

weight g

age weeks

anomologies

activity

1

7

6

AT

2

6

6

T

4

5

4

AT

Splenectomy

5

4

4

AT

Surgery and

6

3

2

Donor

7

5

5

Normo

8

3

3

9

10

10

66.7

2

2

0

−

No

No

No

2

2

0

−

No

No

No

chemotherapy 2

34

35

Endometrial

Levonrgestrel

adenoca

intrauterine

100

device 3

34

35

38

40

3

3

0

+

Yes

No

No

2

2

0

−

No

No

No

100

4

2

2

+

Yes

Yes

Yes

3440

40

No

100

2

2

0

+

Yes

No

60.0

2

2

0

+

Yes

Yes

Yes

2850

40

No

Normo

66.7

2

2

0

+

Yes

Yes

Yes

3220

40

No

9

T

77.8

0

0

0

8

8

Donor

87.5

4

2

2

+

Yes

Yes

No

2920

38

No

11

6

6

Normo

50.0

2

2

0

−

No

No

12

8

7

AT

85.7

1

1

0

+

No

No

3 Undifferentiated

Surgery,

breast cancer

chemotherapy

T 50.0

and radiotherapy 4

33

35

Non-Hodgkin lymphoma type B

5

36

41

IDC

chemotherapy 6

30

33

IDC

Surgery, chemotherapy and tamoxifen

7

33

38

IDC

Surgery and chemotherapy

8

41

42

IDC

Surgery and chemotherapy

9

37

40

IDC

Surgery, chemotherapy and tamoxifen

10

31

32

IDC

Surgery and chemotherapy

11

40

44

IDC

Surgery,

No

chemotherapy and tamoxifen AT = asthenoteratozoospermia; IDC = infiltrating ductal carcinoma; HCG = human chorionic gonadotrophin; T = teratozoospermia. bAge

of patient when she underwent oocyte vitrification. of patient when she underwent her IVF cycle.

M Martinez et al.

aAge

Obstetric outcome after oocyte vitrification and warming safe, with similar perinatal outcomes as in age-matched patients (Gelber et al., 2001).

Acknowledgements The authors thank the IVI Foundation and all the embryologists and gynaecologists working in our Fertility Preservation Programme at the IVI clinics. We would also like to express our gratitude to Merck Serono, Spain, for providing the medication for our oncology patients. Supported by FIS PI11/02747 from Ministerio de Ciencia e Innovacion, Madrid, Spain.

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