Progestin-primed ovarian stimulation for fertility preservation in women with cancer: A comparative study.

During the last decade, the improved effectiveness of cancer cures has enhanced the attention on the quality of life of survivors, including the importance of preserving their capacity to have children. Infertility in cancer survivors is an important issue and could be a source of devastating psychological sufferance [ 1 , 2 ]. Accordingly, fertility sparing treatments and technique of fertility preservation have spread. The most relevant Scientific Societies of oncology and reproductive medicine have provided specific guidelines [ 3 – 6 ]. Noteworthy, even women with early endometrial cancer are now considered for conservative management. They are treated hormonally to achieve transient cancer regression, thus allowing pregnancy seeking and postponing hysterectomy after delivery [ 7 – 10 ]. Oocytes cryopreservation plays a crucial role in preserving fertility capacity in young adult women. with malignancies [ 3 – 6 ]. In this setting, enhancing the number of collected oocytes, avoiding a delay in the initiation of oncological treatments, and limiting risks are essential aspects. This can improve effectiveness and safety. Substantial advancements have been obtained with the diffusion of random start protocols (i.e., promptly initiating the ovarian hyperstimulation regardless of the menstrual phase), dual stimulation (performing two hyperstimulation cycles one after the other), the use of aromatase inhibitors in women with hormone-sensitive cancers (to lower peripheral levels of potentially detrimental hormones) and ovulation triggering with Gonadotropin Releasing Hormone (GnRH) agonists rather than human Chorionic Gonadotropin (hCG) (to prevent Ovarian Hyperstimulation syndrome—OHSS) [ 11 – 14 ]. However, there is room for further improvements. There is the need to optimize the regimen of ovarian stimulation, to make treatments more patient-friendly and to reduce costs [ 15 ]. Following a local audit at the end of 2019 among physicians engaged in fertility preservation in our Unit, a new policy of ovarian stimulation for women with cancer was decided and implemented from the beginning of 2020. To reduce costs, we replaced GnRH antagonists with a progestin (progestin-primed ovarian stimulation—PPOS) protocol and substituted corrifollitropin and recombinant FSH with human menopausal gonadotropin (hMG). In addition, we introduced the dual trigger, associating low dose hCG to GnRH agonists to improve oocytes yield [ 16 , 17 ]. In the present ‘before and after’ study (we compared two study periods of one year, before and after January 01 st 2020), we evaluated ovarian stimulation outcomes in 2019 and in 2020 to draw some inferences on the effectiveness of the new regimen.

Overall, 124 women were selected, 46 cryopreserved oocytes in 2019 while 78 on the following year. Twelve women performed dual stimulation and only their first hyperstimulation cycle was accounted for the study purposes. Baseline characteristics of the two groups are shown in Table 1 . Indications to oocytes cryopreservation were similar in the two study periods. However, a statistically significant difference emerged for serum AMH, levels being lower in the second period. AFC: Antral Follicle Count. AMH: Anti-mullerian hormone PPOS: Progestin Primed Ovarian Stimulation Data are reported as mean ± SD or median [interquartile range] or number (percentage) In the first part, three women did not complete their fertility preservation cycle: two decided to anticipate the beginning of chemotherapy and one was stopped because of inadequate follicular growth. In the second part, five women dropped out: one due to personal choice and four for inadequate follicular growth. Therefore, a total of 43 and 73 cycles were available for oocytes retrieval outcomes in 2019 and 2020, respectively. Cycle outcome of the two study periods is illustrated in Table 2 . In the first period, the median [IQR] number of GnRH antagonist injections was 4 [3–5]. In both study periods, we did not observe spontaneous ovulations during the stimulation. Some variables significantly differed. The number of oocytes retrieved and the number of mature oocytes frozen were significantly lower in the second period. However, the ratio between number of mature oocytes and AMH did not differ ( Fig 2 ). Specifically, the median [IQR] rate of retrieved oocytes adjusted per AMH in the first and second period was 5.2 [3.3–8.1] and 5.5 [3.6–8.6], respectively (p = 0.76). The median [IQR] rate of mature oocytes adjusted per AMH in the first and second period was 4.0 [2.3–7.1] and 4.0 [2.7–6.8], respectively (p = 0.80). The left part of the figure refers to the total number of oocytes retrieved. The right part refers to the total number of mature oocytes retrieved and thus cryopreserved. The first study period (simplified in the figure with GnRH ant, to underline the use of GnRH antagonists) is depicted in red. The second study period (simplified in the figure with PPOS, corresponding to progesterone primed ovarian stimulation) is depicted in blue. No statistically significant difference emerged for both comparisons (p = 0.76 and p = 0.80, respectively). Data are reported as mean ± SD or median [interquartile range] or number (percentage) PPOS: Progestin-primed ovarian stimulation a Refers to women who completed the cycle (43 and 73 in the GnRH antagonist and PPOS groups, respectively) b Corrifollitropin alfa 100 and 150 mcg were considered equivalent to 150 and 200 IU per day for 7 days, respectively. The number of ultrasounds was significantly lower in women treated in the second period. The median [IQR] number of scans was 3 [3–4] and 3 [2–3] in the first and second period, respectively (p<0.001). The median [IQR] number of injections (including those needed for gonadotropins, GnRH antagonists, GnRH agonists, and hCG) was 12 [10–14] and 13 [12–15] in the first and second period, respectively (p = 0.01). The total costs of the drugs used for ovarian hyperstimulation (including those needed for gonadotropins, GnRH antagonists, GnRH agonists, hCG, and MPA) in the two study periods were 940 € [IQR: 774–1,096 €] and 520 € [IQR: 434–564 €], respectively (p<0.001). Table 3 shows sex steroids levels in the two study groups, considering separately those who did and did not receive letrozole. Some statistically significant differences emerged. For women who did not receive letrozole, PPOS-treated subjects (second period) had higher levels of Aldosterone, 11-DeoxyCortisol, Androstenedione, Testosterone and lower levels of Cortisol, Corticosterone, DHEAS, 17-OH-Progesterone. For those who received letrozole, PPOS-treated subjects had higher levels of Aldosterone, 11-DeoxyCortisol, testosterone and lower levels of 17-OH-Progesterone.

