- Email: [email protected]
PCOS patients can benefit from in vitro maturation (IVM) of oocytes
European Journal of Obstetrics & Gynecology and Reproductive Biology 165 (2012) 53–56
Contents lists available at SciVerse ScienceDirect
European Journal of Obstetrics & Gynecology and Reproductive Biology journal homepage: www.elsevier.com/locate/ejogrb
PCOS patients can benefit from in vitro maturation (IVM) of oocytes Einat Shalom-Paz a,*, Hananel Holzer a, Weon- Young Son a, Ishai Levin b, Seang Lin Tan a, Benny Almog a a b
Department of Obstetrics and Gynecology, McGill University, Montreal, Quebec, Canada Department of Obstetrics and Gynecology, Lis Medical Center, Tel-Aviv, Israel
A R T I C L E I N F O
A B S T R A C T
Article history: Received 16 September 2011 Received in revised form 8 June 2012 Accepted 1 July 2012
Objective: Our aim was to compare treatment outcome following in vitro maturation (IVM) compared with IVF in patients with polycystic ovarian syndrome (PCOS). Study design: Retrospective evaluation of treatment in women with PCOS who underwent IVM (108) and IVF (108). Results: We found a significant difference in outcome between IVM and IVF, with an increase in the number of mature oocytes derived (10.5 6.5 vs. 15.3 8.8, p < 0.0001) and the cleavage rate (92.4 13.0 vs. 95.2 11.7, p = 0.03) in IVM cycles. Due to the lower implantation rate (16.1% vs. 21.6%, p = 0.07) we tend to transfer more embryos in the IVM group (3.4 0.8 vs. 2.8 1.0, p < 0.0001), but the multiple pregnancy rate in that group was not higher. Importantly, the delivery rate was similar in both groups (26.8% vs. 25%). We also report a yearly change in the success rate of IVM during this period. Conclusions: IVM treatment for PCOS patients may be a valid alternative treatment to IVF with the advantage of eliminating the risk of OHSS and reducing the cost of medication, whilst maintaining high clinical pregnancy rate. ß 2012 Published by Elsevier Ireland Ltd.
Keywords: PCOS In vitro maturation In vitro fertilization Delivery rate
1. Introduction
2. Materials and methods
IVF is a well-established treatment for women with infertility, which leads to enhanced pregnancy and delivery rate using protocols of controlled ovarian hyperstimulation (COH). However, ovarian hyperstimulation syndrome (OHSS) may occur, especially in women with polycystic ovarian syndrome (PCOS). A potentially useful alternative for women with PCOS is earlier retrieval of immature oocytes from small antral follicles, without using hormonal stimulation, followed by in vitro maturation (IVM) of those oocytes. Substituting IVM in PCOS patients eliminates the risk of OHSS and lowers the cost of treatment, but a lower success rate of IVM compared with IVF has been reported [1]. IVM for PCOS patients has been performed in our center for the last ten years. Previously, and similarly to other published date, we reported lower success rates in IVM compared to IVF. Our clinical impression, however, and preliminary data have indicated that the success rate in IVM is consistently improving, approaching results of conventional IVF [2]. Our aim was to compare the treatment outcome of cycles of IVM and IVF in PCOS patients. Additionally, our centre’s five-year IVM outcome is reported.
2.1. Comparison of PCOS IVM patients to PCOS IVF patients
* Corresponding author. Tel.: +1 514 934 1934x36862; fax: +1 514 843 1448. E-mail address: [email protected] (E. Shalom-Paz). 0301-2115/$ – see front matter ß 2012 Published by Elsevier Ireland Ltd. http://dx.doi.org/10.1016/j.ejogrb.2012.07.001
The records of all patients with a diagnosis of PCOS treated by IVM (n = 310 cycles) or IVF (n = 108 patients) between January 2005 to December 2009 were extracted. Each woman in the IVF group (n = 108) was matched by age, date of treatment (as closest) and number of attempt (as possible), to one woman in the IVM group (n = 310), creating two groups of 108 PCOS patients differing by the method of treatment (IVM or IVF) for comparison. During this time period, IVM was offered as first-line treatment for women with PCOS. The advantages and disadvantages of the treatment were discussed with the patients. IVF was offered as an alternative, and patients declining IVM proceeded to IVF. All patients were defined as having PCOS according to the Rotterdam consensus criteria [3]. This study is a retrospective analysis of our centre’s results, and institutional review board approval was therefore deemed unnecessary. However, the protocols for IVM treatment were approved by the Institutional Research Ethics Board of the McGill University Health Center (MUHC) and all patients provided written informed consent. IVF was performed as previously described [1,4–6]. In the IVM arm, the aim of treatment was to achieve endometrial thickness of at least 6 mm or a leading follicle of 10 mm to 12 mm. When either condition was met, 10,000 IU hCG (Profasi; Serono, Oakville,
54
E. Shalom-Paz et al. / European Journal of Obstetrics & Gynecology and Reproductive Biology 165 (2012) 53–56
