Open versus closed oocyte vitrification in an oocyte donation programme: a prospective randomized sibling oocyte study

Human Reproduction, Vol.31, No.2 pp. 377 384, 2016 Advanced Access publication on January 2, 2016 doi:10.1093/humrep/dev321 ORIGINAL ARTICLE Infertility Open versus closed oocyte vitrification in an oocyte donation programme: a prospective randomized sibling oocyte study N. De Munck 1,2, *, S. Santos-Ribeiro 1,3, D. Stoop 1, H. Van de Velde 1,2, and G. Verheyen 1 1 Centre for Reproductive Medicine, UZ Brussel, Laarbeeklaan 101, Brussels, Belgium 2 Reproduction and Genetics, Vrije Universiteit Brussel (VUB), Laarbeeklaan 101, Brussels, Belgium 3 Department of Obstetrics, Gynaecology and Reproductive Medicine, Hospital Universitário de Santa Maria, Avenida Professor Egas Moniz, Lisbon 1649-035, Portugal *Correspondence address. Tel: +32-477-66-94; E-mail: neelke.demunck@uzbrussel.be Submitted on October 7, 2015; resubmitted on November 20, 2015; accepted on November 27, 2015 study question: Is the survival of donor oocytes with the CryotopSC device superior to the survival with the closed CBSvit device? summary answer: The CryotopSC device and the CBSvit device showed similar survival rates. what is known already: Health authorities are cautious about possible cross contamination during liquid nitrogen storage or handling when working with open vitrification devices. At present, the use of open devices is still allowed since little information is available on the efficiency of closed devices. study design, size, duration: A prospective randomized sibling oocyte study was performed in the Centre for Reproductive Medicine (UZBrussel) between January 2014 and July 2015. The survival after warming and the embryological outcome of donor oocytes vitrified using two devices was compared: the CBSvit device (closed vitrification and closed storage) and the CryotopSC device (open vitrification and closed storage). A difference of 10% was defined to prove the superiority of the CryotopSC device. In total, 250 warmed oocytes were needed in each arm. participants/materials, setting, methods: Oocytes from 48 donors were included in the study: 253 vitrified with the CBSvit device and 257 with the CryotopSC device. Equal numbers of oocytes from both devices and originated from the same donor cycle were allocated to each of 78 recipients, in order to exclude donor and recipient (male factor) effects. main results and the role of chance: There were no differences found between the CBSvit and the CryotopSC in terms of survival after warming (93.7 versus 89.9%) or fertilization per injected oocyte (74.3 versus 81.4%). The degeneration rate after ICSI was significantly higher for the CBSvit device: 11.4 versus 6.1% (P ¼ 0.041). A significantly higher number of zygotes in the CryotopSC group finished their first mitosis 25 27 h post-injection (34.1 versus 52.1%, P ¼ 0.001). On Day 3, the overall embryo quality distribution did not vary between groups, but a significantly higher cell number was obtained in the CryotopSC device: 6.8 + 2.8 versus 7.6 + 2.8 (P ¼ 0.01). The utilization rate per mature oocyte, per surviving oocyte or per fertilized oocyte did not differ. The embryos with the highest quality were selected for transfer on Day 3. The clinical pregnancy rate per transfer cycle was 36.5%. limitations, reasons for caution: The results of this study should not be extrapolated to other female groups, since oocytes from young fertile donors were used in this study. wider implications of the findings: In many countries, the use of open devices is still allowed due to the limited reports on the efficiency of closed devices. Knowing the caution of health authorities about the use of open devices, there is an urgent need for efficiency studies with closed devices. The results obtained in the current study shows the efficiency of a safe closed vitrification device, leaving behind any concern about possible cross contamination during handling or storage. study funding/competing interest(s): No funding was obtained. The authors have no conflict of interest to declare. trial registration number: NCT01952184. & The Author 2016. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please email: journals.permissions@oup.com

378 De Munck et al. trial registration date: 24 September 2013. date of first patient s enrolment: 23 January 2014. Key words: open vitrification / closed vitrification / oocytes / CBSvit / CryotopSC Introduction Oocyte vitrification is an established procedure that has replaced the slow freezing procedure in many centres (Gardner et al., 2007; Gook and Edgar, 2007; Edgar and Gook, 2012). It has been clearly proven that the survival rates, embryo developmental capacity and pregnancy rates after vitrification are higher compared with slow freezing (Gardner et al., 2007; Cao et al., 2009, Smith et al., 2010; Cobo and Diaz, 2011). Furthermore, limited but thus far reassuring data are available regarding the obstetric and neonatal outcomes after slow freezing and vitrification of oocytes (Borini et al., 2007; Chian et al., 2008; Noyes et al., 2009; Cai et al., 2012; Cobo et al., 2014). Since the introduction of oocyte vitrification, most successes have been reported with the use of open devices, while closed devices are often still considered experimental (Vajta et al., 2015). However, safety considerations have made a favourable case for the use of closed instead of open devices for oocyte vitrification (Bielanski and Vajta, 2009). There are two major differences between open and closed devices. Firstly, open devices allow direct contact of the oocytes with the liquid nitrogen (LN 2 ), thereby generating very high cooling rates and preventing chilling injury (Vajta and Kuwayama, 2006). In closed devices, the oocytes are not in direct contact with the LN 2 resultingindecreasedcoolingratesdueto thermal isolation by the formation of a nitrogen vapour coat, thereby sometimes