- Email: [email protected]
Priming in vitro maturation cycles with gonadotropins: salvage treatment for nonresponding patients
Priming in vitro maturation cycles with gonadotropins: salvage treatment for nonresponding patients Einat Shalom-Paz, M.D.,a Benny Almog, M.D.,a Amir Wiser, M.D.,a Ishai Levin, M.D.,b Shauna Reinblatt, M.D.,a Mousmi Das, M.D.,a Weon-Young Son, Ph.D.,a and Holzer Hananel, M.D.a a Department of Obstetrics and Gynecology, McGill University, Montreal, Quebec, Canada; and b Department of Obstetrics and Gynecology, Lis Maternity Hospital, Tel Aviv Medical Center, Tel Aviv, Israel
Objective: To compare the treatment outcomes in in vitro maturation (IVM) cycles primed with human menopausal gonadotropin with those for pure IVM cycles in patients with polycystic ovary syndrome. Design: Prospective observational. Setting: University-based tertiary medical center. Patient(s): Patients undergoing IVM cycles (primed IVM, 47; pure IVM, 118). Intervention(s): IVM treatment with and without human menopausal gonadotropin stimulation. Main Outcome Measure(s): Pregnancy rates. Result(s): The clinical pregnancy rate demonstrated a tendency toward improvement in the primed IVM group (53.1% versus 43.2%, respectively) with better implantation and delivery rates (20.1% versus 14.4%; 95% confidence intervals 1.0–3.06 and 40.4% versus 24.6%; 95% confidence intervals 0.1–0.8, respectively). We found no significant difference in pure IVM compared with primed IVM in the number of eggs collected, size of leading follicle, fertility rate, cleavage rate, and the number of embryos transferred. Total mature eggs and maturation rate were significantly higher in the group of pure IVM (11 2.1 versus 8.7 0.5 and 68.5% 17.5% versus 60.9% 0.4%, respectively). Importantly, the endometrial thickness was significantly improved in primed IVM cycles (7.9 1.9 mm versus 7.1 0.8 mm), possibly leading to better implantation and pregnancy rates. Conclusion(s): Patients who fail to demonstrate endometrial or follicular growth during IVM cycles may benefit from gonadotropin priming during the same cycle. (Fertil Steril 2011;96:340–3. 2011 by American Society for Reproductive Medicine.) Key Words: In vitro maturation, minimal stimulation, PCOS, pregnancy rate
In vitro maturation (IVM) has emerged in the last few years as a technique that offers advantages over controlled ovarian hyperstimulation cycles mainly because of reduced costs and the elimination of ovarian hyperstimulation syndrome (1). The addition of gonadotropins to IVM cycle protocols (priming IVM cycles) has been suggested (2). Theoretically, this addition may improve embryologic factors such as oocyte maturation rate in vivo and in vitro and endometrial thickness leading to improved implantation and pregnancy rates. Several studies have been performed, with conflicting results: an increased oocyte maturation rate was demonstrated in human menopausal gonadotropin (hMG)–primed IVM cycles in monkeys (3) and humans (4), whereas other studies failed to show such improvement (5–8). Our unit has been performing IVM treatment cycles for patients with polycystic ovary syndrome (PCOS) for the last 6 to 7 years. Some patients with PCOS have demonstrated a ‘‘steady state’’ cycle, in which the follicles do not develop, the endometrium does not thicken, and salvage is needed to improve the poor outcomes of the cycle. In these patients, we use hMG priming during the same IVM cycle (2). Received March 3, 2011; revised May 30, 2011; accepted June 1, 2011; published online June 30, 2011. E.S-P. has nothing to disclose. B.A. has nothing to disclose. A.W. has nothing to disclose. I.L. has nothing to disclose. S.R. has nothing to disclose. M.D. has nothing to disclose. W-Y.S. has nothing to disclose. H.H. has nothing to disclose. The first two authors contributed equally to this work. Reprint requests: Einat Shalom-Paz, M.D., Department of Obstetrics and Gynecology, McGill University, 687 Pine Avenue West, Montreal, Quebec, Canada, H3A 1A1 (E-mail: [email protected]).
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Our aim was to compare the treatment outcomes from IVM cycles primed with hMG with pure IVM cycles in patients with PCOS.
