Ovarian stimulation to cryopreserve fertilized oocytes in cancer patients can be started in the luteal phase

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1 Ovarian stimulation to cryopreserve fertilized oocytes in cancer patients can be started in the luteal phase Michael von Wolff, M.D., a Christian J. Thaler, M.D., b Torsten Frambach, M.D., c Cosima Zeeb, M.D., e Barbara Lawrenz, M.D., d Roxana M. Popovici, M.D., a and Thomas Strowitzki, M.D. a a Department of Gynecologic Endocrinology and Reproductive Medicine, Women s University Hospital, University of Heidelberg, Heidelberg; b Gynecologic Endocrinology and Reproductive Medicine, Women s Hospital, Ludwig- Maximilians-University of Munich, Campus Grosshadern, Grosshadern; c Department of Obstetrics and Gynecology, University of Wuerzburg, Wuerzburg; d Women s Hospital, University of Tuebingen, Tuebingen, Germany; and e Department of Gynecologic Endocrinology and Reproduction, Women s Hospital of St. Gallen, St. Gallen, Switzerland Objective: To analyze if oocytes can be obtained in all patients before cancer treatment within 2 weeks by initiating ovarian stimulation during the follicular or luteal phase. Design: Prospective controlled multicenter trial. Setting: Four university-based centers. Patient(s): Forty cancer patients before chemotherapy. Intervention(s): Twenty-eight patients were stimulated with gonadotropins in the follicular phase (group I). In 12 patients (group II), ovarian stimulation was initiated in the luteal phase, and these received GnRH antagonists and recombinant FSH. In 14 patients, 143 oocytes were further processed for fertilization by intracytoplasmic sperm injection (ICSI). Main Outcome Measure(s): Number of oocytes aspirated after ovarian stimulation, cumulative FSH/hMG dosage, viability and maturity of oocytes, and fertilization rate by ICSI. Result(s): Patients in group I (age yrs) were stimulated on average for 10.6 days, and patients in group II (age yrs) for 11.4 days. Total amount of FSH was on average 2,255 IU (I) and 2,720 IU (II) per patient. Average and median numbers of aspirated oocytes were, respectively, 13.1 and 11.5 (I) versus 10.0 and 8.5 (II); 83.7% (I) and 80.4% (II) of the oocytes were mature and viable and could be treated by ICSI. Fertilization rate was 61.0% (I) versus 75.6% (II). Conclusion(s): This pilot study suggests that oocytes can be obtained before cancer treatment efficiently irrespective of the phase of the menstrual cycle. (Fertil Steril Ò 2009;92: Ó2009 by American Society for Reproductive Medicine.) Key Words: Cancer, ovarian stimulation, fertility preservation, luteal phase Increasing survival rates in cancer, new reproductive techniques, and the growing interest in life quality after cancer therapy has put fertility preservation into the focus of oncologists and patients. Long-term survival rates in Hodgkin lymphoma and breast cancer have reached 90% and 80%, respectively. However, many of these women will experience infertility and early menopause due to ovarian toxicity of the chemotherapies or radiation of the pelvis. About 70% of cancer patients within the reproductive life span wish to have children after completion of oncologic therapy (1, 2), and this urges oncologists and reproductive specialists to offer efficient treatment options of fertility protection for cancer therapy. Recent progress in reproductive medicine has led to development of several new fertility-preserving techniques. Patients can be offered ovarian stimulation followed Received April 2, 2008; revised August 2, 2008; accepted August 7, 2008; published online October 20, M.v.W. has nothing to disclose. C.T. has nothing to disclose. T.F. has nothing to disclose. C.Z. has nothing to disclose. B.L. has nothing to disclose. R.P. has nothing to disclose. T.S. has nothing to disclose. Reprint requests: Michael von Wolff, Department of Gynecologic Endocrinology and Reproductive Medicine, University of Heidelberg, Voßstraße 9, Heidelberg, Germany (FAX: ; michael. von.wolff@med.uni-heidelberg.de). by cryopreservation of fertilized oocytes or unfertilized oocytes, cryopreservation of in vitro matured oocytes, cryopreservation and retransplantation of ovarian tissue, and the potential of ovarian protection in vivo by application of GnRH analogues (GnRH-a) (3). Most of these techniques are still experimental or their efficacy is limited. The efficacy of ovarian stimulation, followed by IVF or intracytoplasmic sperm injection (ICSI) and cryopreservation of 2-pronuclear (PN) stages or embryos, is also limited, but this technique is well established and can be performed by all IVF centers. Therefore, this technique can be