STUDY DESIGN
5.1. PURPOSE
Among different techniques for female fertility preservation, oocyte cryopreservation is, for the moment the only one not experimental and effective available in Italy (37). However this strategy may require a time frame too long between the first examinations and the start of cancer treatment.
The aim of this analysis is to compare the results obtained using different stimulation protocol started in two different menstrual cycle phases, follicular or late follicular/luteal phase, in order to evaluate the possibility of starting the procedure, independently from menstrual cycle moment at time of first examination, so reducing time frame until chemo- and/or radiotherapy beginning.
Moreover we analyze the effect and safety of controlled ovarian stimulation using a combination protocol of letrozole-gonadotropins in breast cancer patients.
5.2. MATERIALS AND METHODS
performed at the Infertility and Assisted Reproduction Centre of University of Pisa, from December 2010 to May 2014.
Among the 21 patients examined, 20 have a cancer diagnosis waiting for treatment, whereas one is affected by multiple sclerosis and she need gonadotoxic therapy. All patients consent to treatment, as well as to the release of all information concerning their treatment. Women includ in the analysis have regular menstrual cycle. Patients are divided in two group homogeneous for age, basal AMH and FSH levels, depending on the phase of menstrual cycle, follicular or late follicular/luteal phase, in which the begin ovarian stimulation. In both group ovarian stimulation is carried out with antagonist protocol.
Women, who are stimulated during follicular phases, after waiting spontaneous menses, receive a controlled ovarian stimulation starting on cycle day 2 with recombinant FSH (Gonal F®, Serono, Italy) or with highly purified urinary FSH (Fostimon®, IBSA, Italy). Initial dosage depended on age, basal levels of FSH, AMH levels and AFC ultrasound and it is adjusted according to follicular growth and estradiol levels. Once follicles reached a diameter of 14 mm, Cetrorelix (Cetrotide®, Serono, Italy) is added in order to prevent premature. Patients in the late follicular/luteal phase receive a modified antagonist protocol. They are administered GnRH antagonist to induce immediate luteolysis and menses in few days. Then they started ovarian stimulation with gonadotropins on cycle day 2 as described above. When dominant follicle reached a diameter of 14 mm, in order to prevent premature ovulation GnRh antagonist is administered.
After 36-38 hours, oocyte retrieval is performed by transvaginal ultrasound under local anaesthesia and sedation. Oocytes are kept in a specific incubation medium (Sydney IVF Gamete buffer, Cook Ireland ltd, Limerick, Eire) and they are observed by a stereomicroscope in order to evaluate oocytes quality. Viable and mature oocytes (metaphase II) are subsequently cryopreserved by vitrification method.
In the late follicular/luteal phase are included three patients affected by breast cancer. Two of them receive a combined stimulation protocol with letrozole and gonadotropins. Patients received 5 mg letrozole daily starting on cycle day 2 or 3 and after 2 days gonadotropins until hCG administration. If estradiol levels remained elevated after oocyte retrieval, letrozole is restarted and continued until E2 levels fell to <50 pg/ml
The main outcome measure is the number of days between first gynaecological examination and the beginning of oocyte cryopreservation. We also evaluate the duration of ovarian stimulation, cumulative dosage of gonadotropins, number of oocyte collected, the percentage of mature and vitrified oocytes.
Statistical analysis: the results expressed in percentage are evaluated with the Fischer test. Student’ T test is used to compare data in both groups and results. A p<0.05 iss considered statistically significant.