MATERIALS AND METHODS Our study group was composed of 165 IVM cycles that were performed for patients with PCOS in our clinic between January 2008 and June 2010. PCOS was defined according to the Rotterdam criteria (9). Another 125 IVM cycles were performed for other indications (fertility preservation, poor responder’s repeated IVF failures) but were not included in the study group. The Institutional Research Ethics Board of the McGill University Health Center approved the IVM protocols, and all patients provided written informed consent. The IVM cycles were performed as previously described (10, 11). In accordance with our IVM protocol (2), the first ultrasound was performed on day 3 of the cycle for follicular count and endometrial thickness assessment. A second scan was scheduled on day 7 to 9 of the cycle. All of our patients were scheduled to start treatment with pure IVM cycles without hMG support. At the second ultrasound (day 7–9 cycle) scan, patients who did not show any follicular development or endometrial thickening (dominant follicle <10 mm or endometrium <6 mm; n ¼ 47 cycles and n ¼ 45 unique patients) were prescribed 2 to 5 days of gonadotropins. The other group of patients (n ¼ 118 cycles; n ¼ 107 unique patients) continued according to our protocol (2) with no hMG or other support until one of the criteria for hCG stimulation was achieved, either an endometrium of 6 mm or a leading follicle of 10 to 12 mm. Six women from the pure IVM group had a second treatment with primed hMG because of low metaphase II on the day of the collection on the first cycle. There was no cancellation of cycles in either group.
Fertility and Sterility Vol. 96, No. 2, August 2011 Copyright ª2011 American Society for Reproductive Medicine, Published by Elsevier Inc.
0015-0282/$36.00 doi:10.1016/j.fertnstert.2011.06.003
For ovarian stimulation, menotropins 150 IU (FSH 75 IU and LH 75 IU; Repronex; Ferring) or follitropin alfa 150 IU (Gonal F; EMD Serono) was used, starting on day 7 to 12 of the cycle and for a duration of 2 to 5 days. The aim of the treatment was to achieve endometrial thickness of at least 6 mm or a leading follicle of 10 to 12 mm. When either condition was met, 10,000 IU of hCG (Profasi; EMD Serono) was administered. Ovum pickup was scheduled for 38 hours after hCG administration (11). The number of follicles and measurement of follicle diameters were calculated on the day of hCG administration. Collection started with the largest follicle, and subsequent collection of oocytes was performed according to accessibility of the follicles. This was followed by examination of oocyte maturity. To avoid the possibility of missing small cumulus-oocyte complexes, the remaining follicular aspirates were filtered using a cell strainer with 70-mm mesh (BD Biosciences) and washed 3 times with oocyte washing medium (Cooper Surgical). The medium contained HEPES buffer supplemented with recombinant human serum albumin. The oocytes collected were assessed for nuclear maturity under the dissecting microscope with high magnification (80) using the sliding method.If no germinal vesicle was found in the oocyte cytoplasm, the cumulus masses were removed with hyaluronidase and mechanical pipetting, followed by reassessment of maturity. Mature oocytes on the collection day (day 0, 0–6 hours) were inseminated on the same day, whereas the immature oocytes (germinal vesicle or germinal vesicle breakdown stage) were cultured in IVM medium (Cooper Surgical) supplemented with 75 mIU/mL FSH and LH. Following culture on day 1 (24–30 hours), the oocytes were denuded from cumulus cells with hyaluronidase and mechanical pipetting. Immature oocytes remaining at the germinal vesicle or germinal vesicle breakdown stage were further cultured in the same medium, and the meiotic status was reexamined on day 2 (48- to 52-hour culture). Mature oocytes were inseminated by intracytoplasmic sperm injection. The zygotes were cultured in embryo maintenance medium (Cooper Surgical). Embryonic development was assessed on day 2 (41–43 hours) and day 3 (65–67 hours) after insemination according to the regularity of blastomeres, the percentage and pattern of anucleate fragments, and all dysmorphic characteristics of the embryos. We defined embryos as good quality if they had at least 3 cells on day 2 and 6 cells on day 3, contained <20% anucleate fragments, and exhibited no apparent morphological abnormalities. Embryos
showing blastomere multinucleation, poor cell adhesion, uneven cell division, and cytoplasmic abnormalities were defined as low quality. The best-quality embryos were transferred on day 2 or 3 after intracytoplasmic sperm injection. All women underwent serum b-hCG measurement 14 days after ovum pickup. In those with positive hCG, we performed a transvaginal ultrasound examination 2 weeks later. Clinical pregnancy was defined as the presence of an intrauterine gestational sac with fetal heart activity. The clinical pregnancy rate was calculated out of the total cycles started. We collected demographic and treatment outcome data such as the number of oocytes collected and matured in vivo, maturation rate, fertilization and cleavage rate, number of embryos transferred, implantation rate, and pregnancy rate.
Statistics The statistical analysis was carried out using the SPSS software package (version 18; SPSS Inc.). Differences between parameters in the different patient groups were evaluated using t tests and Mann-Whitney tests, as appropriate. Differences between proportions were evaluated using Fisher’s exact test. P<.05 was considered statistically significant.