offered to many patients who ask for fertilitypreserving techniques before chemo- and/or radiotherapy. However, this procedure may require a total of up to 6 weeks, particularly, if patients happen to be in the luteal phase. Conventional stimulation regimes are started in the early follicular phase or after >10 days of GnRH-a pretreatment to ensure complete luteolysis. Madrigrano et al. (4) described in 23 patients treated for breast cancer a time frame of on average 31.3 days (range days) to collect oocytes. Because the time frame until the initiation of the chemo- and/or radiotherapy is limited, this time frame is 1360 Fertility and Sterility â Vol. 92, No. 4, October /09/$36.00 Copyright ª2009 American Society for Reproductive Medicine, Published by Elsevier Inc. doi: /j.fertnstert

2 unacceptable in many patients. Therefore, we developed and tested a stimulation protocol allowing ovarian stimulation within the luteal phase to ensure oocyte collection within 2 weeks in all patients. Cancer patients in the luteal phase were started on GnRH antagonists to down-regulate LH and initiate luteolysis. Simultaneously, follicular stimulation was initiated with recombinant FSH only, thus avoiding exogenous LH activity which might support the luteal body and prevent luteolysis. To systematically analyze the efficacy of this luteal protocol, oocyte numbers and oocyte quality were assessed and compared with cancer patients stimulated in the follicular phase. MATERIALS AND METHODS The prospective controlled study was performed at four university-affiliated women s hospitals, all of which are members of Fertiprotekt (5), a network for fertility preservation before chemo- and radiotherapy. The patients were recruited between January and December Nineteen patients were treated in Heidelberg (Germany), ten in Munich, seven in Tuebingen, and four in Wuerzburg (all in Germany). All patients provided informed consent after counseling for alternative options of fertility preservation techniques according to the guidelines of the network. Patient Population All patients were diagnosed with cancer and were prepared for chemotherapy. Time frame until the initiation of chemotherapy was at least 2 weeks. All patients had regular menstrual cycles. The phase of menstrual cycle was evaluated by the onset of the last menstrual period, by ultrasound, and/or by progesterone concentrations. Study Protocol Patients who were stimulated during the follicular phase received either a short flare-up protocol or an antagonist protocol. In patients who started ovarian stimulation in the late follicular phase, ovulation and formation of a luteal body were excluded by progesterone analysis. For the short protocol, patients received GnRH-a, followed by stimulation with recombinant FSH (Puregon; Organon, Oberschleißheim, Germany; Gonal F; Merck Serono, Munich, Germany) or with highly purified hmg (Menogon HP; Ferring, Kiel, Germany). Initial dosage depended on the age, body mass index, and smoking habits of the patient and was adjusted according to the follicle growth and the E 2 levels. Once an adequate ovarian response was confirmed and mean follicular diameter was R18 mm, FSH and hmg were discontinued and hcg was administered. For the antagonist protocol, patients received first FSH and hmg without GnRH-a. The GnRH antagonists (Cetrorelix; Serono; or Orgalutran; Organon) were added when the ovaries responded to the stimulation (follicular diameter R14 mm or LH >8 IU/L). Ovulation induction was performed by application of hcg. Women in the luteal phase received a modified antagonist protocol, which was first tested in the study center (Heidelberg) and was then adopted by the other participating centers. Patients received GnRH antagonists to induce immediate luteolyis. At the same time, ovarian stimulation was initiated by daily injection of recombinant FSH. The combination of both medications was continued until follicular maturity was assessed and ovulation induction was initiated (as described above). The combination of GnRH antagonists and recombinant FSH resulted in luteolysis within 2 (midluteal phase) to 4 (early luteal phase) days, followed by follicle growth, as documented by progesterone analysis. Transvaginal oocyte retrieval was scheduled h after hcg administration and was performed under general anesthesia. Oocytes were cryopreserved either immediately or after fertilization by IVF or ICSI. A total of 81 oocytes from six patients stimulated in the follicular phase and 47 oocytes from five patients stimulated in the luteal phase were further processed for ICSI by denudation and microscopic evaluation. Only those oocytes that were viable and