questioning whether complete vitrification is indeed obtained. Previous authors have claimed that extremely high cooling rates are a prerequisite to obtain high survival rates for oocytes (Vajta and Kuwayama, 2006). Nevertheless, some centres prefer to use closed devices in order to safeguard the stored human body material. Secondly, open devices have the potential risk of cross contamination during vitrification and LN 2 storage, while the heat sealing of closed devices before vitrification efficiently prevents contamination (Bielanski and Vajta, 2009). However, the use of sterilized LN 2 during vitrification followed by storage in LN 2 vapour offers a solution to this potential risk of contamination. Only two studies have previously reported on the comparison between open and closed devices for oocyte vitrification (Paffoni et al., 2011; Papatheodorou et al., 2013). These studies reported contradictory results for the survival rate, fertilization rate and clinical pregnancy rate. As only scarce data are available on the success rates obtained with closed oocyte vitrification (Stoop et al., 2012; De Munck et al., 2013, 2015a; Papatheodorou et al., 2013), clear evidence from larger prospective trials is currently lacking (Vajta et al., 2015). The purpose of this study was to test the benefit of open vitrification characterized by an extremely high cooling rate. A prospective trial was designed comparing closed (CBSvit) and open (CryotopSC) vitrification on sibling donor oocytes. Materials and Methods For this study, approval was obtained by the Local Ethical Committee of Universitair Ziekenhuis Brussel. This study was registered at clinicaltrials.gov with identification number NCT01952184 (accessed in September 2013). Study design This prospective trial was designed as a superiority trial to find a 10% higher survival rate after open vitrification using CryotopSC (open vitrification, closed storage; Kitazato, Tokyo, Japan) as compared with closed vitrification using the High Security CBSvit device (closed vitrification, closed storage; CryoBiosystems, France). A total of 250 oocytes were needed in each arm to find a significant difference in survival rate of at least 10% in favour of the CryotopSC device: from 75 to 85% (a ¼ 0.05 and power of 80%). The 75% was based on the average results obtained in our laboratory. The policy at our IVF centre is to give at least 6 mature oocytes to each recipient, therefore each oocyte donor with at least six mature oocytes was eligible for this sibling study. According to a computer-generated randomization list, the first half of the donor oocytes were vitrified with one device and the other half with the other device. To exclude the influence of any male factor, all recipients received ideally an equal number of oocytes from each system (unless an odd number of oocytes was assigned or available) (Fig. 1). To avoid inter-operator variability, all vitrification and warming procedures were carried out by a single embryologist. In order to minimize the potential for additional confounding, the same vitrification/warming protocols and media were used for both devices. Main outcome measures The primary end-point was survival rate after warming and the secondary end-points were degeneration and fertilization after ICSI and embryo quality. Finally, pregnancy outcomes were also recorded. Donor and recipient information Fresh oocytes were obtained from 42 donors in 48 donation cycles between January 2014 and November 2014. The following data were collected from oocyte donors: basic donor characteristics, number of donation cycles, pregnancy history, ovarian stimulation characteristics and number of retrieved Figure 1 Flowchart of the randomization. The flowchart gives an example of the distribution of 12 mature oocytes (MII) obtained in one donor cycle to 2 different oocyte recipients. Half of the oocytes were vitrified with the closed CBSvit device (standard device) and the other half with the CryotopSC device. Both recipients received six MII oocytes: three from the CBSvit device and three from the CryotopSC device.

Open versus closed oocyte vitrification 379 oocytes. From oocyte recipients, the following data were obtained: basic demographic characteristics, pregnancy history, endometrium preparation characteristics, semen source used and embryo transfer characteristics. All mature oocytes were vitrified for the bank; for 25 donors multiples of 6 mature oocytes were vitrified for the bank and surplus oocytes were donated to research. Multifollicular ovarian stimulation For 47 out of the 48 donation cycles, a gonadotrophin-releasing hormone (GnRH)-antagonist protocol with recombinant FSH was used. On Day 2 of the menstrual cycle (day 1 of stimulation), injections of recombinant FSH, follitropin alfa (Elonva, MSD) or follitropin beta (Puregon, MSD) were initiated. On Day 7 of the cycle (day 6 of stimulation), subcutaneous administration of GnRH antagonist ganirelix (Orgalutran, MSD) was started at a daily dose of 0.25 mg. From Day 7 of the cycle onwards, ovarian ultrasound scans and blood sampling for estradiol, progesterone, FSH and LH concentrations were performed to monitor and control follicular growth. Oocyte retrieval by transvaginal needle aspiration was performed 36 h after ovulation triggering with triptoreline 0.2 mg injection (Gonapeptyl, Ferring or Decapeptyl, Ipsen). One donor underwent stimulation under a long GnRH-agonist protocol followed by 5000 IU hcg (Pregnyl, MSD) for ovulation triggering. Recipient preparation Preparation of recipients was performed using a standard protocol of GnRH agonist, estrogen and progesterone as detailed elsewhere (Stoop et al., 2012). Summarily, a daily dosage of 0.6 mg Busereline (Suprefact, Sanofi- Aventis, Belgium) was initiated in the midluteal phase of the cycle preceding the embryo transfer cycle. After confirming down-regulation by measuring serum estradiol and progesterone concentrations, estrogen was