RESULTS A total of 165 cycles were included in the study group, which was divided according to the cycle approach and treatment into 2 subgroups: pure IVM (n ¼ 118) and primed IVM (n ¼ 47). Of the 47 primed IVM cycles, 17 cycles had thin endometrium, 21 cycles were primed owing to small follicles below 10 mm, and 9 cycles had both endometrial width and follicular size that did not match our criteria. The age of the patients was similar in both groups (32.9 4.9 years versus 32.5 1.1 years for the pure and primed groups, respectively; P¼.57). Other basic characteristics of the patients in each group are shown in Table 1. The number of oocytes collected, fertilization rate, cleavage rate, and the rate of goodquality embryos were comparable (Table 1). We did find, however, significant differences in other embryologic and clinical variables (Table 1). The total number of matured
TABLE 1 Characteristics of clinical and embryologic outcome.
Age (y) Duration of cycle (days) FSH (IU/L) LH (IU/L) BMI (kg/m2) Leading follicle (mm) Endometrial thickness on day of hCG (mm) No. of follow-up days until hCG No. of oocytes collected Total no. of mature oocytes Total no. of day 0 MII oocytes (%) Total no. of day 1 MII oocytes (%) Total no. of day 2 MII oocytes (%) Maturation rate (% SD) Fertilization rate (% SD) Good-quality embryos (% SD) Cleavage rate (% SD) No. of embryos transferred
Pure IVM (n [ 118)
Stimulated IVM (n [ 47)
P value
32.9 4.9 38.1 2.2 6.5 4.1 9.1 4.0 23.4 1.3 11.1 4.1 7.1 0.3 8.8 0.5 17.1 1.5 11 0.7 2.5 1.1 (22.7) 5.5 0.4 (50) 3.0 1.4 (27.2) 68.5 3.9 70.5 0.4 43.5 4.5 92.6 5.3 3.4 0.3
32.5 1.1 44.8 8.0 5.9 1.4 5.5 3.5 25.7 4.4 11.1 2.7 7.9 0.3 10.3 2.8 15.3 5.4 8.9 3.6 1.9 4.3 (21.3) 4.6 2.4 (51.6) 2.6 0.4 (29.2) 61.7 4.1 71.5 2.2 47.5 3.2 91.3 6.2 3.3 0.2
NS NS NS NS NS NS .01 NS NS .04 NS NS NS .04 NS NS NS NS
Mean difference (95% CI)
0.8 (0.1–1.5)
4.0 (3.0–6.0)
6.5 (0.0–13.5)
Note: Data are presented as mean SD, unless otherwise indicated. BMI ¼ body mass index; CI ¼ confidence intervals; IVM ¼ in vitro maturation; MII ¼ metaphase II; NS ¼ nonsignificant. Shalom-Paz. Priming of in vitro maturation cycles. Fertil Steril 2011.
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TABLE 2 Pregnancy outcome.
Implantation rate (%) Clinical pregnancy rate per cycle (%) Early miscarriage per cycle (%) Early miscarriage per clinical pregnancy (%) Late miscarriage (12–23 weeks) (%) Delivery rate from OPU (%) Multiple pregnancy rate (%)
Pure IVM (n [ 118)
Stimulated IVM (n [ 47)
P value
56/400 (14.4) 51/117 (43.6) 14/117 (12) 14/51 (27.4) 1/117 (0.9) 36/117 (30.8) 13/51 (25.5)
31/154 (20.1) 25/47 (53.1) 5/47 (10.6) 5/25 (20) 1/47 (2.1) 19/47 (40.4) 6/25 (24)
.02 NS NS NS NS .04 NS
Odds ratio (95% CI) 1.8 (1.0–3.06)
0.3 (0.1–0.8)
Note: CI ¼ confidence intervals; IVM ¼ in vitro maturation; OPU ¼ ovum pickup. Shalom-Paz. Priming of in vitro maturation cycles. Fertil Steril 2011.
oocytes and the maturation rate were higher in pure IVM cycles than in the primed IVM cycles (11 0.7 versus 8.9 3.6; P¼.04; 95% confidence intervals [CI] 4.0 [3.0–6.0] and 68.5% 3.9% versus 61.7% 4.1%; P¼.05; 95% CI 6.5 [0.0–13.5], respectively). Importantly, although the size of the leading follicle did not seem to be different (11.1 4.1 mm versus 11.1 2.7 mm; P¼.99), the endometrial thickness was significantly higher in the primed IVM group compared with that in the pure IVM group (7.9 0.3 mm versus 7.1 0.3 mm; P¼.01; 95% CI 0.8 [0.1–1.5]). Interestingly, although the number of transferred embryos was comparable between the 2 groups, the implantation rate and delivery rate were higher in primed IVM cycles compared with that in pure IVM cycles (20.1% versus 14.4%; P¼.02; odds ratio [95% CI] 1.8 [1.0–3.06] and 40.4% versus 24.5%; P¼.04; odds ratio [95% CI] 0.3 [0.1–0.8]) (Table 2).