Ontario, Canada) was administrated. Oocyte retrieval was scheduled 8 h after hCG administration. The number and diameter of the follicles were recorded on the day of hCG administration. Collection began 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. In order to avoid the possibility of missing a small cumulus–oocyte complex (COC), the remaining follicular aspirates were filtered using a cell strainer with 70 mm mesh (Falcon, Becton–Dickinson & Company, NJ, USA) and washed three times with oocyte washing medium (Cooper Surgical, CT, USA). The medium contained HEPES buffer supplemented with recombinant human serum albumin. The oocytes collected were assessed for nuclear maturity under a dissecting microscope with high magnification (80) using the sliding method. If no germinal vesicle (GV) 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 h) were inseminated on the same day, whereas the immature oocytes (GV- or GV breakdown (GVBD)-stage) were cultured in IVM medium (Cooper Surgical, CT, USA) supplemented with 75 mIU/ml FSH and LH. Following culture on day 1 (24–30 h), the oocytes were denuded of cumulus cells with hyaluronidase and mechanical pipetting. Immature oocytes remaining at GV- or GVBD-stage were further cultured in the same medium and the meiotic status was reexamined on day 2 (48–52 h culture). Matured oocytes were inseminated by intracytoplasmic sperm injection (ICSI). The zygotes were cultured in Embryo Maintenance Medium (Cooper Surgical, CT, USA). Embryonic development was assessed on day 2 (41–43 h) and on day 3 (65–67 h) 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 three cells on day 2 and six cells on day 3, contained <20% anucleate fragments and did not exhibit any apparent morphological abnormalities. Embryos showing blastomere multi-nucleation, 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 ICSI. All women underwent serum b-human chorionic gonadotropin (b-hCG) measurement 14 days after ovum pick-up (OPU). In those with positive hCG, we performed a transvaginal ultrasound examination 2 weeks later. Clinical pregnancy was defined as the presence of intrauterine gestational sac with fetal heart activity. The clinical pregnancy rate was calculated for the total number of cycles started. Data collected included the following: demographics, concomitant cause of infertility, treatment protocol for IVF patients, and treatment outcome including: number of eggs collected, maturation, fertilization and cleavage rate, number of embryos transferred, implantation, pregnancy and live birth rate. 2.2. Summary of IVM results 2005–2009 All IVM PCOS (n = 310) cycles from 2005 to 2009 were analyzed in terms of cycle outcome. All other etiologies for IVM in our center, such as fertility preservation for cancer patients, poor ovarian reserve, and previous maturation failure in IVF cycles, were not included in the summary results. 2.3. Statistics Statistical analysis was carried out using the SPSS software package (SPSS Inc., Chicago, IL). Differences between parameters in the different patient groups were evaluated using the t-test and Mann Whitney test where appropriate. Differences between
proportions were evaluated using the Fisher exact test. p < 0.05 was considered as statistically significant. 3. Results A total of 108 patients were included in each group. In the IVM group all patients were in their first treatment; in the IVF group 75% were in their first cycle and 25% in their second cycle of IVF. In the IVF group 23.1% of women had male factor infertility in addition to PCOS, while in the IVM group 12.03% of women presented with more than one infertility cause (p = 0.03). In the IVF group 14.81% were treated with an antagonist protocol and the remainder with a long agonist protocol. The number of oocytes collected was comparable between the two groups, but there were significant differences in most embryological variables including: total number of matured oocytes (10.5 6.5 vs. 15.3 8.8, p < 0.0001 respectively), fertilization rate (70.2 20.2 vs. 68.7 22.1, p < 0.0001 respectively), cleavage rate (92.4 13.0 vs. 95.2 11.7; p < 0.03 respectively). The number of embryo transferred was significantly higher in the IVM group (3.4 0.8 vs. 2.8 1.0, p < 0.0001 respectively) due to previous experience in our unit [1] with lower implantation rate (16.1 vs. 21.6, p = 0.07 in present study, respectively). The pregnancy rate and live birth rate were comparable (Table 1). 3.1. Analyzing IVM results 2005–2009 Table 2 presents year by year embryological and cycle outcome data of IVM treatments performed during the years 2005–2009 for PCOS patients in our center. Number of oocytes collected, rates of maturation, fertilization and cleavage, number of embryos transferred, and pregnancy and live birth rates are shown. Most of the data regarding embryological variables did not change over the years, but the important outcome parameters such as pregnancy rate and implantation rate showed consistent improvement, with significant change comparing 2005 with 2009 (38.4% vs. 59% p = 0.04, and 7.7% vs. 18.0% p = 0.005 respectively) without an increase in the number of embryos transferred (3.6 0.9 vs. 3.5 0.3). 4. Comments Our study shows comparable cycle outcome for PCOS patients with IVF and IVM. These results were achieved after consistent improvement in IVM outcome in PCO patients during the period of 2005–2009. Table 1 Comparison of IVM to IVF cycles in PCOS patients.
Age No. of oocytes collected* Total No. of MII (matured) oocytes* Day 0 MII oocytes* Day 1 MII oocytes* Day 2 MII oocytes* Fertilization rate (%, SD) Cleavage rate (%, SD) No. of embryo transferred* Implantation rate (%) Pregnancy rate (%) Clinical pregnancy rate (%) Twin pregnancy rate (%) Triplets or quadruplets rate (%) Clinical miscarriage rate Delivery rate (%) *
Mean SD.