administered orally using estradiol valerate (Progynova, Bayer) at 2 mg twice a day over 6 days, then increased to 2 mg three times a day for another 7 days. Endometrial thickness was measured on Day 13 of estradiol valerate administration. If the endometrial thickness was at least 7 mm, a daily administration of 600 mg progesterone (Utrogestan, Bessins, Belgium) was started the day after. Oocytes were warmed on the second day of progesterone administration. Oocyte vitrification and warming Cumulus oocyte complexes (COCs) were denuded from surrounding cumulus and corona cells with Cumulase w (80 USP Units/ml, Halozyme Therapeutics Inc., San Diego, CA, USA) (De Vos et al., 2008). After denudation, oocytes were cultured in fertilization medium (Origio Sequential Fert TM, Denmark) until they were vitrified. Vitrification and warming was carried out as previously described (De Munck et al., 2015a) with minor adaptations. The two above described vitrification devices were used within each donation cycle. Oocytes were vitrified individually or in pairs. The Irvine Scientific w Vitrification Freeze Kit (Irvine Scientific, United States) was used for vitrification. The equilibration solution (ES) contained 7.5% (v/v) ethylene glycol (EG), 7.5% (v/v) dimethylsulfoxide (DMSO) in Hepes-buffered M-199 medium supplemented with 20% dextran serum supplement (DSS); the vitrification solution (VS) contained 15% (v/v) EG, 15% (v/v) DMSO and 0.5 M sucrose in supplemented M-199 medium. Oocytes were placed in a droplet of 25 ml HTF-Hepes supplemented with HSA, which was immediately merged with 25 ml ES1 for 2 min at room temperature, followed by a second merging with 25 ml ES2 for 2 min. Subsequently oocytes were transferred into a new 50 ml ES3 droplet for 10 min. The following step was completed within 60 s: two consecutive washes in 50 ml VS, loading of the straw with minimal volume, followed by vitrification. For the CBS vit straw, after loading the oocytes, straws were thermosealed before being plunged into LN 2. For the CryotopSC: after loading the oocytes, straws were immediately plunged into liquid nitrogen, followed by insertion into the straw cap and sealing of the cap. Insertion of the straw into the cap was performed in the vapour phase (21508C) above the LN 2. For warming, the Irvine Scientific w Vitrification Thaw Kit (Irvine Scientific, United States) was used. The thawing solution (TS) contained 1 M sucrose in Hepes-buffered M-199 medium supplemented with 20% DSS. The dilution solution (DS) contained 0.5 M sucrose in the supplemented M-199 medium and the washing solution (WS) was supplemented M-199 medium. Oocytes were immediately placed in 200 ml preheated TS at 378C for 1 min, followed by 3 min in 50 ml DS, and a wash for 5 min in 50 ml WS1 followed by a short wash in WS2; all steps were performed at room temperature. After washing, oocytes were transferred into individual 25 ml droplets of fertilization medium under oil and scored for survival. Subsequently, they were cultured in an incubator with 5% O 2,6%CO 2 and 89% N 2 at 378C. Evaluation of survival Warmed oocytes were considered morphologically survived if they had no dark/degenerated or contracted ooplasm and no cracked zona pellucida. Fertilization and embryo quality After 1.5 h incubation in fertilization medium, ICSI was performed on all surviving oocytes (Van Landuyt et al., 2005) with fresh or frozen partner sperm or donor sperm. The injected oocytes were cultured individually in 25 ml droplets of cleavage medium (Origio Sequential Cleav TM, Denmark) under oil (Ovoil, Vitrolife, Sweden) until Day 3. Fertilization was checked 16 20 h after injection, early cleavage was checked 26 h after injection and embryo development was evaluated on Day 2 and Day 3. The following parameters were scored: number of cells and cell size symmetry, fragmentation, nuclear state of the blastomeres, compaction and cytoplasmic abnormalities (vacuoles, granulation). Based on those parameters, an embryo score was given to each embryo as previously described by De Munck et al. (2015b): excellent, good, moderate or poor. Briefly, excellent embryos had at least 7 cells with,10% fragments; good embryos had at least 6 cells and/or,20% fragments; moderate embryos had at least 4 cells and/or 20 50% fragmentation and/or multinucleation in,50% of the blastomeres and poor embryos showed 50% fragmentation and/or multinucleation. The presence of cytoplasmic anomalies (vacuoles, granulation) and size of the blastomeres were also taken into account. In the morning of Day 3, the embryos selected for intrauterine transfer were placed in 25 ml droplets of blastocyst medium (Origio Sequential Blast TM, Denmark). Supernumerary excellent and good-quality embryos were vitrified on Day 3. Exceptionally, vitrification was performed after extended embryo culture to Day 5 or 6. All fresh embryo transfers were performed on Day 3. The morphologically best embryos either derived from the Cryotop or from the CBSvit arm, were always selected for transfer. The embryologist was blinded for embryo selection. The utilization rate was calculated as the total number of embryos transferred and cryopreserved. Additionally, pregnancy rates per started cycle and per transfer were analyzed. Pregnancy was defined as a positive bhcg blood test 14 days after transfer. Clinical pregnancy was defined as the presence of at least one gestational sac at ultrasonographic visualization; multiple gestational sacs were counted as one clinical pregnancy. Ongoing pregnancy was defined as the presence of at least one viable fetus 9 12 weeks after embryo transfer. Implantation rate was the number of gestational sacs per number of embryos transferred. Statistical analysis Univariate statistical analysis was performed using either the Fisher s Exact test (for categorical variables) or the paired Student s t-test (for continuous variables). P-values below 0.05 were considered statistically significant.