DISCUSSION Our study demonstrated a rescue procedure for patients enrolled to pure IVM cycles but who failed to accomplish a leading follicle or thick endometrium. Previously, such patients were deemed to cycle cancellation. The addition of minimal-dose hMG or follitropin alfa stimulation changed the outcomes for these patients, as we have demonstrated, and allowed them to achieve ovum pickup with good results. We were also able to report a tendency toward higher pregnancy rates in the primed IVM subgroup. It was suggested that embryos derived from IVM cycles have demonstrated lower implantation rates than those derived from IVF (10). One strategy to overcome this incongruity in implantation rate is to increase the number of embryos transferred in IVM cycles (12). In the present study, we demonstrated a significant improvement in implantation rate and delivery rate by priming IVM cycles with gonadotropins (14.4% versus 20.1%; P¼.02 and 24.6% versus 40.4%; P¼.04). Also, an improvement in endometrial thickness was shown in primed IVM cycles (7.9 0.3 mm versus 7.1 0.3 mm; P¼.01). This significant increase in endometrial thickness may have contributed to the better outcomes of the primed IVM subgroup. The miscarriage rate in total was comparable between the 2 subgroup and represented 10% to 12% of the cycles, a result that is in agreement with previous studies that presented a miscarriage rate of up to 53% in patients with PCOS (13). Our results corresponded to those of previous studies demonstrating that short and minimal FSH priming did not increase the number of oocytes obtained per aspiration and did not improve oocyte maturation, cleavage rates, or embryonic development (2, 6). They did
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not pose added risk to patients with ovarian hyperstimulation syndrome. Mikkelsen and Lindenberg (7) reported also that oocyte maturation and implantation rates were significantly improved by recombinant FSH priming before harvest of immature oocytes from patients with PCOS. We did not demonstrate better maturation rates in the primed IVM group, but the implantation rate was significantly improved. There is conflicting evidence regarding FSH priming before immature oocyte retrieval. Animal and human studies have suggested that administration of FSH for few days in the early follicular phase or late luteal phase would increase the number of competent oocytes retrieved (5, 6, 14, 15). According to our data, the addition hMG for a short duration (2–5 days) before hCG administration may enhance endometrial development and implantation rate to a degree that will contribute to enhancement of outcomes of pure IVM cycles. In this study, we did not demonstrate any difference in the duration of the cycle, and the 2 groups had oligomenorrhea (38.1 2.2 days versus 44.8 8.0 days) (Table 1). Both groups met the criteria for ovum pickup, and then ET was completed. There were no dropouts from the treatment. We showed as well that despite the unfavorable prognosis of the group who needed priming, they had better clinical results. Priming with gonadotropins was shown to have no side effect and could improve the clinical outcome. We so far use the pure IVM treatment for patients with PCOS, but it makes sense to consider adding the minimal stimulation with gonadotropins to this group, mainly because of the improvement in endometrial thickness. It is of utmost importance to define criteria for the selection of specific treatment protocols. The morphological and endocrinological characteristics may suggest that the patient is a good candidate for IVM, but it is the objective indicators of success, such as the number of growing follicles, number of dominant follicles, and endometrial thickness, that actually affect the end points of the treatment such as the number of retrieved oocytes, maturation rate, fertilization rate, and mainly implantation and pregnancy outcomes. We are sometimes faced with situations for which the selected protocol was not ideal. For patients who did not respond as we anticipated, we have to find a quick and efficient solution. Priming IVM cycles with hMG may change the unwanted results. In conclusion, our results suggested that priming IVM cycles with hMG may serve to salvage pure IVM cycles destined to fail. We demonstrated in such patients that instead of canceling the cycle, the addition of minimal-dose FSH resulted in significantly better implantation and pregnancy rates. Vol. 96, No. 2, August 2011
REFERENCES 1. Shalom-Paz E, Almog B, Shehata F, Huang J, Holzer H, Chian RC, et al. Fertility preservation for breast-cancer patients using IVM followed by oocyte or embryo vitrification. Reprod Biomed Online 2010;21:566–71. 2. Fadini R, Dal Canto MB, Mignini Renzini M, Brambillasca F, Comi R, Fumagalli D, et al. Effect of different gonadotrophin priming on IVM of oocytes from women with normal ovaries: a prospective randomized study. Reprod Biomed Online 2009;19:343–51. 3. Younis AI, Sehgal PK, Biggers JD. Antral follicle development and in-vitro maturation of oocytes from macaques stimulated with a single subcutaneous injection of pregnant mare’s serum gonadotrophin. Hum Reprod 1994;9:2130–4. 4. Wynn P, Picton HM, Krapez JA, Rutherford AJ, Balen AH, Gosden RG. Pretreatment with follicle stimulating hormone promotes the numbers of human oocytes reaching metaphase II by in-vitro maturation. Hum Reprod 1998;13:3132–8. 5. Suikkari AM, Tulppala M, Tuuri T, Hovatta O, Barnes F. Luteal phase start of low-dose FSH priming