mature (metaphase II) were fertilized by ICSI. Fertilization was confirmed the following day by microscopic evaluation of the PN stages. Outcome Measures The main outcome measure was number of oocytes collected after ovarian stimulation during the follicular versus luteal phase of the menstrual cycle. Secondary outcome measures were cumulative dosage of recombinant FSH and/or highly purified hmg needed for ovarian stimulation until induction of ovulation, number of days needed for ovarian stimulation until induction of ovulation, percentage of viable (not degenerated) and mature (metaphase II) oocytes allowing fertilization by ICSI, and fertilization rate (percentage of 2PN stage 16 h after ICSI treatment). Statistical Analysis Fisher exact test for independence was used to compare data in both study groups. RESULTS Characteristics of Trial Participants Patients were diagnosed with Hodgkin lymphoma (n ¼ 18), non-hodgkin lymphoma (n ¼ 2), breast cancer (n ¼ 13), and other malignancies, such as rectum carcinoma, astrocytoma, sarcoma, and vasculitis (n ¼ 7) (Table 1). After counseling and offering alternative fertility-preserving techniques, such as cryopreservation of ovarian tissue, patients decided to perform ovarian stimulation and oocyte collection. Average age of patients was 27.6 years (range years) in the follicular phase group and 31.2 years (range years) Fertility and Sterility â 1361

3 1362 von Wolff et al. Luteal phase stimulation Vol. 92, No. 4, October 2009 TABLE 1 Characteristics of trial participants. No. Day of cycle Diagnosis at initiation of gonadotropin administration Age (yrs) Duration of gonadotropin administration (days) Dosage (IU) Aspirated oocytes Fertilization procedure by IVF or ICSI Fertilized oocytes/ oocytes treated Follicular phase group 1 1 HL , ICSI 17/ HL ,625 7 ICSI 4/6 3 1 HL , IVF 7/ Breast ,250 3 ø 5 2 Vasculitis ,250 8 ø 6 2 Breast , ø 7 3 HL ,025 8 IVF 5/8 8 3 Breast ,950 6 IVF HL , IVF 13/ Rectum canc ,350 4 ICSI 2/ Breast ,700 3 ICSI 3/ Sarcoma ,946 2 ø 13 5 NHL , ø 14 5 Astrocytoma , ø 15 5 HL ,025 7 ø 16 6 HL ,800 4 ø 17 7 HL , ø 18 7 HL , ICSI/ø 3/ HL , ICSI 4/ Astrocytoma , IVF/ø 7/ HL ,825 9 ø 22 9 NHL ,175 6 ø 23 9 Breast , IVF/ø 2/ Breast , ø HL , ø HL , ICSI/ø 4/ Breast , ø Sarcoma , ICSI 10/13 Luteal phase group 1 14 Breast ,250 3 IVF 3/ HL ,575 7 ICSI 4/ HL , ø

4 TABLE 1 Continued. Fertilized oocytes/ oocytes treated Fertilization procedure by IVF or ICSI Aspirated oocytes Dosage (IU) Duration of gonadotropin administration (days) Diagnosis at initiation of gonadotropin administration Age (yrs) Day of cycle No Breast ,175 3 ICSI 1/ Breast , ICSI 9/ HL ,911 9 ø 7 20 Breast ,375 7 ICSI/ø 3/ Sarcoma ,911 9 ø 9 22 Breast , ICSI 10/ Breast ,000 7 ICSI 4/ HL ,974 8 ø HL , ø Note: HL ¼ Hodgkin lymphoma; ICSI ¼ intracytoplasmic sperm injection; NHL ¼ non-hodgkin lymphoma; Breast ¼ breast cancer; ø ¼ cryopreservation of unfertilized oocytes. in the luteal phase group (Table 2). Cryopreservation of unfertilized oocytes was performed in six patients in the follicular phase group and five patients in the luteal phase group. Cryopreservation of fertilized oocytes was performed in 18 of the follicular phase group and 6 of the luteal phase group patients, and mixed cryopreservation was performed in 4 of the follicular phase group and 1 of the luteal phase group patients. In most patients, ICSI instead of IVF was performed to reduce the risk of fertilization failure. Outcome of Study Groups First, the number of collected oocytes was evaluated to analyze the efficacy of ovarian stimulation, and second, the viability and maturity of oocytes fertilized by ICSI were evaluated to compare the quality of oocytes in both groups. Patients stimulated in the follicular phase received on average in 10.6 days 2,255 IU FSH/hMG. The dosage and the duration of gonadotropin administration were slightly but not significantly higher in those patients stimulated in the luteal phase (2,720 IU FSH in 11.4 days) (Fig. 1). In contrast, the average number of oocytes was slightly higher in the follicular phase group (13.1 vs. 10.0). The difference of 3.1 oocytes was not significant (P¼.22). Ninety-two oocytes in the follicular phase group and 51 oocytes in the luteal phase group were further processed for fertilization by ICSI. Denudation revealed 83.7% versus 80.4% viable and mature oocytes. Fertilization rate was 61.0% versus 75.6%, and fertilization rate per collected oocytes was 51.1% versus 60.8%. All differences in the study groups were not significant, indicating an equal quality of oocytes in both groups. The data also revealed slightly higher dosage and duration of gonadotropin administration as