IVM
IVF
(n = 108)
(n = 108)
32.9 4.1 17.09 13.6 10.5 6.5 2.2 2.9 5.2 3.6 3.0 3.7 70.2 20.2 92.4 13.0 3.4 0.8 60 (16.1) 52 (48.1) 48 (44.4) 8 (15.3) 2 (3.8) 19(17.5) 29 (26.8)
33.5 4.7 19.4 9.5 15.3 8.8 15.3 8.8 – – 68.7 22.1 95.2 11.7 2.8 1.0 64 (21.6) 48 (44.4) 45 (41.6) 17 (35.4) 1 (2.0) 18(16.6) 27(25.0)
p
NS NS <0.0001 <0.0001 – – NS 0.03 <0.0001 0.07 NS NS 0.02 NS NS NS
E. Shalom-Paz et al. / European Journal of Obstetrics & Gynecology and Reproductive Biology 165 (2012) 53–56
55
Table 2 IVM embryologic and cycle outcome data 2005–2009.
Number of cycles Age* Eggs collected* Matured eggs* M2 at collection day* M2 at day1* M2 at day 2* Fertilization rate (% SD) Cleavage rate (% SD) Embryos transferred Implantation rate (%) Chemical pregnancy rate (%) Live birth rate (%)
2005
2006
2007
2008
2009
p**
39 32 2 19.4 4.5 13.3 3.7 1.2 0.8 7.1 0.1 4.8 2.7 49.9 2.6 89 7.7 3.6 0.9 11/142 (7.7) 15/39 (38.4) 6/39 (15.3)
77 33.1 2.2 16.6 0.4 10.8 1.5 1.3 0.9 5.5 3.1 4 0.7 70.3 4.6 88.4 1.0 3.6 0.9 27/281 (9.6) 27/77 (35) 12/77 (15.5)
60 32.9 0.6 19.8 14.2 13.8 10 2.3 1.6 7.2 5.1 4.2 5.5 71.5 12.5 91 0.6 3.6 0.9 25/214 (11.6) 28/59 (47.4) 15/59 (25.4)
66 33.2 0.2 18.6 8.9 11.7 4.7 1.8 1.2 4.2 0.9 1.9 0.6 67.2 9.0 92.4 5.3 3.5 0.3 26/231 (11.2) 26/65 (40) 14/65 (21.5)
68 32.2 3.7 16.8 9.4 10.0 4.8 2.1 1.9 5.1 3.5 2.7 2.8 69.9 24.6 93.2 13.2 3.4 0.6 36/199 (18) 36/61 (59) 23/61(37.7)
– NS NS NS 0.003 0.021 0.007 0.001 NS NS 0.005 0.04 0.01
NA: not available; NS: not significant. * Mean SD. ** When comparing 2005 and 2009 only.
We are aware of the drawback in the matching methods which led to 25% of the IVF cycles being the second attempt. We prioritized the age and date of the treatment before the attempt. Embryos derived from IVM have demonstrated lower implantation rates than those derived from IVF [7]. One strategy to overcome this challenge is to increase the number of embryos transferred in IVM cycles [1]. However, the implantation rate of IVM embryos in our center has increased over the years from 7.7% (2005) to 18% (2009) (Table 2). In spite of the higher number of embryos transferred after IVM, the rate of twin pregnancy is lower compared to IVF (15.3% vs. 35.4%), due to lower overall implantation rate (16.1% (IVM) vs. 21.6%(IVF)) (Table 1). Importantly, our data show that the most important single outcome – the delivery rate – has improved to parallel the delivery rate of IVF (26.8% vs. 25%, respectively). We are not aware of other studies which have evaluated IVM results over a period of time. We were unable to specify a particular cause for the improvement observed in our results, but small changes in the methods and techniques used may have a considerable effect over time. The following paragraphs will discuss a few minor modifications in the clinical and embryological protocols that have been implemented in our center over this period of time, which may explain the trend observed. 4.1. Size of dominant follicle We recently reported that dominant follicle size of 10–12 mm on the day of hCG administration has been found to be an important factor associated with a higher number of oocytes matured in vivo, higher clinical pregnancy rate and higher implantation rate (compared to other dominant follicle size). IVM cycles were retrospectively analyzed according to dominant follicle (DF) diameter as follows; Group 1: DF diameter 14 mm. We found a positive correlation between DF size and number of in vivo matured oocytes collected (Group 1, 2 and 3 = 6.9, 10.6 and 15.1%, respectively). The rates of IVM, fertilization and embryo development were similar among the sibling immature oocytes collected from the three groups [8]. Accordingly, since 2009 our internal protocols have been altered and we administer hCG when the dominant follicle is 10–12 mm. 4.2. Endometrial preparation There are two optional protocols used to accelerate endometrial growth in IVM cycles—the addition of estradiol or the addition of hMG are reported to have dual effects, priming the follicles and accelerating the endometrial growth [9,10].