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of follicles results in an efficient recovery, maturation and fertilization of immature human oocytes. Hum Reprod 2000;15:747–51. Mikkelsen AL, Smith SD, Lindenberg S. In-vitro maturation of human oocytes from regularly menstruating women may be successful without follicle stimulating hormone priming. Hum Reprod 1999;14: 1847–51. Mikkelsen AL, Lindenberg S. Benefit of FSH priming of women with PCOS to the in vitro maturation procedure and the outcome: a randomized prospective study. Reproduction 2001;122:587–92. Lin YH, Hwang JL, Huang LW, Mu SC, Seow KM, Chung J, et al. Combination of FSH priming and hCG priming for in-vitro maturation of human oocytes. Hum Reprod 2003;18:1632–6. Thessaloniki ESHRE/ASRM-Sponsored PCOS Consensus Workshop Group. Consensus on infertility treatment related to polycystic ovary syndrome. Fertil Steril 2008;89:505–22. Son WY, Chung JT, Herrero B, Dean N, Demirtas E, Holzer H, et al. Selection of the optimal day for oo-
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cyte retrieval based on the diameter of the dominant follicle in hCG-primed in vitro maturation cycles. Hum Reprod 2008;23:2680–5. Son WY, Chung JT, Chian RC, Herrero B, Demirtas E, Elizur S, et al. A 38 h interval between hCG priming and oocyte retrieval increases in vivo and in vitro oocyte maturation rate in programmed IVM cycles. Hum Reprod 2008;23:2010–6. Reinblatt SL, Buckett W. In vitro maturation for patients with polycystic ovary syndrome. Semin Reprod Med 2008;26:121–6. Balen AH, MacDougall J, Tan SL. The influence of the number of embryos transferred in 1060 in-vitro fertilization pregnancies on miscarriage rates and pregnancy outcome. Hum Reprod 1993;8:1324–8. Dizerega GS, Hodgen GD. Follicular phase treatment of luteal phase dysfunction. Fertil Steril 1981;35: 428–32. Gougeon A, Testart J. Influence of human menopausal gonadotropin on the recruitment of human ovarian follicles. Fertil Steril 1990;54:848–52.
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Objective: To compare the treatment outcomes in in vitro maturation (IVM) cycles primed with human menopausal gonadotropin with those for pure IVM cycles in patients with polycystic ovary syndrome. Design: Prospective observational. Setting: University-based tertiary medical center. Patient(s): Patients undergoing IVM cycles (primed IVM, 47; pure IVM, 118). Intervention(s): IVM treatment with and without human menopausal gonadotropin stimulation. Main Outcome Measure(s): Pregnancy rates. Result(s): The clinical pregnancy rate demonstrated a tendency toward improvement in the primed IVM group (53.1% versus 43.2%, respectively) with better implantation and delivery rates (20.1% versus 14.4%; 95% confidence intervals 1.0–3.06 and 40.4% versus 24.6%; 95% confidence intervals 0.1–0.8, respectively). We found no significant difference in pure IVM compared with primed IVM in the number of eggs collected, size of leading follicle, fertility rate, cleavage rate, and the number of embryos transferred. Total mature eggs and maturation rate were significantly higher in the group of pure IVM (11 2.1 versus 8.7 0.5 and 68.5% 17.5% versus 60.9% 0.4%, respectively). Importantly, the endometrial thickness was significantly improved in primed IVM cycles (7.9 1.9 mm versus 7.1 0.8 mm), possibly leading to better implantation and pregnancy rates. Conclusion(s): Patients who fail to demonstrate endometrial or follicular growth during IVM cycles may benefit from gonadotropin priming during the same cycle. (Fertil Steril 2011;96:340–3. 2011 by American Society for Reproductive Medicine.) Key Words: In vitro maturation, minimal stimulation, PCOS, pregnancy rate
In vitro maturation (IVM) has emerged in the last few years as a technique that offers advantages over controlled ovarian hyperstimulation cycles mainly because of reduced costs and the elimination of ovarian hyperstimulation syndrome (1). The addition of gonadotropins to IVM cycle protocols (priming IVM cycles) has been suggested (2). Theoretically, this addition may improve embryologic factors such as oocyte maturation rate in vivo and in vitro and endometrial thickness leading to improved implantation and pregnancy rates. Several studies have been performed, with conflicting results: an increased oocyte maturation rate was demonstrated in human menopausal gonadotropin (hMG)–primed IVM cycles in monkeys (3) and humans (4), whereas other studies failed to show such improvement (5–8). Our unit has been performing IVM treatment cycles for patients with polycystic ovary syndrome (PCOS) for the last 6 to 7 years. Some patients with PCOS have demonstrated a ‘‘steady state’’ cycle, in which the follicles do not develop, the endometrium does not thicken, and salvage is needed to improve the poor outcomes of the cycle. In these patients, we use hMG priming during the same IVM cycle (2). Received March 3, 2011; revised May 30, 2011; accepted June 1, 2011; published online June 30, 2011. E.S-P. has nothing to disclose. B.A. has nothing to disclose. A.W. has nothing to disclose. I.L. has nothing to disclose. S.R. has nothing to disclose. M.D. has nothing to disclose. W-Y.S. has nothing to disclose. H.H. has nothing to disclose. The first two authors contributed equally to this work. Reprint requests: Einat Shalom-Paz, M.D., Department of Obstetrics and Gynecology, McGill University, 687 Pine Avenue West, Montreal, Quebec, Canada, H3A 1A1 (E-mail: [email protected]).