5.3 RESULTS
TABLE 5.1PATIENTS AFFECTED BY OVARIAN BORDERLINE TUMOR PRESENT ONLY ONE OVARY P Age Diagnosis Day of the menstrual cycle at the medical interview Number of days to wait for start
ovarian stimulation Duration of gonadotropin administratio n (days) Dosage (IU) Aspirated oocytes Metaphase II oocytes Vitrified oocytes Follicular phase group
1 26 Nasopharyn K 25 5 8 1200 12 6 6 2 22 Ovarian borderline Tumour* 6 24 12 2700 5 4 4 3 32 LH 7 30 7 3400 27 9 18 4 31 LH 2 0 12 2100 7 4 6 5 37 MS 15 15 11 4425 6 3 6 6 40 Ovarian borderline Tumour* 9 19 8 2100 4 3 3 7 30 LH 10 18 10 3600 4 3 3 8 34 LH 1 0 13 775 12 10 10 9 29 colon 16 17 11 33 10 8 9 10 36 LH 20 8 13 4950 7 7 7 11 28 Ovarian borderline Tumour* 1 0 8 2100 4 4 4
Late follicular/luteal phase
12 22 LH 16 6 (VS 12) 9 2400 16 12 12 13 30 K mammella 16 6 (VS 12) 10 2700 12 6 11 14 26 LH 13 12 (VS 15) 8 2400 15 12 12 15 28 LH 16 10 (vs 12) 10 2700 18 10 15 16 29 osteosarcoma periostale 18 13 (VS 10) 11 3000 21 11 11 17 36 k mammella 10 11 (vs 18) 9 1350 10 8 8 18 40 k mammella 15 5 (vs 13) 12 3150 7 5 5 19 22 LH 20 6 (VS 10) 9 2400 16 12 12 20 31 melanoma 16 6 (VS 12) 9 1567 14 10 10 21 24 LH 13 7 (VS 15) 9 1234 12 9 11
Cancer diagnoses are: Hodgkin lymphoma (n=10), ovarian borderline tumour (n=3), breast cancer (n=3), melanoma (n=1), osteosarcoma (n=1), colon cancer (n=1) and nasopharyngeal tumour (n=1). At the moment of examinations, patients with ovarian borderline tumour present only one ovary due to the previous laparoscopic unilateral oophorectomy.
Average age of patients is 31.00 ± 5.51 in follicular group and 28.8 ± 5.84 (p 0.3). The cumulative dosage of gonadotropins, duration of ovarian stimulation and the number of oocyte retrieved and quality oocyte, are evaluated in order to analyze the efficafy of stimulation protocols (table 5.2). Patients stimulated in follicular phase receive on average of 2489. 36 ± 1516.5 UI in 10.27 days. The other group receive 2490 UI of FSH in period of time of 9.1 days. Both parameters result similar in the two groups.
The average number of retrieved oocyte is higher in the late follicula/luteal phase (8.90 ± 6.71 vs. 14.1 ± 4.04). To analyze this result we have also to considerate that in the follicular group are included three patients affected by ovarian borderline tumor already treated with laparoscopic unilateral oophorectomy. This parameter is calculated considering the number of oocytes obtained from single ovary and not from single patients. Difference is statistically significant (p= 0.04). However the number of mature oocytes (metaphase II) and the number of vitrified oocyte are similar in both group (61,5% vs. 67.38% and (76.84% vs 75.89%).
Considering time from first examination until the beginning of ovarian stimulation, and consequently start of oncological therapy after about 10 days, (table 5.3), we don’ found any statistically difference between the two examinated group. Women in follicular
TABLE 5.2
group wait an average time was 12.36 ± 10.4 days (with a range from no waiting time for until 28 days) against 8.2 ± 2.97 days in the second group. We have to notice that in the late follicular/luteal phase group we don’t wait for spontaneous menstrual cycle but we try to induce them using GnRh antagonist to have a rapid luteolysis. So comparing days that women had really waited to days that women should have waited to start stimulation on day two on their spontaneous menses, there is a benefit of average waiting time of 7.45 ± 3.75 vs. 12.9 ± 2.46 (table 5.3). This result is statistically significant (p= 0.01).
We focus our attention on estradiol level on hCG administration day and values are similar in both group (respectively 1248,90 ± 1190 vs. 1271,70 ± 1032). Two patients receiv a combined protocol with letrozole and gonadotropins because the are affected by breast cancer. We can notice that estradiol levels are lower than in other group but with a good oocyte retrieval (respectively 174 pg/mL and 210 pg/mL) (table 5.4). Due to the small size of group we can not do a statistical analyses, but results were encouraging to affirm the safety as well as the efficacy in this kind of patients.