380 De Munck et al. Results A total of 48 donation cycles (42 donors) were carried out in order to obtain the 500 warmed oocytes needed for this prospective study. The donor characteristics are presented in Table I. The oocytes were vitrified within a period of 10 months (January 2014 until November 2014). Out of the 675 mature oocytes obtained in 48 cycles and 599 oocytes were vitrified for the egg donation programme, 298 with the CBSvit device (standard vitrification device) and 301 with the CryotopSC device. The recipient characteristics are presented in Table II. A total of 74 recipients received one or two embryos for transfer. Four recipients did not receive a fresh embryo transfer. Three transfer cancellations were due to a medical reason and the embryos were cryopreserved. One recipient had no embryo transfer because of poor embryo quality. Survival after warming and embryological outcome per vitrification device is shown in Table III. The survival rate obtained with the CBSvit device (93.7%) was not different (P ¼ 0.119) from the survival rate obtained with the CryotopSC device (89.9%). All surviving oocytes were injected. The number of normally fertilized oocytes per surviving/injected oocyte (74.3 and 81.4%, respectively) was comparable (P ¼ 0.064), but the degeneration rate after ICSI was significantly higher (P ¼ 0.041) after vitrification with the CBSvit device (11.4 versus 6.1%). A significantly higher proportion (P ¼ 0.001) of zygotes in the CryotopSC arm showed early cleavage on Day 1 (34.1 versus 52.1%). On Day 3 of development, no difference was found in the distribution of the four embryo qualities (P ¼ 0.338), nor in the distribution of the cell number (P ¼ 0.057) between the devices. However, a significant Table I Donor characteristics. Donor Information No. of donor cycles 48 No. of donors 42 1 donation 36 2 donations 6 Age (average + SD) [range] 28.1 + 3.9 [21 35] BMI 23.7 + 3.6 Ovarian stimulation characteristics Days of stimulation [range] 11.5 + 1.7 [8 15] Total gonadotrophin dose (IU) 2064.1 + 532.0 E 2 peak (pg/ml) 1837.6 + 1038.9 Oocyte retrieval No. of cumulus oocyte complexes 833 Average + SD 17.4 + 9.1 No. of mature oocytes (%) 675 (81.0) Average + SD 14.1 + 6.8 No. of oocytes donated to research* 76 No. of vitrified oocytes (%) 599 (88.8) Average + SD 12.5 + 6.4 IU, internal units; SD, standard deviation. *25 oocyte donors signed the research consent: all multiples of six mature oocytes were vitrified for the bank and the remainingoocytes (1 5) were donatedto research. difference (P ¼ 0.01) in the average cell number on Day 3 was observed: 6.8 in the CBSvit device versus 7.6 in the CryotopSC device. When comparing the utilization rate per warmed oocyte, per surviving oocyte or per fertilized oocyte, no differences were found between the devices. Supplementary Figure S1 shows the oocytes of one recipient during the different steps of the warming procedure for both devices. The results on the pregnancy outcome, stratified per device and per fresh or frozen (FrET) embryo transfer, are presented in Supplementary Table SI. For all recipients with a fresh embryo transfer, a pregnancy rate of 47.3% (35/74) was obtained, with a clinical pregnancy rate of 36.5% (27/74), an ongoing pregnancy rate of 31.1% (23/74) and an implantation rate of 25.4% (31/122). Of the 55 recipients with surplus embryos frozen, 19 were pregnant from the fresh embryo transfer. They had a total of 50 embryos cryopreserved and so far none of those patients have come back for a FrET. For the 36 remaining recipients without an ongoing pregnancy, a total of 83 surplus embryos were cryopreserved and 30 have returned for a FrET. The results of the all FrET cycles are also presented in Supplementary Table SI. The cumulative pregnancy outcomes for all 78 recipients are the following: a pregnancy rate of 55.1% (43/78) with a clinical pregnancy rate of 47.4% (37/78) and an ongoing pregnancy rate of 39.7% (31/78). At this moment, 11 out of 47 recipients have not had an ongoing pregnancy; still they have 27 surplus embryos cryopreserved. There have been 16 live births from 14 recipients; the delivery outcome of 12 recipients is still unknown and 5 pregnancies are still ongoing. Table II Recipient characteristics. Recipient information No. of recipient cycles 78 No. of recipients 78 Age (average + SD) [range] 38.8 + 5.4 [23 46] Partner age (average + SD) 39.4 + 7.6 BMI 24.2 + 4.5 Semen source Fresh autologous sperm 74 Frozen autologous sperm 2 Donor sperm 2 Embryo transfer SET 26 CBSvit 6 CryotopSC 20 DET 48 CBSvit 11 Mixed 24 CryotopSC 13 Average number of embryos transferred 1.6 Cycles with embryo transfer 74 Cycles without embryo transfer 4 Medical reason 3 Poor embryo quality 1 SET, single embryo transfer; DET, double embryo transfer; SD, standard deviation.