340
Our aim was to compare the treatment outcomes from IVM cycles primed with hMG with pure IVM cycles in patients with PCOS.
MATERIALS AND METHODS Our study group was composed of 165 IVM cycles that were performed for patients with PCOS in our clinic between January 2008 and June 2010. PCOS was defined according to the Rotterdam criteria (9). Another 125 IVM cycles were performed for other indications (fertility preservation, poor responder’s repeated IVF failures) but were not included in the study group. The Institutional Research Ethics Board of the McGill University Health Center approved the IVM protocols, and all patients provided written informed consent. The IVM cycles were performed as previously described (10, 11). In accordance with our IVM protocol (2), the first ultrasound was performed on day 3 of the cycle for follicular count and endometrial thickness assessment. A second scan was scheduled on day 7 to 9 of the cycle. All of our patients were scheduled to start treatment with pure IVM cycles without hMG support. At the second ultrasound (day 7–9 cycle) scan, patients who did not show any follicular development or endometrial thickening (dominant follicle <10 mm or endometrium <6 mm; n ¼ 47 cycles and n ¼ 45 unique patients) were prescribed 2 to 5 days of gonadotropins. The other group of patients (n ¼ 118 cycles; n ¼ 107 unique patients) continued according to our protocol (2) with no hMG or other support until one of the criteria for hCG stimulation was achieved, either an endometrium of 6 mm or a leading follicle of 10 to 12 mm. Six women from the pure IVM group had a second treatment with primed hMG because of low metaphase II on the day of the collection on the first cycle. There was no cancellation of cycles in either group.
Fertility and Sterility Vol. 96, No. 2, August 2011 Copyright ª2011 American Society for Reproductive Medicine, Published by Elsevier Inc.
0015-0282/$36.00 doi:10.1016/j.fertnstert.2011.06.003
For ovarian stimulation, menotropins 150 IU (FSH 75 IU and LH 75 IU; Repronex; Ferring) or follitropin alfa 150 IU (Gonal F; EMD Serono) was used, starting on day 7 to 12 of the cycle and for a duration of 2 to 5 days. The aim of the treatment was to achieve endometrial thickness of at least 6 mm or a leading follicle of 10 to 12 mm. When either condition was met, 10,000 IU of hCG (Profasi; EMD Serono) was administered. Ovum pickup was scheduled for 38 hours after hCG administration (11). The number of follicles and measurement of follicle diameters were calculated on the day of hCG administration. Collection started with the largest follicle, and subsequent collection of oocytes was performed according to accessibility of the follicles. This was followed by examination of oocyte maturity. To avoid the possibility of missing small cumulus-oocyte complexes, the remaining follicular aspirates were filtered using a cell strainer with 70-mm mesh (BD Biosciences) and washed 3 times with oocyte washing medium (Cooper Surgical). The medium contained HEPES buffer supplemented with recombinant human serum albumin. The oocytes collected were assessed for nuclear maturity under the dissecting microscope with high magnification (80) using the sliding method.If no germinal vesicle was found in the oocyte cytoplasm, the cumulus masses were removed with hyaluronidase and mechanical pipetting, followed by reassessment of maturity. Mature oocytes on the collection day (day 0, 0–6 hours) were inseminated on the same day, whereas the immature oocytes (germinal vesicle or germinal vesicle breakdown stage) were cultured in IVM medium (Cooper Surgical) supplemented with 75 mIU/mL FSH and LH. Following culture on day 1 (24–30 hours), the oocytes were denuded from cumulus cells with hyaluronidase and mechanical pipetting. Immature oocytes remaining at the germinal vesicle or germinal vesicle breakdown stage were further cultured in the same medium, and the meiotic status was reexamined on day 2 (48- to 52-hour culture). Mature oocytes were inseminated by intracytoplasmic sperm injection. The zygotes were cultured in embryo maintenance medium (Cooper Surgical). Embryonic development was assessed on day 2 (41–43 hours) and day 3 (65–67 hours) after insemination according to the regularity of blastomeres, the percentage and pattern of anucleate fragments, and all dysmorphic characteristics of the embryos. We defined embryos as good quality if they had at least 3 cells on day 2 and 6 cells on day 3, contained <20% anucleate fragments, and exhibited no apparent morphological abnormalities. Embryos
showing blastomere multinucleation, poor cell adhesion, uneven cell division, and cytoplasmic abnormalities were defined as low quality. The best-quality embryos were transferred on day 2 or 3 after intracytoplasmic sperm injection. All women underwent serum b-hCG measurement 14 days after ovum pickup. In those with positive hCG, we performed a transvaginal ultrasound examination 2 weeks later. Clinical pregnancy was defined as the presence of an intrauterine gestational sac with fetal heart activity. The clinical pregnancy rate was calculated out of the total cycles started. We collected demographic and treatment outcome data such as the number of oocytes collected and matured in vivo, maturation rate, fertilization and cleavage rate, number of embryos transferred, implantation rate, and pregnancy rate.