Follicular phase group Late follicular/luteal phase P-value Age 31.00 ± 5.51 28.8 ± 5.84 0.3859 NS Duration of gonadotropin administration (days) 10.27 ±2.19 9.6±1.17 0.3993 NS Dosage (IU) 2489.36 ± 1516.5 2490 ± 485.7 0.999 NS E2 at the day of ovulation induction 1248.9 ± 1129.2 1150.1 ± 1057.75 0.83872 NS Aspirated oocytes 8.90 ± 6.71 14.1 ± 4.04 0.04 * Metaphase II oocytes % 61.5 % 67.38% 0.39 NS Vitrified oocytes % 76.84 % 75.89 % 0.98 NS
TABLE 5.3
TABLE 5.4
P
Day of the menstrual cycle at the medical
interview
Number of days to wait for start ovarian stimulation
Number of days gained Follicular phase group
1 25 5 / 2 6 24 / 3 7 30 / 4 2 0 / 5 15 15 / 6 9 19 / 7 10 18 / 8 1 0 / 9 16 17 / 10 20 8 / 11 1 0 /
Late follicular(/luteal phase
12 16 6 (VS 12) 6 13 16 6 (VS 12) 6 14 13 12 (VS 15) 3 15 16 10 (vs 12) 2 16 18 13 (VS 10) +3* 17 10 11 (vs 18) 7 18 15 5 (vs 13) 8 19 20 6 (VS 10) 4 20 16 6 (VS 12) 6 21 13 7 (VS 15) 8
P Age Diagnosis E2 peak at ovulation induction Aspirated oocytes Metaphase II oocytes Vitrified oocytes 36 k mammella 174 10 8 8 40 k mammella 210 7 5 5
5.4DISCUSSION
Nowadays considerable advances have been done in cancer diagnosis and cancer treatments. This has determined an increasing rate s of patients survivors.
Loss of fertility is one of the most devastating consequences of radio- or cytotoxic therapy for young patients who have expectations of a normal reproductive life. Oncofertility has emerged as a new interdisciplinary field to address the issue of gonadotoxicity associated with cancer therapies and to facilitate fertility preservation.
Various strategies have been used for fertility preservation.
To date embryo cryopreservation is the first to provide a tangible and widely
documented chance of success (33) among fertility preservation options, but it is prohibited in Italy by 40/2004 law.
Oocytes cryopreservation is, for the moment, the only not experimental technique (37) actionable in Italy, for young cancer women who desire to preserve their fertility. However, oocyte cryopreservation put the problem of time necessary to complete the whole procedure, that may be too long for cancer patients who have to start oncological therapy. The procedure may require a total of up to 6 weeks, particularly if patients happen to be in the luteal phase at the moment of first examination. Conventional stimulation protocols are started in the early follicular phase. Madrigano et al. stated an average an average time frame of 31.3 days (range 10-65 days) to collect oocytes in 23 patients treated for breast cancer (68).
Because the time frame until the initiation of the chemo- and/or radiotherapy is limited, this waiting time is unacceptable in many patients. Therefore it is fundamental to
even in the luteal phase, ensuring the oocyte withdrawal as soon as possible, without compromising the results of the whole procedure.
The concept of using GnRH-antagonists to induce luteolysis in the mid-luteal phase is described first by Anderson (58) and then considered again by von Wolff (59). The first one describes two patients with breast cancer examinated in the mid-luteal phase and who receive GnRH-antagonists, with the result of a rapid fall of progesterone followed by menses 2-4 days later. Ovarian stimulation is carried out 4 days after application of GnRH-antagonists. Patients are stimulated for 9 and 11 days and 8 and 6 oocytes are collected. Because ovarian stimulation is preceded by a 4-day pre-treatment with GnRH-antagonists, oocyte collection needed 16 days. The protocol used by von Wolff is based on the employment of both GnRH-antagonist and recombinant FSH at the same time, irrespective of the day of menstrual cycle. Although the number of oocytes results slightly but not significantly lower in patients stimulated during the luteal phase (13.1 vs 10.0), as well as the number of mature oocytes (83.7% vs 80.4%) and the fertilization rate (61.0% vs 75.6%), this stimulation protocol allows oocyte retrieval in cancer patients within 2 weeks irrespective of the day of menstrual cycle at first consultation.
In our analyses we compare the results between two groups of patients, in the first one we wait for menses before starting ovarian stimulation and in the other one we try to induce a rapid luteolysis and then we start with gonadotropin administration. Both groups show similar results about duration of stimulation, total dosage of gonadotropins administered, number of oocyte collected and percentage of mature (M II) oocytes and then vitrified. The real difference between the two groups is the number of days needed to start the procedure, lower in the luteal group, than in the follicular one where spontaneous
menses have been expected. The most important datum is that, despite of time advantage, the yield in terms of number of oocyte collected and, above all, their good quality, was not lower than in case of stimulation started waiting spontaneous menses.
Our analysis shows the possibility to start oocyte cryopreservation procedure, anytime without considering menstrual cycle’s phase.
5.5 CONCLUSION
This study suggests that controlled ovarian stimulation for fertility preservation in cancer patients can be started soon, both for women who are in follicular phase and for whom are in luteal phase at first examination. This allows to reduce significantly the time frame until starting cancer therapies, ensuring oocytes collection within a time frame acceptable by the most of oncological diseases.