Open versus closed oocyte vitrification 381 Table III Embryological data. CBSvit CryotopSC P value... No. of recipient cycles 78 No. of warmed oocytes 510 Survival No. of warmed oocytes 253 257 No. of survived oocytes (%) 237 (93.7) 231 (89.9) 0.119 No. of oocytes not found (%) 0 6 (2.3) Fertilization Fertilized/survived (%) 176 (74.3) 188 (81.4) 0.064 Degenerated (%) 27 (11.4) 14 (6.1) 0.041 Fertilized/warmed (%) 176/253 (69.6) 188/257 (73.2) 0.370 Day 1 Early cleavage 2 cell 60/176 (34.1) 98/188 (52.1) 0.001 Day 3 Embryo Quality 0.338 Excellent (%) 82 (46.6) 91 (48.4) Good (%) 35 (19.9) 34 (18.1) Moderate (%) 30 (17.0) 42 (22.3) Poor (%) 29 (16.5) 21 (11.2) Day 3 cell number 0.057 Fragmented 0 2 1 cell 15 6 2 5 cells 29 30 6 7 cells 39 36 8 cells 61 54.8 cells 30 57 Compacting 2 3 Mean cell number 6.8 + 2.8 7.6 + 2.8 0.01 Embryo utilization No. of embryos transferred on Day 3 52 70 No. of embryos cryopreserved on Day 3 65 63 No. of embryos cryopreserved on Day 5 2 1 No. of embryos cryopreserved on Day 6 1 1 Per mature oocyte (%) 120/253 (47.4) 135/257 (52.5) 0.250 Per surviving oocyte (%) 120/237 (50.6) 135/231 (58.4) 0.090 Per fertilized oocyte (%) 120/176 (68.2) 135/188 (71.8) 0.450 Discussion This prospective comparative trial aimed to show the superiority of the CryotopSC device (open vitrification/closed storage) over the CBSvit device (closed vitrification/closed storage) for oocyte vitrification. No differences were observed regarding the survival rate after oocyte warming, fertilization rate, embryo quality on Day 3 and utilization rate between the two devices. The only significant differences obtained were a higher degeneration rate after ICSI with the CBSvit device, a higher early cleavage rate with the CryotopSC device and a higher cell number on Day 3 with the CryotopSC device. The current results failed to show the superiority of the CryotopSC device over the CBSvit device regarding the primary outcome (survival rate). The study tested the effect of a single variable, the vitrification device, on oocyte survival rate. The study design shows several strengths due to careful elimination of other variables which may cause bias: (i) use of the same vitrification media and protocol for both devices and (ii) exclusion of any influence of a male factor: every recipient received half of the oocytes from the closed device and the other half from the open device, (iii) exclusion of inter-operator variability for the vitrification procedure as well as the warming procedure: all procedures were performed by one single operator, and finally, (iv) exclusion of inter-donor and inter-cycle variability by vitrifying the oocytes of one donor cycle with both devices. This sibling oocyte study, powered to detect a 10% difference in survival rate, failed to show a significant difference in survival rate between the devices: 93.7% in the closed CBSvit device versus 89.9% in the open

382 De Munck et al. CryotopSC device. When compared with the only two reported studies comparing open and closed devices (Paffoni et al., 2011; Papatheodorou et al., 2013), this is the first report showing no difference in survival rate. The two previous reports found a significantly lower survival rate for the closed device: 57.9 versus 82.8% (Paffoni et al., 2011) and 82.9 versus 91.0% (Papatheodorou et al., 2013). Not only is the survival rate in our study very high, it also confirms the reproducibility of the results obtained in our centre: a previous study with the CBSvit device reported a survival rate of 90.2% (Stoop et al., 2012). Moreover, survival rates are comparable to the ones obtained with the open storage Cryotop device, ranging from 84.7 to 96.8% (Nagy et al., 2009; Cobo et al., 2010, 2015; Rienzi et al., 2010, 2012; Trokoudes et al., 2011) and to the survival rates (87.3%) that are published online by the company (www.kitazato-dibimed.com) regarding the efficiency of the CryotopSC device. When considering fertilization rates of the oocytes that survived warming using closed devices, the 74.3% in our study is in between the fertilization rates obtained in the previous studies: 57.6% (Paffoni et al., 2011) and 82.5% (Papatheodorou et al., 2013). In our study, the fertilization rate of the open CryotopSC device (81.4%) seemed higher than in the previous reports (73.0 and 73.4%, respectively). The significantly higher degeneration rate after ICSI in the CBSvit arm (11.4 versus 6.1%) might be related to a higher impact of closed vitrification on the intrinsic oocyte quality after warming, and invisible by light microscopy. This high degeneration rate was also observed with the closed device in the study of Paffoni et al. (2011), 10.6 versus 6.3%, but was not significantly different. In literature, the degeneration rate after ICSI is not always mentioned for the open Cryotop device. Rienzi et al. (2010) reported a low degeneration rate of 3.3%. Since the day of embryo transfer and the embryo scoring systems differ between centres, it is difficult to make comparisons regarding embryo quality. In the current study, the number of excellent and good quality embryos was 46.6 and 19.9% for the CBSvit device and 48.4 and 18.1% for the CryotopSC device. Again, these results are comparable with our previous report showing 66.3% excellent or good quality embryos (Stoop et al., 2012). In this study, the clinical pregnancy could not be stratified per device since mixed double embryo transfers were performed. For all cycles with an embryo transfer, a clinical pregnancy rate of 36.5% (27/74) was obtained. These results are in line with the report by Papatheodorou et al. (2013): 36.0% clinical pregnancy rate per cycle in the closed Vitrisafe device and 28.0% in the open Vitrisafe