Statistics The statistical analysis was carried out using the SPSS software package (version 18; SPSS Inc.). Differences between parameters in the different patient groups were evaluated using t tests and Mann-Whitney tests, as appropriate. Differences between proportions were evaluated using Fisher’s exact test. P<.05 was considered statistically significant.
RESULTS A total of 165 cycles were included in the study group, which was divided according to the cycle approach and treatment into 2 subgroups: pure IVM (n ¼ 118) and primed IVM (n ¼ 47). Of the 47 primed IVM cycles, 17 cycles had thin endometrium, 21 cycles were primed owing to small follicles below 10 mm, and 9 cycles had both endometrial width and follicular size that did not match our criteria. The age of the patients was similar in both groups (32.9 4.9 years versus 32.5 1.1 years for the pure and primed groups, respectively; P¼.57). Other basic characteristics of the patients in each group are shown in Table 1. The number of oocytes collected, fertilization rate, cleavage rate, and the rate of goodquality embryos were comparable (Table 1). We did find, however, significant differences in other embryologic and clinical variables (Table 1). The total number of matured
TABLE 1 Characteristics of clinical and embryologic outcome.
Age (y) Duration of cycle (days) FSH (IU/L) LH (IU/L) BMI (kg/m2) Leading follicle (mm) Endometrial thickness on day of hCG (mm) No. of follow-up days until hCG No. of oocytes collected Total no. of mature oocytes Total no. of day 0 MII oocytes (%) Total no. of day 1 MII oocytes (%) Total no. of day 2 MII oocytes (%) Maturation rate (% SD) Fertilization rate (% SD) Good-quality embryos (% SD) Cleavage rate (% SD) No. of embryos transferred
Pure IVM (n [ 118)
Stimulated IVM (n [ 47)
P value
32.9 4.9 38.1 2.2 6.5 4.1 9.1 4.0 23.4 1.3 11.1 4.1 7.1 0.3 8.8 0.5 17.1 1.5 11 0.7 2.5 1.1 (22.7) 5.5 0.4 (50) 3.0 1.4 (27.2) 68.5 3.9 70.5 0.4 43.5 4.5 92.6 5.3 3.4 0.3
32.5 1.1 44.8 8.0 5.9 1.4 5.5 3.5 25.7 4.4 11.1 2.7 7.9 0.3 10.3 2.8 15.3 5.4 8.9 3.6 1.9 4.3 (21.3) 4.6 2.4 (51.6) 2.6 0.4 (29.2) 61.7 4.1 71.5 2.2 47.5 3.2 91.3 6.2 3.3 0.2
NS NS NS NS NS NS .01 NS NS .04 NS NS NS .04 NS NS NS NS
Mean difference (95% CI)
0.8 (0.1–1.5)
4.0 (3.0–6.0)
6.5 (0.0–13.5)
Note: Data are presented as mean SD, unless otherwise indicated. BMI ¼ body mass index; CI ¼ confidence intervals; IVM ¼ in vitro maturation; MII ¼ metaphase II; NS ¼ nonsignificant. Shalom-Paz. Priming of in vitro maturation cycles. Fertil Steril 2011.
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TABLE 2 Pregnancy outcome.
Implantation rate (%) Clinical pregnancy rate per cycle (%) Early miscarriage per cycle (%) Early miscarriage per clinical pregnancy (%) Late miscarriage (12–23 weeks) (%) Delivery rate from OPU (%) Multiple pregnancy rate (%)
Pure IVM (n [ 118)
Stimulated IVM (n [ 47)
P value
56/400 (14.4) 51/117 (43.6) 14/117 (12) 14/51 (27.4) 1/117 (0.9) 36/117 (30.8) 13/51 (25.5)
31/154 (20.1) 25/47 (53.1) 5/47 (10.6) 5/25 (20) 1/47 (2.1) 19/47 (40.4) 6/25 (24)
.02 NS NS NS NS .04 NS
Odds ratio (95% CI) 1.8 (1.0–3.06)
0.3 (0.1–0.8)
Note: CI ¼ confidence intervals; IVM ¼ in vitro maturation; OPU ¼ ovum pickup. Shalom-Paz. Priming of in vitro maturation cycles. Fertil Steril 2011.