In conclusion, random start PPOS with hMG and dual trigger represents an easy and affordable ovarian stimulation protocol for fertility preservation in women with cancer, showing similar results to random start with GnRH antagonist plus recombinant FSH protocol but being potentially more friendly and cheap. Additional evidence is needed to rule out a clinical relevance of the detected changes in local steroids hormones and to demonstrate the safety for hormone sensitive cancers. More in general, future efforts are needed to identify the most suitable protocol to be used in this clinical setting and to better tailor treatments according to the clinical characteristics of the patients.

This ‘before and after’ study included all consecutive women with a first diagnosis of cancer who underwent ovarian hyperstimulation for oocytes cryopreservation at the Infertility Unit of the Fondazione IRCCS Ca’ Granda Ospedale Maggiore Policlinico of Milan, Italy between January 2019, and December 2020. Two groups were compared, chronologically divided based on the date of implementation of the new policy of ovarian hyperstimulation (January 1 st , 2020). During the same study period (2019–2020), our unit was also engaged in a parallel study aimed at evaluating follicular fluid steroids concentrations in women with cancer undergoing random start ovarian stimulation protocols. This situation offered us the additional opportunity to obtain information on the local levels of steroids hormones detected for the two different regimens under study. The study was approved by the local Ethical Committee (Milano area B). Women signed an informed consent to participate to the study on follicular fluid steroids concentrations. Conversely, since the ‘before and after’ part of the study was decided a posteriori , they did not sign a specific informed consent for the comparisons of clinical outcomes. However, all women undergoing fertility preservation in our Unit routinely signed an informed consent for their anonymized data to be used for research purposes. During the whole study period, ovarian hyperstimulation was performed according to a random start approach. Gonadotropins were initiated on the day of referral regardless of the phase of the menstrual cycle. In hormone-sensitive cancers, in all the studied period, women were also given oral letrozole 5 mg (Letrozolo ® , TEVA, Italy) daily for the whole duration of ovarian hyperstimulation and up to 5–7 days after oocytes retrieval. The dose of gonadotropins was decided based on the expected ovarian response, i.e., considering clinical data (age, menstrual cycle duration), antral follicular count (AFC) assessed with an ultrasound scan at first referral and serum anti-mullerian hormone (AMH). Women were monitored with serial transvaginal ultrasounds (US) and peripheral hormonal dosages if needed [ 15 ]. Serum AMH concentration was tested in all women prior to initiate the ovarian hyperstimulation. The commercially available Beckman Coulter Gen II ELISA assay on the automated GEMINI platform (STRATEC Biomedical AG, Germany) after dilution with assay buffer was used. In the first study period (January to December 2019), women initiated ovarian hyperstimulation with the long-acting corifollitropin alfa (Elonva ® , Merck Sharp & Dohme, UK) 100 or 150 mcg s.c. according to body weight (< or ≥ 60 kg). Five-seven days later, recombinant FSH daily s.c. (150/225 IU) (Gonal-F ® , Merck-Serono, Italy) was administered, if needed. Pituitary suppression was obtained with GnRH antagonist s.c. (Fyremadel ® 0.25 mg Ferring, Denmark) administered daily, initiating when the leading follicle reached a mean diameter of 13–14 mm, and continued up to the time of ovulation trigger. Final oocyte maturation was triggered with GnRH agonists (Fertipeptyl ® 0.2 mg, Ferring, Denmark) when at least three follicles had a mean diameter of 18 mm ( Fig 1 ). In the upper part of the figure , the regimen used in 2019. Women received corifollitropin-alfa and 6–7 days later recombinant FSH daily, if necessary. Since the