well as a slightly lower number of oocytes in the luteal phase group. The differences were not significant. DISCUSSION The concept of using GnRH antagonists to induce luteolysis in the midluteal phase was described by Anderson et al. (6). The two described patients had breast cancer and were presented in the midluteal phase. They received GnRH antagonists, resulting in a rapid fall of progesterone followed by menses 2 4 days later. Ovarian stimulation was followed 4 days after application of GnRH antagonists, using hmg 150 IU/day. The patients were stimulated for 9 and 11 days. After induction of ovulation, 8 and 6 oocytes were collected 2 days later, and 6 and 4 oocytes were successfully fertilized by IVF, resulting in a fertilization rate of 71% per collected oocyte. The present case report is to our knowledge the only publication describing initiation of ovarian stimulation during the luteal phase of the cycle in cancer patients. Because ovarian stimulation was preceded by a 4-day pretreatment with Fertility and Sterility â 1363

5 TABLE 2 Outcome of study groups. Follicular phase group Luteal phase group Age of patients (yrs) Aspirated oocytes, average (n) Aspirated oocytes, median (n) Days of stimulation Total dosage (IU) 2, , Oocytes further processed for ICSI treatment (n) Viable metaphase II oocytes (%) a Fertilization rate/icsi treatment (%) Fertilization rate/aspirated oocytes (%) a Note: ICSI ¼ intracytoplasmic sperm injection. a Data are limited to oocytes which were further processed for fertilization by ICSI. GnRH antagonists, the time frame needed for oocyte collection was 16 days. The concept of inducing luteolysis by GnRH antagonists, followed by ovarian stimulation a few days later at the onset of menses has been addressed by other studies involving healthy infertility patients to increase efficacy of conventional IVF. The so-called CRASH protocol (7) avoided midluteal flare up by GnRH-a to reduce differences in size and follicular diameters. The synchronization of the follicles was also expected to increase number of oocytes in poor responders, as described by Humaidan et al. (8). Those authors found in 72 IVF/ICSI cycles significantly more oocytes by using the CRASH protocol compared with the preceding long protocol (4.3 vs. 2.4 oocytes). The concept of our protocol differs from the described CRASH protocol (7) and the protocol by Anderson et al. (6). We did not await luteolysis before starting ovarian stimulation, but we initiated both GnRH antagonist and recombinant FSH at the same time, irrespective of the day of menstrual cycle. This protocol was designed to reduce the time frame needed until oocyte collection without significantly reducing the number of the collected oocytes. Our study revealed oocyte numbers slightly but not significantly lower in patients stimulated during the luteal phase. The study was further extended to analyze the quality of the retrieved oocytes in both groups. To differentiate between the maturity and viability of the retrieved oocytes and the potential of the oocytes to be fertilized, we focused on those oocytes which were fertilized by ICSI. Denudation of oocytes allowed evaluation of maturity and viability before ICSI. Again, both groups showed similar results, indicating that high progesterone concentrations at the beginning of ovarian stimulation in the luteal phase group did not affect oocyte quality. Irrespective of these results, it needs to be addressed that the number of oocytes was too low in several patients to provide a realistic chance to become pregnant after freezing, thawing, and embryo transfer. In 25% of patients, the number of retrieved oocytes was only between two and seven. Assuming a fertilization rate of 50% 60% per retrieved oocyte, as shown in this study, and including the degeneration of the pronucleus stages or embryos during the process of thawing (9) only 1 2 embryos will be transfered in these 25% of patients, with an implantation rate/embryo of around 10% (10). It is therefore of imperative importance to increase the number of retrieved oocytes. Furthermore, it needs to be stressed that these data are only related to fertilized oocytes. The FIGURE 1 Number of oocytes collected after initiation of ovarian stimulation in the follicular (n ¼ 28) versus the luteal (n ¼12) phase. Oocytes (n) n = 28 n = 12 Follicular phase (42) (23) = average Luteal phase 1364 von Wolff et al. Luteal phase stimulation Vol. 92, No. 4, October 2009