device. The study of Paffoni et al. (2011) reported an extremely low clinical pregnancy rate per cycle of 7.8% in the closed CryoTip device and 26.4% in the open CryoTop device. Other reports with the Cryotop have reported clinical pregnancy rates per transfer of 55.4% (Cobo et al., 2010), 55.6% (Trokoudes et al., 2011) and 38.5% (Rienzi et al., 2010). It should be mentioned that in all of the above mentioned reports, except for one (1.7; Cobo et al., 2010), on clinical pregnancies, the average number of embryos (2.0) transferred was very high. This number was the lowest (1.6) in our study. A recent publication by Cobo et al. (2015) reported a very high cumulative delivery rate per donation cycle of 78.8%. By looking at the number of oocytes consumed (32 460) and the number of live births (2102), the oocyte-to-baby rate (6.5%) and the number of oocytes needed to achieve a baby (15.4) were calculated. They also showed a tremendous increase in the cumulative live birth as the number of oocytes consumed increases. This rate is rather low if only 5 oocytes are consumed (6.1%) but increases to 39.4% if 10 oocytes are consumed. In our current study a cumulative rate of 39.7% could be obtained with on average 6.5 oocytes consumed per recipient and still 86 embryos are cryopreserved. It is difficult to compare our data with literature reports, as different types of open and closed devices are being used: closed CryoTip versus open CryoTop (with open storage; Paffoni et al., 2011), closed versus open Vitrisafe device (Papatheodorou et al., 2013), and the closed CBSvit versus open CryotopSC (with closed storage) in the current study. Moreover, the study designs were different: the study by Paffoni et al. (2011) was a retrospective inter-patient analysis while the study by Papatheodorou et al. (2013) was a prospective randomized oocyte study. In the latter study, all oocytes from one donor were vitrified with the one type of device and every recipient received oocytes from one type of device, which has the advantage that pregnancy outcomes can be compared between devices. This is different from our prospective study where each recipient received oocytes from both devices. As our study was not powered to detect differences in pregnancy outcome, this parameter is not fully informative. It has been claimed that extremely high cooling rates are a prerequisite for successful survival of oocytes after vitrification (Vajta and Kuwayama, 2006). This is the reason why the present study was designed as a superiority trial. While open devices are able to generate a very high cooling rate (.20 0008C/min) due to the direct contact with LN 2, closed devices are unable to reach this cooling rate due to the thermal isolation. It has been reported that the cooling rate obtained with the closed devices generates a comparable survival rate if the warming rate is sufficiently high (Mazur and Seki, 2011; Seki and Mazur, 2012). This has been confirmed in our previous study (De Munck et al., 2013). A major prerequisite in obtaining high cooling rates is the minimal volume cooling. It is therefore important to aspirate the excess vitrification medium around the oocytes, after having them transferred to the device. Upon warming, the oocytes should stick to the device; oocytes that are immediately released from the device into the thawing solution have the tendency to degenerate. In the current study, oocytes were loaded with a minimal volume of vitrification solution on both devices and high survival rates were obtained. Nevertheless, it appeared that the shrinkage and re-expansion pattern of the oocytes during warming appeared to be different in the two devices. CryotopSC oocytes showed a less extensive shrinkage with the minimal oocyte volume, mostly obtained 1 min earlier than the CBSvit oocytes. Therefore, complete re-expansion was also 1 min earlier in the CryotopSC oocytes. Since oocytes are only visible in a two-dimensional plane, we were unable to calculate the oocyte volume dynamics during the warming procedure. The present observations are only based on microscopic images interpreted by a single operator performing the vitrification and warming procedures. The way of shrinkage and re-expansion does not seem to affect survival, fertilization and embryo quality. However, it might explain the significantly higher degeneration rate with the CBSvit device, even though the oocytes seemed morphologically normal before ICSI. Moreover, it might influence the cleavage rate, since a higher cell number was obtained on Day 3 when oocytes were vitrified with the CryotopSC device. This difference was already visible on Day 1, where oocytes cryopreserved with the CryotopSC device appeared to develop faster to syngamy or first mitosis around 25 27 h post-injection. Whether early cleavage is an important parameter associated with embryo quality and implantation is still debatable, since conflicting data have been reported (Ciray et al., 2006; de los Santos et al., 2014). Further studies are needed to