We recently compared hMG to micronized 17b estradiol supplementation in IVM cycles. We found equal effects on the thickness of the endometrium, but in the hMG group there were significantly more in vivo matured oocytes, and a trend of higher implantation and clinical pregnancy rate was observed. [9] Wynn et al. reported that hMG given during the early follicular phase increases the number of oocytes retrieved and the maturation rate [10], while others did not show a benefit from pre-treatment with hMG [11,12]. During the time period discussed, hMG was implemented in our internal protocols and presumably contributes to the increasing pregnancy rate observed [9]. We add 150 IU/day of hMG for women with endometrial thickness less than 6 mm on the second cycle scan (cycle day 7–10) for 3–5 days [9]. 4.3. Priming with hCG We have previously published our data concerning the use of hCG to prime the follicle for oocyte retrieval [7,13,14]. We believe that priming with hCG prior to IVM collection has two advantages: it increases the maturation rate of oocytes in vitro, and it increases the number of in vivo matured oocytes. Moreover, extending the interval between hCG administration and the collection to 38 h (instead of 34–36) has been found to increase the implantation rate and the clinical pregnancy rate [7], and has been put into practice in our clinical protocols during this time period. 4.4. Identifying in vivo mature oocytes (mature oocytes on the day of collection) Identifying and fertilizing mature oocytes on the day of IVM collection may also have played a role in improving the outcome of our cycles. It has been shown that identifying mature oocytes on the day of collection (i.e. matured in vivo) yields higher pregnancy rates than oocytes matured after 24 h [7,15]. Prior to 2004–2005, collected IVM oocytes were incubated for 24 h before fertilization. Oocytes that have already matured in vivo might become aged as a result of incubation and thus lose their potential. Implementing our modified laboratory protocol in 2005, which included meticulously searching for any mature oocytes right at the time of collection (under a stereomicroscope using a ‘‘spreading’’ technique [5]), has enabled us to identify and fertilize those high potential oocytes. Additionally, as more experience was gained by our embryology team, extra mature oocytes in vivo were identified i.e., from 1.2 0.8 in vivo matured oocytes on 2005 to 2.1 1.9 on 2009 (p = 0.003).
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4.5. The learning curve effect Introducing new procedures or techniques is usually accompanied by a learning curve. It is clear that over the past 10 years of practising IVM in our clinic, better clinical management and better embryological work have been achieved by learning and accumulating experience. The improvement associated with the learning effect is usually hard to assess or quantify [16]. It is most likely composed of taking small steps, laboratory technique ‘‘secrets’’ and clinical judgment, each contributing its small share. It is important to note that over the same time frame we could not detect any clinical or laboratory changes in our IVF protocols. This is reflected by stability in our IVF program results over the same years. In previous reports [1] clinical miscarriage rates of IVM pregnancies were higher than IVF pregnancies. Our results are in agreement with those previous reports, but without statistical significance (19.3 vs. 12.5 for IVM and IVF respectively). The weaknesses of our study are its retrospective nature and our inability to control for the number of embryos transferred. Our results suggest that IVM may serve as a promising alternative to conventional IVF treatment for women with PCOS, with comparable pregnancy rate, implantation rate and delivery rate. Reaching the desired success rate with IVM, however, will be achieved through continuous learning, perseverance and improvement. References [1] Reinblatt SL, Buckett W. In vitro maturation for patients with polycystic ovary syndrome. Seminars in Reproductive Medicine 2008;26:121–6. [2] Child TJ, Phillips SJ, Abdul-Jalil AK, Gulekli B, Tan SL. A comparison of in vitro maturation and in vitro fertilization for women with polycystic ovaries. Obstetrics and Gynecology 2002;100:665–70. [3] Chian RC. In vitro maturation of immature oocytes for infertile women with PCOS. Reproductive Biomedicine Online 2004;8:547–52.
[4] Consensus on infertility treatment related to polycystic ovary syndrome. Fertility and Sterility 2008; 89:505–22. [5] Son WY, Yoon SH, Lim JH. Effect of gonadotrophin priming on in vitro maturation of oocytes collected from women at risk of OHSS. Reproductive Biomedicine Online 2006;13:340–8. [6] Buckett WM, Chian RC, Dean NL, Sylvestre C, Holzer HE, Tan SL. Pregnancy loss in pregnancies conceived after in vitro oocyte maturation, conventional in vitro fertilization, and intracytoplasmic sperm injection. Fertility and Sterility 2008;90:546–50. [7] Son WY, Chung JT, Chian RC, 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. Human Reproduction 2008;23:2010–6. [8] Son WY, Chung JT, Herrero B, et al. Selection of the optimal day for oocyte retrieval based on the diameter of the dominant follicle in hCG-primed in vitro maturation cycles. Human Reproduction 2008;23:2680–5. [9] Elizur SE, Son WY, Yap R, et al. Comparison of low-dose human menopausal gonadotropin and micronized 17beta-estradiol supplementation in in vitro maturation cycles with thin endometrial lining. Fertility and Sterility 2009;92:907–12. [10] 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. Human Reproduction 1998;13:3132–8. [11] Mikkelsen AL, Smith SD, Lindenberg S. In vitro maturation of human oocytes from regularly menstruating women may be successful without follicle stimulating hormone priming. Human Reproduction 1999;14:1847–51. [12] Trounson A, Anderiesz C, Jones G. Maturation of human oocytes in vitro and their developmental competence. Reproduction 2001;121:51–75. [13] Chian RC, Buckett WM, Tulandi T, Tan SL. Prospective randomized study of human chorionic gonadotrophin priming before immature oocyte retrieval from unstimulated women with polycystic ovarian syndrome. Human Reproduction 2000;15:165–70. [14] Chian RC, Gulekli B, Buckett WM, Tan SL. Priming with human chorionic gonadotropin before retrieval of immature oocytes in women with infertility due to the polycystic ovary syndrome. New England Journal of Medicine 1999;341(1624):1626. [15] Son WY, Chung JT, Demirtas E, et al. Comparison of in vitro maturation cycles with and without in vivo matured oocytes retrieved. Reproductive Biomedicine Online 2008;17:59–67. [16] Ramsay CR, Wallace SA, Garthwaite PH, Monk AF, Russell IT, Grant AM. Assessing the learning curve effect in health technologies. Lessons from the nonclinical literature. International Journal of Technology Assessment in Health Care 2002;18:1–10.