observation of a leading follicle with a mean diameter above 13–14 mm, they were prescribed also GnRH antagonist daily. The final maturation was obtained with GnRH agonist 0.2 mg. In the lower part of the figure , the regimen used in 2020 (progestin-primed ovarian stimulation). Women received both hMG and oral MPA daily. Final oocytes maturation was obtained with the concomitant administration of GnRH agonist 0.2 mg, plus hCG 1,000 IU. GnRH: Gonadotropin Releasing hormone. Cor-α: Corifollitropin-alfa. GnRH ant: GnRH antagonist. GnRHa: GnRH agonist. rFSH: recombinant FSH. MPA: medroxy-progesterone acetate. hMG: human Menopausal Gonadotropin. hCG: human Choriogonadotropin. Starting from January 2020, ovarian hyperstimulation was obtained using hMG s.c. (Meropur ® , Ferring, Denmark) 150/225 IU, daily. Contemporary, medroxy-progesterone acetate (MPA) tablets 10 mg (Farlutal, Pfeizer, US) were administered daily until the day of trigger. The dose of MPA was chosen according to previous publications to ensure pituitary inhibition [ 18 ]. Final oocyte maturation was triggered with a combination of GnRH agonists (Fertipeptyl ® 0.2 mg, Ferring, Denmark) and Chorionic gonadotrophin (hCG) 1,000 IU (Gonasi HP, Ibsa, Switzerland) when at least three follicles had a mean diameter of 18 mm ( Fig 1 ). During the whole study period (2019–2020), oocytes were collected 36 hours after triggering through transvaginal ultrasound-guided oocyte retrieval. The protocols of oocytes aspiration, oocytes handling and cryostorage were standardized and reported elsewhere in detail [ 19 , 20 ]. The methodology of follicular fluids collection and steroids assessment is also reported in detail elsewhere [ 21 ]. Briefly, the pool of follicular fluids was collected, and then centrifugated and cryopreserved at -20°C. The samples were concomitantly thawed and levels of 15 steroids were then assayed by liquid chromatography tandem mass spectrometry. The following 15 steroids were tested: 11-deoxycorticosterone, 11-deoxycortisol, 17-OH-progesterone, 21-deoxycortisol, aldosterone, androstenedione, corticosterone, cortisol, cortisone, dehydroepiandrosterone (DHEA), dehydroepiandrosterone sulfate (DHEAS), dihydrotestosterone (DHT), estradiol, progesterone, testosterone. Results were presented separately for women who did and did not receive letrozole because of the well-known relevant effects of this drug on steroidogenesis [ 21 ]. Clinical information regarding patients’ history, cancer diagnosis, US monitoring and oocytes retrieved was obtained from patients’ charts. The costs of the drugs were obtained as described in detail elsewhere [ 22 ]. Gonadotropins were fully refunded in Lombardy. GnRH agonists and antagonists, hCG and MPA were conversely paid out of pocket by the patients. For the analyses, we did not distinguish between those that were or were not refunded and considered them globally. Only drugs used for ovarian hyperstimulation were included (i.e., gonadotropins, GnRH antagonists, GnRH agonists, hCG, and MPA). The costs of the drugs were obtained referring to the local Italian Agency of drugs—Agenzia Italiana del farmaco (AIFA) ( Farmadati sftw access 02/22 ). Data collected were transferred in Statistical Package for Social Science (SPSS 26.0, IL, USA) database for subsequent analyses. Differences between the two study groups were tested using Student’s t test or Mann-Whitney test or Fisher Exact test, as appropriate. The primary outcome was the ratio between the number of mature oocytes retrieved and serum AMH. This outcome was chosen to protect the findings from possible differences in ovarian reserve between the two study periods. To further limit confounders, we set the whole duration of the study period to two years (one year per group). Given an expected number of about 60 cases per year, a mean ± SD number of oocytes stored (10 ± 6) and the setting of type I and II errors at 0.05 and 0.20 respectively, our sample size could allow detection of differences > 3 oocytes.

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