Open versus closed oocyte vitrification 383 confirm whether these differences in morphokinetics after warming may influence pregnancy outcomes. The potential risk of contamination during liquid nitrogen handling and storage is another point of discussion between proponents of open or closed devices. For open devices, vitrification and warming should be performed in sterile LN 2 and should be changed between patients. So far, the only reported infection in LN 2 occurred between blood samples stored in leaky plastic containers (Tedder et al., 1995). In reproductive biology, however, no single report on disease transmission by LN 2 mediated cross-contamination has been published (Vajta et al., 2015). In closed devices, the risk of cross contamination during storage is completely avoided by safe heat sealing. However, some closed devices, such as CryoTip (Kuwayama et al., 2005) or Cryopette (Parmegiani et al., 2012), are warmed in water baths, which may introduce another source of contamination (Vajta et al., 2015). For warming with the CBSvit device, the straw carrying the oocyte is inside a cover straw that is heat-sealed. Before warming, the lower part containing the oocytes is left in LN 2 while the upper part of the cover straw is removed outside the LN 2 by using a scissor (Knipex Stripper, Knipex, Germany). This reveals a part of the inner straw, which allows it to be taken and moved quickly into the thawing solution. The only possible source of contamination for the CBSvit device may come from the Knipex Stripper to cut the straws (Vajta et al., 2015). A limitation in this study is the sample size calculation. The original improvement was based on average survival rates obtained in our laboratory. The increase from 75 to 85% with the CryotopSC device would therefore be feasible. It is known that the success rates of oocyte cryopreservation programmes are extremely dependent on the skills of the operator. The fact that a single operator performed all vitrification and warming cycles in this study, which is a strength of the study, could explain why the 75% survival rate in the CBSvit device had been an underestimation. This superiority trial was set up to find differences in the survival rate between both devices. Although no differences were found for the primary end-point, it should be mentioned that this is not the only parameter reflective on the efficiency of a vitrification method. The differences obtained in the further development (higher degeneration after ICSI, more early cleavage on Day 1 and a higher cell number on Day 3) in the CryotopSC device cannot be neglected. It is important, and our duty, to detect the origin and further implications of these differences between both devices. Knowing the caution of health authorities regarding the possible contamination with open devices, even though it has never been reported (Vajta et al., 2015), the ultimate goal is to provide a safe and efficient protocol for oocyte donations through vitrification. By applying the current protocol for oocyte vitrification in a closed device, it is possible that alterations to the oocyte s structure and function are induced, that may affect further development and even pregnancy outcome. The application of a three step vitrification protocol (5, 10, 20%) for closed devices, as proposed by Vanderzwalmen et al. (2009), where the oocytes are exposed to a gradual, but higher final concentrations of the cryoprotectants in a shorter time period, could counterbalance the effects of the lower cooling rate in closed devices. To be able to understand these differences, further research is needed. The results obtained in the current prospective controlled trial clearly failed to show the superiority of the open CryotopSC device over the closed CBSvit device. These results are of utmost importance for oocyte vitrification allowing successful use of a safe closed vitrification device, at least in terms of survival. As oocyte survival and embryo quality are not the end-points of infertility treatment, large multicentre RCTs powered to detect differences in live birth rates between open and closed devices are needed. Supplementary data Supplementary data are available at http://humrep.oxfordjournals.org/. Acknowledgements The authors wish to thank the clinical embryologists, laboratory technologists, nurses and clinicians of the Centre for Reproductive Medicine. Authors roles N.D.M.: conception and design, vitrification and warming procedures, data acquisition, analysis and interpretation of data, writing the article. S.-R.S.: statistical analysis and interpretation of data, critical review of the article. D.S.: study design, critical review of the article. H.V.d.V.: study design, critical review of the article. G.V.: study design, interpretation of the data, critical review of the article. Funding No funding was obtained for this study. Conflict of interest None declared. References Bielanski A, Vajta G. Risk of contamination of germplasm during cryopreservation and cryobanking in IVF units. Hum Reprod 2009; 24:2457 2467. Borini A, Bianchi V, Bonu MA, Sciajno R, Sereni E, Cattoli M, Mazzone S, Trevisi MR, Iadarola I, Distratis V et al. Evidence-based clinical outcome of oocyte slow cooling. Reprod Biomed Online 2007;15:175 181. Cai LB, Qian XQ, Wang W, Mao YD, Yan ZJ, Liu CZ, Ding W, Huang J, Chai DC, Chian RC et al. Oocyte vitrification technology has made eggsharing donation easier in China. Reprod Biomed Online 2012;24:186 190. Cao YX, Xing Q, Li L, Cong L, Zhang ZG, Wei ZL, Zhou P. Comparison of survival and embryonic development in human oocytes cryopreserved by slow-freezing and vitrification. Fertil Steril 2009;92:1306 1311. Chian RC, Huang JY, Tan SL, Lucena E, Saa A, Rojas A, Ruvalcaba Castellon LA, Garcia Amador MI, Montoya Sarmeinto JE. Obstetric and perinatal outcome in 200 infants conceived from vitrified oocytes. Reprod Biomed Online 2008;16:608 610. Ciray HN, Karagenç L, Ulug U, Bener F, Bahçeci M. Early cleavage morphology affects the quality and implantation potential of day 3 embryos. Fertil Steril 2006;85:358 365. Cobo A, Diaz C. Clinical application of oocyte vitrification: a systematic review and meta-analysis of randomized controlled trials. Fertil Steril 2011;96:277 285. Cobo A, Meseguer M, Remohí J, Pellicer A. Use of cryo-banked oocytes in an ovum donation programme: a prospective, randomized, controlled, clinical trial. Hum Reprod 2010;25:2239 2246.