Contents lists available at SciVerse ScienceDirect
European Journal of Obstetrics & Gynecology and Reproductive Biology journal homepage: www.elsevier.com/locate/ejogrb
PCOS patients can benefit from in vitro maturation (IVM) of oocytes Einat Shalom-Paz a,*, Hananel Holzer a, Weon- Young Son a, Ishai Levin b, Seang Lin Tan a, Benny Almog a a b
Department of Obstetrics and Gynecology, McGill University, Montreal, Quebec, Canada Department of Obstetrics and Gynecology, Lis Medical Center, Tel-Aviv, Israel
A R T I C L E I N F O
A B S T R A C T
Article history: Received 16 September 2011 Received in revised form 8 June 2012 Accepted 1 July 2012
Objective: Our aim was to compare treatment outcome following in vitro maturation (IVM) compared with IVF in patients with polycystic ovarian syndrome (PCOS). Study design: Retrospective evaluation of treatment in women with PCOS who underwent IVM (108) and IVF (108). Results: We found a significant difference in outcome between IVM and IVF, with an increase in the number of mature oocytes derived (10.5 6.5 vs. 15.3 8.8, p < 0.0001) and the cleavage rate (92.4 13.0 vs. 95.2 11.7, p = 0.03) in IVM cycles. Due to the lower implantation rate (16.1% vs. 21.6%, p = 0.07) we tend to transfer more embryos in the IVM group (3.4 0.8 vs. 2.8 1.0, p < 0.0001), but the multiple pregnancy rate in that group was not higher. Importantly, the delivery rate was similar in both groups (26.8% vs. 25%). We also report a yearly change in the success rate of IVM during this period. Conclusions: IVM treatment for PCOS patients may be a valid alternative treatment to IVF with the advantage of eliminating the risk of OHSS and reducing the cost of medication, whilst maintaining high clinical pregnancy rate. ß 2012 Published by Elsevier Ireland Ltd.
Keywords: PCOS In vitro maturation In vitro fertilization Delivery rate
1. Introduction
2. Materials and methods
IVF is a well-established treatment for women with infertility, which leads to enhanced pregnancy and delivery rate using protocols of controlled ovarian hyperstimulation (COH). However, ovarian hyperstimulation syndrome (OHSS) may occur, especially in women with polycystic ovarian syndrome (PCOS). A potentially useful alternative for women with PCOS is earlier retrieval of immature oocytes from small antral follicles, without using hormonal stimulation, followed by in vitro maturation (IVM) of those oocytes. Substituting IVM in PCOS patients eliminates the risk of OHSS and lowers the cost of treatment, but a lower success rate of IVM compared with IVF has been reported [1]. IVM for PCOS patients has been performed in our center for the last ten years. Previously, and similarly to other published date, we reported lower success rates in IVM compared to IVF. Our clinical impression, however, and preliminary data have indicated that the success rate in IVM is consistently improving, approaching results of conventional IVF [2]. Our aim was to compare the treatment outcome of cycles of IVM and IVF in PCOS patients. Additionally, our centre’s five-year IVM outcome is reported.
2.1. Comparison of PCOS IVM patients to PCOS IVF patients
* Corresponding author. Tel.: +1 514 934 1934x36862; fax: +1 514 843 1448. E-mail address: [email protected] (E. Shalom-Paz). 0301-2115/$ – see front matter ß 2012 Published by Elsevier Ireland Ltd. http://dx.doi.org/10.1016/j.ejogrb.2012.07.001
The records of all patients with a diagnosis of PCOS treated by IVM (n = 310 cycles) or IVF (n = 108 patients) between January 2005 to December 2009 were extracted. Each woman in the IVF group (n = 108) was matched by age, date of treatment (as closest) and number of attempt (as possible), to one woman in the IVM group (n = 310), creating two groups of 108 PCOS patients differing by the method of treatment (IVM or IVF) for comparison. During this time period, IVM was offered as first-line treatment for women with PCOS. The advantages and disadvantages of the treatment were discussed with the patients. IVF was offered as an alternative, and patients declining IVM proceeded to IVF. All patients were defined as having PCOS according to the Rotterdam consensus criteria [3]. This study is a retrospective analysis of our centre’s results, and institutional review board approval was therefore deemed unnecessary. However, the protocols for IVM treatment were approved by the Institutional Research Ethics Board of the McGill University Health Center (MUHC) and all patients provided written informed consent. IVF was performed as previously described [1,4–6]. In the IVM arm, the aim of treatment was to achieve endometrial thickness of at least 6 mm or a leading follicle of 10 mm to 12 mm. When either condition was met, 10,000 IU hCG (Profasi; Serono, Oakville,
54
E. Shalom-Paz et al. / European Journal of Obstetrics & Gynecology and Reproductive Biology 165 (2012) 53–56
