Combining Interventions of Fertility Preservation to Mitigate Fertility Loss After Breast Cancer (Coimbra)

This clinical prospective randomised controlled trial will evaluate the impact of an ovarian biopsy on the oocyte yield after controlled ovarian stimulation before chemotherapy in view of breast cancer. The purpose of this trial is to learn about the possibility to combine these two fertility preservation procedures without decreasing the number of oocytes collected after an ovarian stimulation.

All patients will undergo a biopsy of the ovarian cortex via laparoscopy, after patient eligibility has been established and patient consent has been obtained. The side of the ovarian biopsy will be randomized on the day of the laparoscopy either from the right side (group 1) or from the left side (group 2). The ovarian tissue will be cryopreserved. A small part will also be evaluated by the pathologist to screen for malignant contamination and follicle density. All patients will then continue with a controlled ovarian stimulation. In both groups, a first blood analysis with hormonal assessment (E2, P, LH, FSH, hCG and AMH) will be performed at first visit, independent of their cycle. At that time a transvaginal ultrasound (frequency ≥ 7 MHz) will be done as well to assess the antral follicle count and the ovarian volume. The antral follicle count will be assessed using real-time 2D-US evaluation, the standard use in clinical practice. The ovarian volume will be measured using the prolate ellipsoid formula (volume = length x width x height x 0.523). If a dominant follicle is noted, choriongonadotropin (Pregnyl ® 5000 IU), choriongonadotropin Alfa (Ovitrelle ® 250 µg) or triptorelin (Gonapeptyl ® or Decapeptyl ® 0.2 mg) will be given to the patient to trigger ovulation. Thereafter, the controlled ovarian stimulation can be initiated in the luteal phase. The size of the ovarian cortex biopsy will be calculated as 20% of the ovarian volume measured at the initial ultrasound. Immediately after the ovarian biopsy, the objective measurements of the biopsy will be noted in weight (gram) and volume (length x width x height mm³) because the biopsy fragment can be considered a rectangular box. A correction factor will be used afterwards in the statistical analysis if the volume of the ovarian biopsy is not equal to 20%. The laparoscopic procedure by which the ovarian cortex biopsy will be performed, will be standardised: The technique developed by ProFam will be used and adapted if needed at the surgeon's discretion. A three to four-port laparoscopy will be used. The ovarian cortex biopsy will be performed using a curved scissor. Bipolar or unipolar cauterisation will be avoided as much as possible. If necessary for approximation or for hemostatic reasons, the ovarian edges can be stitched or Surgicel® Absorbable Hemostat can be used. During the course of the study, there will be a number of blood analyses and ultrasounds, to evaluate follicular growth. This will be arranged at specific time points starting from day 6 of the stimulation and will be performed every other day, until the day of trigger. If necessary and based on clinician's decision, a supplementary blood analysis and/or ultrasound can be scheduled. At the follow-up ultrasounds the follicular growth will be noted for each ovary separately to assess difference in reaction and/or growth after the ovarian biopsy. The controlled ovarian stimulation will start the day of the laparoscopy. It can start either in basal circumstances, or in the early follicular or luteal phase. A fixed GnRH antagonist protocol will be used. Ovarian stimulation will be started with Corifollitropin alfa 0.15 mg (Elonva®). On day six the antagonist, Ganirelix (Orgalutran®), will be added to prevent a premature LH surge. If needed ovarian stimulation can be continued after seven days using follitropin beta (Puregon®). The dosage of Puregon® is dependent on the AMH level at the first visit. Patients with AMH > 2 µg/L, will be started with 200-225 IE Puregon® daily. If the initial AMH level is <2 µg/L, patients will receive 225-300 IE Puregon® daily. Elonva ® and Puregon ® will be administered in the evening, whereas Orgalutran ® will be injected in the morning. Agonist trigger Triptorelin 0.2 mg (Gonapeptyl®) will be ministered as soon as at least three follicles reach a mean diameter of 18 mm or wider, with intermediary follicles 14 mm or wider. In case of LH levels <2 IU/L at the start of ovarian stimulation, a dual ovulation strategy will be adapted: Triptorelin 0.2mg and hCG 2500 IU (or ovitrelle 250 µg) will be given. Oocyte retrieval will be planned 36 hours after triggering. This will be performed as a transvaginal oocyte pick-up. Oocyte vitrification will be carried out after denudation and assessment of maturity.