6 efficacy of cryopreservation of unfertilized oocytes needs to be further evaluated. The risk of developing a clinically relevant ovarian hyperstimulation syndrome (OHSS) can be expected to be low, because all oocytes will be cryopreserved. In 2007, the registry of the Fertiprotekt network (5) included only 1 in 54 patients (0.5%) in which the chemotherapy needed to be postponed because of severe OHSS. The network has therefore modified its recommendations and suggests ovarian stimulation of patients with high initial dosage of IU of FSH or hmg. Our protocol combined GnRH antagonists and recombinant FSH, because LH, a constituent in hmg, was expected to delay luteolysis. A few days after a luteal body has been formed, luteolysis is delayed by low basal levels of serum LH (11). This implies that luteolyis, induced by reducing LH levels, takes longer during the initial stages of luteal formation. Natural luteal regression is induced by the postreceptor loss of the LH signaling efficiency in the late luteal phase luteal body. To sustain luteal function, as in early pregnancy, high doses of LH/hCG are needed (12). A few days after formation of the luteal body, artificial luteal regression can be initiated by administering GnRH antagonists, reducing the low serum basal levels of LH. Direct effects of GnRH antagonists on the luteal body seem unlikely (13). According to the physiology of luteal formation and luteolyis we expected and confirmed in selected patients a longer time frame needed to induce luteolysis in the early luteal phase (around 4 days) compared with the midluteal phase (around 2 days). This study describes a protocol which allows oocyte retrieval in cancer patients within 2 weeks irrespective of the day of menstrual cycle at first consultation. It also demonstrates that oocyte number is slightly but not significant lower in patients stimulated during the luteal phase. This protocol therefore substantially contributes to the discussion about fertility preservation in cancer patients, because it allows cryopreservation of fertilized oocytes within a time frame acceptable in many cancer diseases. It can be offered to patients with, e.g., Hodgkin lymphoma or estrogen-independent breast cancer. In estrogen-dependant breast cancer it should be considered to combine this protocol with tamoxifen or letrozole to reduce estrogen concentrations (14). REFERENCES 1. Schover LR, Rybicki LA, Martin BA, Bringelsen KA. Having children after cancer. A pilot survey of survivors attitudes and experiences. Cancer 1999;86: Schover LR, Brey K, Lichtin A, Lipshultz LI, Jeha S. Knowledge and experience regarding cancer, infertility, and sperm banking in younger male survivors. J Clin Oncol 2002;20: Lee SJ, Schover LR, Partridge AH, Patrizio P, Wallace WH, Hagerty K, et al. American Society of Clinical Oncology recommendations on fertility preservation in cancer patients. J Clin Oncol 2006;24: Madrigrano A, Westphal L, Wapnir I. Egg retrieval with cryopreservation does not delay breast cancer treatment. Am J Surg 2007;194: Fertiprotekt. Network for fertility preservation before chemo- and radiotherapy. Available at: Accessed 15 March Anderson RA, Kinniburgh D, Baird DT. Preliminary experience of the use of a gonadotrophin-releasing hormone antagonist in ovulation induction/in-vitro fertilization prior to cancer treatment. Hum Reprod 1999;14: Friden BE, Nilsson L. Gonadotrophin-releasing hormone-antagonist luteolysis during the preceding mid-luteal phase is a feasible protocol in ovarian hyperstimulation before in vitro fertilization. Acta Obstet Gynecol Scand 2005;84: Humaidan P, Bungum L, Bungum M, Hald F, Agerholm I, Blaabjerg J, et al. Reproductive outcome using a GnRH antagonist (cetrorelix) for luteolysis and follicular synchronization in poor responder IVF/ICSI patients treated with a flexible GnRH antagonist protocol. Reprod Biomed Online 2005;11: Menezo Y. Cryopreservation of IVF embryos: which stage? Eur J Obstet Gynecol Reprod Biol 2004;113(Suppl 1):S Deutsches IVF-Register. Available at: Accessed 15 March Niswender GD, Juengel JL, Silva PJ, Rollyson MK, McIntush EW. Mechanisms controlling the function and life span of the corpus luteum. Physiol Rev 2000;80: Zeleznik AJ. In vivo responses of the primate corpus luteum to luteinizing hormone and chorionic gonadotropin. Proc Natl Acad Sci U S A 1998;95: Del Canto F, Sierralta W, Kohen P, Mu~noz A, Strauss JF 3rd, Devoto L. Features of natural and gonadotropin-releasing hormone antagonist-induced corpus luteum regression and effects of in vivo human chorionic gonadotropin. J Clin Endocrinol Metab 2007;92: Azim AA, Costantini-Ferrando M, Lostritto K, Oktay K. Relative potencies of anastrozole and letrozole to suppress estradiol in breast cancer patients undergoing ovarian stimulation before in vitro fertilization. J Clin Endocrinol Metab 2007;92: Fertility and Sterility â 1365