Ontario, Canada) was administrated. Oocyte retrieval was scheduled 8 h after hCG administration. The number and diameter of the follicles were recorded on the day of hCG administration. Collection began 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. In order to avoid the possibility of missing a small cumulus–oocyte complex (COC), the remaining follicular aspirates were filtered using a cell strainer with 70 mm mesh (Falcon, Becton–Dickinson & Company, NJ, USA) and washed three times with oocyte washing medium (Cooper Surgical, CT, USA). The medium contained HEPES buffer supplemented with recombinant human serum albumin. The oocytes collected were assessed for nuclear maturity under a dissecting microscope with high magnification (80) using the sliding method. If no germinal vesicle (GV) 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 h) were inseminated on the same day, whereas the immature oocytes (GV- or GV breakdown (GVBD)-stage) were cultured in IVM medium (Cooper Surgical, CT, USA) supplemented with 75 mIU/ml FSH and LH. Following culture on day 1 (24–30 h), the oocytes were denuded of cumulus cells with hyaluronidase and mechanical pipetting. Immature oocytes remaining at GV- or GVBD-stage were further cultured in the same medium and the meiotic status was reexamined on day 2 (48–52 h culture). Matured oocytes were inseminated by intracytoplasmic sperm injection (ICSI). The zygotes were cultured in Embryo Maintenance Medium (Cooper Surgical, CT, USA). Embryonic development was assessed on day 2 (41–43 h) and on day 3 (65–67 h) 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 three cells on day 2 and six cells on day 3, contained <20% anucleate fragments and did not exhibit any apparent morphological abnormalities. Embryos showing blastomere multi-nucleation, 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 ICSI. All women underwent serum b-human chorionic gonadotropin (b-hCG) measurement 14 days after ovum pick-up (OPU). In those with positive hCG, we performed a transvaginal ultrasound examination 2 weeks later. Clinical pregnancy was defined as the presence of intrauterine gestational sac with fetal heart activity. The clinical pregnancy rate was calculated for the total number of cycles started. Data collected included the following: demographics, concomitant cause of infertility, treatment protocol for IVF patients, and treatment outcome including: number of eggs collected, maturation, fertilization and cleavage rate, number of embryos transferred, implantation, pregnancy and live birth rate. 2.2. Summary of IVM results 2005–2009 All IVM PCOS (n = 310) cycles from 2005 to 2009 were analyzed in terms of cycle outcome. All other etiologies for IVM in our center, such as fertility preservation for cancer patients, poor ovarian reserve, and previous maturation failure in IVF cycles, were not included in the summary results. 2.3. Statistics Statistical analysis was carried out using the SPSS software package (SPSS Inc., Chicago, IL). Differences between parameters in the different patient groups were evaluated using the t-test and Mann Whitney test where appropriate. Differences between
proportions were evaluated using the Fisher exact test. p < 0.05 was considered as statistically significant. 3. Results A total of 108 patients were included in each group. In the IVM group all patients were in their first treatment; in the IVF group 75% were in their first cycle and 25% in their second cycle of IVF. In the IVF group 23.1% of women had male factor infertility in addition to PCOS, while in the IVM group 12.03% of women presented with more than one infertility cause (p = 0.03). In the IVF group 14.81% were treated with an antagonist protocol and the remainder with a long agonist protocol. The number of oocytes collected was comparable between the two groups, but there were significant differences in most embryological variables including: total number of matured oocytes (10.5 6.5 vs. 15.3 8.8, p < 0.0001 respectively), fertilization rate (70.2 20.2 vs. 68.7 22.1, p < 0.0001 respectively), cleavage rate (92.4 13.0 vs. 95.2 11.7; p < 0.03 respectively). The number of embryo transferred was significantly higher in the IVM group (3.4 0.8 vs. 2.8 1.0, p < 0.0001 respectively) due to previous experience in our unit [1] with lower implantation rate (16.1 vs. 21.6, p = 0.07 in present study, respectively). The pregnancy rate and live birth rate were comparable (Table 1). 3.1. Analyzing IVM results 2005–2009 Table 2 presents year by year embryological and cycle outcome data of IVM treatments performed during the years 2005–2009 for PCOS patients in our center. Number of oocytes collected, rates of maturation, fertilization and cleavage, number of embryos transferred, and pregnancy and live birth rates are shown. Most of the data regarding embryological variables did not change over the years, but the important outcome parameters such as pregnancy rate and implantation rate showed consistent improvement, with significant change comparing 2005 with 2009 (38.4% vs. 59% p = 0.04, and 7.7% vs. 18.0% p = 0.005 respectively) without an increase in the number of embryos transferred (3.6 0.9 vs. 3.5 0.3). 4. Comments Our study shows comparable cycle outcome for PCOS patients with IVF and IVM. These results were achieved after consistent improvement in IVM outcome in PCO patients during the period of 2005–2009. Table 1 Comparison of IVM to IVF cycles in PCOS patients.
Age No. of oocytes collected* Total No. of MII (matured) oocytes* Day 0 MII oocytes* Day 1 MII oocytes* Day 2 MII oocytes* Fertilization rate (%, SD) Cleavage rate (%, SD) No. of embryo transferred* Implantation rate (%) Pregnancy rate (%) Clinical pregnancy rate (%) Twin pregnancy rate (%) Triplets or quadruplets rate (%) Clinical miscarriage rate Delivery rate (%) *
Mean SD.