384 De Munck et al. Cobo A, Serra V, Garrido N, Olmo I, Pellicer A, Remohí J. Obstetric and perinatal outcome of babies born from vitrified oocytes. Fertil Steril 2014;102:1006 1015. Cobo A, Garrido N, Pellicer A, Remohi J. Six years experience in ovum donation using vitrified oocytes: report of cumulative outcomes, impact of storage time, and development of a predictive model for oocyte survival rate. Fertil Steril 2015;104:1426 1434. de los Santos MJ, Arroyo G, Busquet A, Calderón G, Cuadros J, Hurtado de Mendoza MV, Moragas M, Herrer R, Ortiz A, Pons C et al. A multicenter prospective study to assess the effect of early cleavage on embryo quality, implantation, and live-birth rate. Fertil Steril 2014;101:981 987. De Munck N, Verheyen G, Van Landuyt L, Stoop D, Van de Velde H. Survival and post-warming in vitro competence of human oocytes after high security closed system vitrification. J Assist Reprod Genet 2013;30:361 369. De Munck N, Petrussa L, Verheyen G, Staessen C, Vandeskelde Y, Sterckx J, Bocken G, Jacobs K, Stoop D, De Rycke M et al. Chromosomal meiotic segregation, embryonic developmental kinetics and DNA (hydroxy) methylation analysis consolidate the safety of human oocyte vitrification. Mol Hum Reprod 2015a;21:535 544. De Munck N, Santos-Ribeiro S, Mateizel I, Verheyen G. Reduced blastocyst formation in reduced culture volume. J Assist Reprod Genet 2015b;32: 1365 1370. De Vos A, Van Landuyt L, Van Ranst H, Vandermonde A, D Haese V, Sterckx J, Haentjens P, Devroey P, Van der Elst J. Randomized siblingoocyte study using recombinant human hyaluronidase versus bovinederived Sigma hyaluronidase in ICSI patients. Hum Reprod 2008; 23:1815 1819. Edgar DH, Gook DA. A critical appraisal of cryopreservation (slow cooling versus vitrification) of human oocytes and embryos. Hum Reprod Update 2012;18:536 554. Gardner DK, Sheehan CB, Rienzi L, Katz-Jaffe M, Larman MG. Analysis of oocyte physiology to improve cryopreservation procedures. Theriogenology 2007;67:64 72. Gook DA, Edgar DH. Human oocyte cryopreservation. Hum Reprod Update 2007;13:591 605. Kuwayama M, Vajta G, Ieda S, Kato O. Comparison of open and closed methods for vitrification of human embryos and the elimination of potential contamination. Reprod Biomed Online 2005;11:608 614. Mazur P, Seki S. Survival of mouse oocytes after being cooled in a vitrification solution to 21968C at 958 to 70,0008C/min and warmed at 6108 to 118,0008C/min: a new paradigm for cryopreservation by vitrification. Cryobiology 2011;62:1 7. Nagy ZP, Chang CC, Shapiro DB, Bernal DP, Elsner CW, Mitchell-Leef D, Toledo AA, Kort HI. Clinical evaluation of the efficiency of an oocyte donation program using egg cryo-banking. Fertil Steril 2009;92:520 526. Noyes N, Porcu E, Borini A. Over 900 oocyte cryopreservation babies born with no apparent increase in congenital anomalies. Reprod Biomed Online 2009;18:769 776. Paffoni A, Guarneri C, Ferrari S, Restelli L, Nicolosi AE, Scarduelli C, Ragni G. Effects of two vitrification protocols on the developmental potential of human mature oocytes. Reprod Biomed Online 2011;22:292 298. Papatheodorou A, Vanderzwalmen P, Panagiotidis Y, Prapas N, Zikopoulos K, Georgiou I, Prapas Y. Open versus closed oocyte vitrification system: a prospective randomized sibling-oocyte study. Reprod Biomed Online 2013;26:595 602. Parmegiani L, Accorsi A, Bernardi S, Arnone A, Cognigni GE, Filicori M. A reliable procedure for decontamination before thawing of human specimens cryostored in liquid nitrogen: three washes with sterile liquid nitrogen (SLN2). Fertil Steril 2012;98:870 875. Rienzi L, Romano S, Albricci L, Maggiulli R, Capalbo A, Baroni E, Colamaria S, Sapienza F, Ubaldi F. Embryo development of fresh versus vitrified metaphase II oocytes after ICSI: a prospective randomized sibling-oocyte study. Hum Reprod 2010;25:66 73. Rienzi L, Cobo A, Paffoni A, Scarduelli C, Capalbo A, Vajta G, Remohí J, Ragni G, Ubaldi FM. Consistent and predictable delivery rates after oocyte vitrification: an observational longitudinal cohort multicentric study. Hum Reprod 2012;27:1606 1612. Seki S, Mazur P. Ultra-rapid warming yields high survival of mouse oocytes cooled to 21968c in dilutions of a standard vitrification solution. PLoS One 2012;7:e36058. Smith GD, Serafini PC, Fioravanti J, Yadid I, Coslovsky M, Hassun P, Alegretti JR, Motta EL. Prospective randomized comparison of human oocyte cryopreservation with slow-rate freezing or vitrification. Fertil Steril 2010;94:2088 2095. Stoop D, De Munck N, Jansen E, Platteau P, Van den Abbeel E, Verheyen G, Devroey P. Clinical validation of a closed vitrification system in an oocyte-donation programme. Reprod Biomed Online 2012;24:180 185. Tedder RS1, Zuckerman MA, Goldstone AH, Hawkins AE, Fielding A, Briggs EM, Irwin D, Blair S, Gorman AM, Patterson KG et al. Hepatitis B transmission from contaminated cryopreservation tank. Lancet 1995; 346:137 140. Trokoudes KM, Pavlides C, Zhang X. Comparison outcome of fresh and vitrified donor oocytes in an egg-sharing donation program. Fertil Steril 2011;95:1996 2000. Vajta G, Kuwayama M. Improving cryopreservation systems. Theriogenology 2006;65:236 244. Vajta G, Rienzi L, Ubaldi FM. Open versus closed systems for vitrification of human oocytes and embryos. Reprod Biomed Online 2015;30:325 333. Vanderzwalmen P, Ectors F, Grobet L, Prapas Y, Panagiotidis Y, Vanderzwalmen AStecher A, Frias P, Liebermann J, Zech N. Aspectic vitrification of blastocysts from infertile patients, egg donors and after IVM. Reprod Biomed Online 2009;19:700 707. Van Landuyt L, De Vos A, Joris H, Verheyen G, Devroey P, Van Steirteghem A. Blastocyst formation in in vitro fertilization versus intracytoplasmic sperm injection cycles: influence of the fertilization procedure. Fertil Steril 2005; 83:1397 1403.