Fertility Preservation, Reproductive Techniques, assisted, Ovulation Induction, Oocyte Retrieval, Cryopreservation, Ovary

All patients will undergo a laparoscopy for an ovarian biopsy (about 20% of the ovarian volume). The side of the ovarian biopsy will be randomized on the day of the laparoscopy either from the right side (group 1) or from the left side (group 2). All patients will then continue with a controlled ovarian stimulation, using corifollitropin alpha and follitropin beta in a GnRH antagonist protocol using ganirelix. The metaphase II oocyte yield will be evaluated between the biopsied versus non-biopsied ovary to proof a non-inferiority (assuming that a difference of 1 metaphase II oocyte yielded from the biopsied ovary compared to the non-biopsied ovary is not clinically relevant).

Patients in group 1 will undergo a laparoscopy for an ovarian biopsy from the right ovary. They will then continue with a controlled ovarian stimulation, using a GnRH antagonist protocol. This will be started with Corifollitropin alfa 0.15 mg in the evening. On day six the antagonist, Ganirelix, will be added in the morning. If needed stimulation can be continued after seven days using follitropin beta daily in the evening (dosage ranging from 200-300 IE depending on AMH levels). Agonist trigger Triptorelin 0.2 mg will be administered for ovulation induction. In case of LH levels <2 IU/L at the start of ovarian stimulation, a dual ovulation strategy will be adapted: Triptorelin 0.2mg and choriongonadotropin 2500 IU (or choriongonadotropin alfa 250 µg) will be given. A transvaginal oocyte retrieval will be planned 36 hours after triggering.

Patients in group 2 will undergo a laparoscopy for an ovarian biopsy from the left ovary. They will then continue with a controlled ovarian stimulation, using a GnRH antagonist protocol. This will be started with Corifollitropin alfa 0.15 mg in the evening. On day six the antagonist, Ganirelix, will be added in the morning. If needed stimulation can be continued after seven days using follitropin beta daily in the evening (dosage ranging from 200-300 IE depending on AMH levels). Agonist trigger Triptorelin 0.2 mg will be administered for ovulation induction. In case of LH levels <2 IU/L at the start of ovarian stimulation, a dual ovulation strategy will be adapted: Triptorelin 0.2mg and choriongonadotropin 2500 IU (or choriongonadotropin alfa 250 µg) will be given. A transvaginal oocyte retrieval will be planned 36 hours after triggering.

A laparoscopy will be performed to achieve an ovarian cortical biopsy of one of both ovaries. Approximately twenty % of the ovarian volume as initially measured on the baseline ultrasound at the first visit will be removed to be processed in the lab for cortex freezing. The laparoscopic procedure in order to take the ovarian cortex biopsy will be standardised as much as possible. The technique developed by ProFam will be followed and adapted if needed by the surgeon's discretion. A three to four port laparoscopy will be used. The ovarian cortex biopsy will be taken using a curved scissor. Bipolar or unipolar cauterisation will be avoided as much as possible. If necessary for approximation or for hemostatic reasons, the ovarian edges can be stitched or Surgicel ® Absorbable Hemostat can be used.

After ovarian stimulation, a transvaginal oocyte retrieval will be performed either under local or general anaesthetics (patient's choice). The oocyte yield between the biopsied and non-biopsied ovary will be evaluated.

Evaluation of the proportion of follicles that were responsive to FSH and is calculated by dividing the number of preovulatory follicles (16-20 mm) × 100 by the antral follicle count (3-8 mm)

From ovarian stimulation until the day of oocyte retrieval (2-3 weeks, depending on patient's response)

Evaluation of proportion of metaphase II oocytes within the COC yield between biopsies and non-biopsied ovary

Inclusion Criteria: Age ≥ 18 and ≤ 35 years BMI ≥ 18 and ≤ 35 kg/m² Diagnosis of breast cancer Presence of 2 ovaries Signed informed consent form Medically fit for general anesthesia (ASA score 1-3) Permission of oncology team (with agreement to postpone chemo/radiotherapy for at least 2 weeks) Random start controlled ovarian stimulation AFC minimum (ie antral follicles measuring between 2-9 mm): 8 antral follicles Exclusion Criteria: Age <18 or >35 years BMI <18 or >35 kg/m² Difference in AFC between the ovaries of more than 7 antral follicles Diagnosis of PCOS Previous radiotherapy and/or chemotherapy (neo-adjuvant chemotherapy included) Endometriose rAFS 3-4 Allergy or reaction to the use of Elonva®, Puregon®, Orgalutran®, Pregnyl® Gonapeptyl®, Decapeptyl® or letrozole in the past

As this is a multicenter study, we plan to share IPD between the study sites. An electronical case report form (eCRF) will be set up and completed in all sites. Data will be handled in accordance with the general data protection regulation (GDPR).

The data will become available over the course of the study until 6 months after ending this study. This will give us the time to analyse the data of the different sites.

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