IVM
IVF
(n = 108)
(n = 108)
32.9 4.1 17.09 13.6 10.5 6.5 2.2 2.9 5.2 3.6 3.0 3.7 70.2 20.2 92.4 13.0 3.4 0.8 60 (16.1) 52 (48.1) 48 (44.4) 8 (15.3) 2 (3.8) 19(17.5) 29 (26.8)
33.5 4.7 19.4 9.5 15.3 8.8 15.3 8.8 – – 68.7 22.1 95.2 11.7 2.8 1.0 64 (21.6) 48 (44.4) 45 (41.6) 17 (35.4) 1 (2.0) 18(16.6) 27(25.0)
p
NS NS <0.0001 <0.0001 – – NS 0.03 <0.0001 0.07 NS NS 0.02 NS NS NS
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Table 2 IVM embryologic and cycle outcome data 2005–2009.
Number of cycles Age* Eggs collected* Matured eggs* M2 at collection day* M2 at day1* M2 at day 2* Fertilization rate (% SD) Cleavage rate (% SD) Embryos transferred Implantation rate (%) Chemical pregnancy rate (%) Live birth rate (%)
2005
2006
2007
2008
2009
p**
39 32 2 19.4 4.5 13.3 3.7 1.2 0.8 7.1 0.1 4.8 2.7 49.9 2.6 89 7.7 3.6 0.9 11/142 (7.7) 15/39 (38.4) 6/39 (15.3)
77 33.1 2.2 16.6 0.4 10.8 1.5 1.3 0.9 5.5 3.1 4 0.7 70.3 4.6 88.4 1.0 3.6 0.9 27/281 (9.6) 27/77 (35) 12/77 (15.5)
60 32.9 0.6 19.8 14.2 13.8 10 2.3 1.6 7.2 5.1 4.2 5.5 71.5 12.5 91 0.6 3.6 0.9 25/214 (11.6) 28/59 (47.4) 15/59 (25.4)
66 33.2 0.2 18.6 8.9 11.7 4.7 1.8 1.2 4.2 0.9 1.9 0.6 67.2 9.0 92.4 5.3 3.5 0.3 26/231 (11.2) 26/65 (40) 14/65 (21.5)
68 32.2 3.7 16.8 9.4 10.0 4.8 2.1 1.9 5.1 3.5 2.7 2.8 69.9 24.6 93.2 13.2 3.4 0.6 36/199 (18) 36/61 (59) 23/61(37.7)
– NS NS NS 0.003 0.021 0.007 0.001 NS NS 0.005 0.04 0.01
NA: not available; NS: not significant. * Mean SD. ** When comparing 2005 and 2009 only.
We are aware of the drawback in the matching methods which led to 25% of the IVF cycles being the second attempt. We prioritized the age and date of the treatment before the attempt. Embryos derived from IVM have demonstrated lower implantation rates than those derived from IVF [7]. One strategy to overcome this challenge is to increase the number of embryos transferred in IVM cycles [1]. However, the implantation rate of IVM embryos in our center has increased over the years from 7.7% (2005) to 18% (2009) (Table 2). In spite of the higher number of embryos transferred after IVM, the rate of twin pregnancy is lower compared to IVF (15.3% vs. 35.4%), due to lower overall implantation rate (16.1% (IVM) vs. 21.6%(IVF)) (Table 1). Importantly, our data show that the most important single outcome – the delivery rate – has improved to parallel the delivery rate of IVF (26.8% vs. 25%, respectively). We are not aware of other studies which have evaluated IVM results over a period of time. We were unable to specify a particular cause for the improvement observed in our results, but small changes in the methods and techniques used may have a considerable effect over time. The following paragraphs will discuss a few minor modifications in the clinical and embryological protocols that have been implemented in our center over this period of time, which may explain the trend observed. 4.1. Size of dominant follicle We recently reported that dominant follicle size of 10–12 mm on the day of hCG administration has been found to be an important factor associated with a higher number of oocytes matured in vivo, higher clinical pregnancy rate and higher implantation rate (compared to other dominant follicle size). IVM cycles were retrospectively analyzed according to dominant follicle (DF) diameter as follows; Group 1: DF diameter
We recently compared hMG to micronized 17b estradiol supplementation in IVM cycles. We found equal effects on the thickness of the endometrium, but in the hMG group there were significantly more in vivo matured oocytes, and a trend of higher implantation and clinical pregnancy rate was observed. [9] Wynn et al. reported that hMG given during the early follicular phase increases the number of oocytes retrieved and the maturation rate [10], while others did not show a benefit from pre-treatment with hMG [11,12]. During the time period discussed, hMG was implemented in our internal protocols and presumably contributes to the increasing pregnancy rate observed [9]. We add 150 IU/day of hMG for women with endometrial thickness less than 6 mm on the second cycle scan (cycle day 7–10) for 3–5 days [9]. 4.3. Priming with hCG We have previously published our data concerning the use of hCG to prime the follicle for oocyte retrieval [7,13,14]. We believe that priming with hCG prior to IVM collection has two advantages: it increases the maturation rate of oocytes in vitro, and it increases the number of in vivo matured oocytes. Moreover, extending the interval between hCG administration and the collection to 38 h (instead of 34–36) has been found to increase the implantation rate and the clinical pregnancy rate [7], and has been put into practice in our clinical protocols during this time period. 4.4. Identifying in vivo mature oocytes (mature oocytes on the day of collection) Identifying and fertilizing mature oocytes on the day of IVM collection may also have played a role in improving the outcome of our cycles. It has been shown that identifying mature oocytes on the day of collection (i.e. matured in vivo) yields higher pregnancy rates than oocytes matured after 24 h [7,15]. Prior to 2004–2005, collected IVM oocytes were incubated for 24 h before fertilization. Oocytes that have already matured in vivo might become aged as a result of incubation and thus lose their potential. Implementing our modified laboratory protocol in 2005, which included meticulously searching for any mature oocytes right at the time of collection (under a stereomicroscope using a ‘‘spreading’’ technique [5]), has enabled us to identify and fertilize those high potential oocytes. Additionally, as more experience was gained by our embryology team, extra mature oocytes in vivo were identified i.e., from 1.2 0.8 in vivo matured oocytes on 2005 to 2.1 1.9 on 2009 (p = 0.003).