oocytes and the maturation rate were higher in pure IVM cycles than in the primed IVM cycles (11 0.7 versus 8.9 3.6; P¼.04; 95% confidence intervals [CI] 4.0 [3.0–6.0] and 68.5% 3.9% versus 61.7% 4.1%; P¼.05; 95% CI 6.5 [0.0–13.5], respectively). Importantly, although the size of the leading follicle did not seem to be different (11.1 4.1 mm versus 11.1 2.7 mm; P¼.99), the endometrial thickness was significantly higher in the primed IVM group compared with that in the pure IVM group (7.9 0.3 mm versus 7.1 0.3 mm; P¼.01; 95% CI 0.8 [0.1–1.5]). Interestingly, although the number of transferred embryos was comparable between the 2 groups, the implantation rate and delivery rate were higher in primed IVM cycles compared with that in pure IVM cycles (20.1% versus 14.4%; P¼.02; odds ratio [95% CI] 1.8 [1.0–3.06] and 40.4% versus 24.5%; P¼.04; odds ratio [95% CI] 0.3 [0.1–0.8]) (Table 2).
DISCUSSION Our study demonstrated a rescue procedure for patients enrolled to pure IVM cycles but who failed to accomplish a leading follicle or thick endometrium. Previously, such patients were deemed to cycle cancellation. The addition of minimal-dose hMG or follitropin alfa stimulation changed the outcomes for these patients, as we have demonstrated, and allowed them to achieve ovum pickup with good results. We were also able to report a tendency toward higher pregnancy rates in the primed IVM subgroup. It was suggested that embryos derived from IVM cycles have demonstrated lower implantation rates than those derived from IVF (10). One strategy to overcome this incongruity in implantation rate is to increase the number of embryos transferred in IVM cycles (12). In the present study, we demonstrated a significant improvement in implantation rate and delivery rate by priming IVM cycles with gonadotropins (14.4% versus 20.1%; P¼.02 and 24.6% versus 40.4%; P¼.04). Also, an improvement in endometrial thickness was shown in primed IVM cycles (7.9 0.3 mm versus 7.1 0.3 mm; P¼.01). This significant increase in endometrial thickness may have contributed to the better outcomes of the primed IVM subgroup. The miscarriage rate in total was comparable between the 2 subgroup and represented 10% to 12% of the cycles, a result that is in agreement with previous studies that presented a miscarriage rate of up to 53% in patients with PCOS (13). Our results corresponded to those of previous studies demonstrating that short and minimal FSH priming did not increase the number of oocytes obtained per aspiration and did not improve oocyte maturation, cleavage rates, or embryonic development (2, 6). They did
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not pose added risk to patients with ovarian hyperstimulation syndrome. Mikkelsen and Lindenberg (7) reported also that oocyte maturation and implantation rates were significantly improved by recombinant FSH priming before harvest of immature oocytes from patients with PCOS. We did not demonstrate better maturation rates in the primed IVM group, but the implantation rate was significantly improved. There is conflicting evidence regarding FSH priming before immature oocyte retrieval. Animal and human studies have suggested that administration of FSH for few days in the early follicular phase or late luteal phase would increase the number of competent oocytes retrieved (5, 6, 14, 15). According to our data, the addition hMG for a short duration (2–5 days) before hCG administration may enhance endometrial development and implantation rate to a degree that will contribute to enhancement of outcomes of pure IVM cycles. In this study, we did not demonstrate any difference in the duration of the cycle, and the 2 groups had oligomenorrhea (38.1 2.2 days versus 44.8 8.0 days) (Table 1). Both groups met the criteria for ovum pickup, and then ET was completed. There were no dropouts from the treatment. We showed as well that despite the unfavorable prognosis of the group who needed priming, they had better clinical results. Priming with gonadotropins was shown to have no side effect and could improve the clinical outcome. We so far use the pure IVM treatment for patients with PCOS, but it makes sense to consider adding the minimal stimulation with gonadotropins to this group, mainly because of the improvement in endometrial thickness. It is of utmost importance to define criteria for the selection of specific treatment protocols. The morphological and endocrinological characteristics may suggest that the patient is a good candidate for IVM, but it is the objective indicators of success, such as the number of growing follicles, number of dominant follicles, and endometrial thickness, that actually affect the end points of the treatment such as the number of retrieved oocytes, maturation rate, fertilization rate, and mainly implantation and pregnancy outcomes. We are sometimes faced with situations for which the selected protocol was not ideal. For patients who did not respond as we anticipated, we have to find a quick and efficient solution. Priming IVM cycles with hMG may change the unwanted results. In conclusion, our results suggested that priming IVM cycles with hMG may serve to salvage pure IVM cycles destined to fail. We demonstrated in such patients that instead of canceling the cycle, the addition of minimal-dose FSH resulted in significantly better implantation and pregnancy rates. Vol. 96, No. 2, August 2011
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