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4.5. The learning curve effect Introducing new procedures or techniques is usually accompanied by a learning curve. It is clear that over the past 10 years of practising IVM in our clinic, better clinical management and better embryological work have been achieved by learning and accumulating experience. The improvement associated with the learning effect is usually hard to assess or quantify [16]. It is most likely composed of taking small steps, laboratory technique ‘‘secrets’’ and clinical judgment, each contributing its small share. It is important to note that over the same time frame we could not detect any clinical or laboratory changes in our IVF protocols. This is reflected by stability in our IVF program results over the same years. In previous reports [1] clinical miscarriage rates of IVM pregnancies were higher than IVF pregnancies. Our results are in agreement with those previous reports, but without statistical significance (19.3 vs. 12.5 for IVM and IVF respectively). The weaknesses of our study are its retrospective nature and our inability to control for the number of embryos transferred. Our results suggest that IVM may serve as a promising alternative to conventional IVF treatment for women with PCOS, with comparable pregnancy rate, implantation rate and delivery rate. Reaching the desired success rate with IVM, however, will be achieved through continuous learning, perseverance and improvement. References [1] Reinblatt SL, Buckett W. In vitro maturation for patients with polycystic ovary syndrome. Seminars in Reproductive Medicine 2008;26:121–6. [2] Child TJ, Phillips SJ, Abdul-Jalil AK, Gulekli B, Tan SL. A comparison of in vitro maturation and in vitro fertilization for women with polycystic ovaries. Obstetrics and Gynecology 2002;100:665–70. [3] Chian RC. In vitro maturation of immature oocytes for infertile women with PCOS. Reproductive Biomedicine Online 2004;8:547–52.
[4] Consensus on infertility treatment related to polycystic ovary syndrome. Fertility and Sterility 2008; 89:505–22. [5] Son WY, Yoon SH, Lim JH. Effect of gonadotrophin priming on in vitro maturation of oocytes collected from women at risk of OHSS. Reproductive Biomedicine Online 2006;13:340–8. [6] Buckett WM, Chian RC, Dean NL, Sylvestre C, Holzer HE, Tan SL. Pregnancy loss in pregnancies conceived after in vitro oocyte maturation, conventional in vitro fertilization, and intracytoplasmic sperm injection. Fertility and Sterility 2008;90:546–50. [7] Son WY, Chung JT, Chian RC, 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. Human Reproduction 2008;23:2010–6. [8] Son WY, Chung JT, Herrero B, et al. Selection of the optimal day for oocyte retrieval based on the diameter of the dominant follicle in hCG-primed in vitro maturation cycles. Human Reproduction 2008;23:2680–5. [9] Elizur SE, Son WY, Yap R, et al. Comparison of low-dose human menopausal gonadotropin and micronized 17beta-estradiol supplementation in in vitro maturation cycles with thin endometrial lining. Fertility and Sterility 2009;92:907–12. [10] 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. Human Reproduction 1998;13:3132–8. [11] Mikkelsen AL, Smith SD, Lindenberg S. In vitro maturation of human oocytes from regularly menstruating women may be successful without follicle stimulating hormone priming. Human Reproduction 1999;14:1847–51. [12] Trounson A, Anderiesz C, Jones G. Maturation of human oocytes in vitro and their developmental competence. Reproduction 2001;121:51–75. [13] Chian RC, Buckett WM, Tulandi T, Tan SL. Prospective randomized study of human chorionic gonadotrophin priming before immature oocyte retrieval from unstimulated women with polycystic ovarian syndrome. Human Reproduction 2000;15:165–70. [14] Chian RC, Gulekli B, Buckett WM, Tan SL. Priming with human chorionic gonadotropin before retrieval of immature oocytes in women with infertility due to the polycystic ovary syndrome. New England Journal of Medicine 1999;341(1624):1626. [15] Son WY, Chung JT, Demirtas E, et al. Comparison of in vitro maturation cycles with and without in vivo matured oocytes retrieved. Reproductive Biomedicine Online 2008;17:59–67. [16] Ramsay CR, Wallace SA, Garthwaite PH, Monk AF, Russell IT, Grant AM. Assessing the learning curve effect in health technologies. Lessons from the nonclinical literature. International Journal of Technology Assessment in Health Care 2002;18:1–10.