Submitted on January 25, 2016; resubmitted on May 4, 2016; accepted on May 10, 2016

Transcription

1 Human Reproduction, Vol.31, No.8 pp , 2016 Advanced Access publication on June 6, 2016 doi: /humrep/dew127 ORIGINAL ARTICLE Embryology Pregnancy and birth outcomes following fresh or vitrified embryo transfer according to blastocyst morphology and expansion stage, and culturing strategy for delayed development B. Wirleitner 1, *, M. Schuff 1, A. Stecher 1, M. Murtinger 1, and P. Vanderzwalmen 1,2 1 IVF Centers Prof. Zech - Bregenz, Römerstrasse 2, A-6900 Bregenz, Austria 2 Centre Hospitalier Inter Régional Edith Cavell (CHIREC), Braine-l alleud, Bruxelles, Belgium *Correspondence address. Tel: ; Fax: ; b.wirleitner@ivf.at Submitted on January 25, 2016; resubmitted on May 4, 2016; accepted on May 10, 2016 study question: How do live birth rates (LBRs), following fresh and vitrified/warmed embryo transfer, compare according to morphological grade, developmental stage and culturing strategy of human blastocysts in vitro? summaryanswer: Equivalent LBRs were obtained after fresh embryo transfer and after vitrified/warmed embryo transfer of blastocysts of top or non-top quality, while vitrification after prolonged embryo culture of blastocysts with delayed development had a positive impact on LBR. what is known already: Blastocyst morphology correlates with clinical outcome; however, few data are available on vitrified/ warmed embryo transfer using non-top quality blastocysts. The aim of this study was to determine clinical outcomes of non-top quality blastocysts and blastocysts with delayed development that underwent vitrified/warmed embryo transfer. study design, size, duration: This retrospective, single-centre study (conducted January 2009 to June 2013) compared 1010 fresh embryo transfer and 1270 vitrified/warmed embryo transfer of blastocysts originating from the same stimulation cycle. Furthermore, 636 fresh embryo transfers and 304 vitrified/warmed embryo transfer after delayed expansion or blastulation in the same period were also analysed. participants/materials, setting, methods: Clinical outcomes after fresh and vitrified/warmed embryo transfer according to blastocyst morphology were compared in both groups. main results and the role of chance: Similar LBRs after fresh embryo transfer or after vitrified/warmed embryo transfer of top or non-top quality blastocysts were observed. A statistically significant improvement in clinical outcomes was obtained after vitrified/warmed embryo transfer of Day 5 embryos with delayed expansion or blastulation when applying prolonged culture. Our study suggests that vitrification of non-top quality blastocysts as well as delayed cavitating and blastulating Day 5 embryos should be considered in autologous IVF cycles. limitations and reasons for caution: Given that the present retrospective study used aseptic vitrification of blastocysts, the results, particularly the survival rates, may not be fully applicable to other vitrification protocols. The retrospective nature of the study has to be mentioned. wider implications of the findings: Restriction of vitrification to top quality blastocysts mayresult in discarding potentially viable embryos. study funding and competing interest(s): This study was not externally funded. There are no conflicts of interest to declare. Key words: freeze-all / live birth rate / cryo-cycle / aseptic blastocyst vitrification / delayed blastocyst development & The Author Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please journals.permissions@oup.com

2 1686 Wirleitner et al. Introduction The introduction of vitrification was an important step towards increased survival rate (SR) and live birth rate (LBR) for embryos after cryopreservation (Devine et al., 2015). In addition to cryopreservation of surplus embryos after fresh embryo transfer, vitrification is nowadays commonly used in a broad range of applications. A freeze-all strategy is becoming increasingly popular, not only for patients at risk of ovarian hyperstimulation syndrome (OHSS), but also to prevent negative interference between ovarian stimulation and endometrial receptivity. Frozen embryo transfer was recently reported to provide higher implantation rates (IRs) and pregnancy rates (PRs) than fresh embryo transfer, suggesting better endometrial preparation for implantation in the absence of ovarian stimulation (Vanderzwalmen et al., 2012; Shapiro et al., 2014). Moreover, frozen embryo transfer has shown promise for decreasing both maternal and perinatal morbidity, indicating that frozen embryo transfer in the absence of ovarian stimulation leads to a more suitable physiological uterine environment (Weinerman and Mainigi, 2014). In light of these findings, it is now being discussed whether or not there still remain indications for fresh embryo transfer, or whether it is now on the time for a freeze-all strategy (Barnhart, 2014). Another approach in ART that has contributed to increasing success rates is blastocyst culture, allowing an improved embryo selection for embryo transfer, in one part due to the fact that embryos at the blastocyst stage show reduced aneuploidy rates (Fragouli and Wells, 2011). Furthermore, blastocyst transfer improves uterine and embryonic synchronicity, resulting in higher IRs (Zech et al., 2007; Papanikolaou et al., 2008). With vitrification and blastocyst culture, two important issues arise regarding the freeze-all strategy. The first issue is which (morphological) type of blastocysts can be selected for vitrification. This question is crucial, as it is well accepted that blastocyst morphology, classified by the degree of expansion and appearance of the inner cell mass (ICM) and trophectoderm (TE), is associated with IR and LBR after fresh and frozen embryo transfer (Gardner and Schoolcraft, 1999; Ahlström et al., 2011; Goto et al., 2011; Van den Abbeel et al., 2013). Because of the correlation between morphology and LBR, blastocyst vitrification is generally restricted to embryos with expansion scores of 3 5 on Day 5 and an ICM or TE with minimum Grade B according to the Gardner score (Gardner et al., 2000). However, available data on the implantation potential of non-top quality blastocysts after vitrification are limited. In autologous IVF cycles, top quality blastocysts are not obtained in all stimulation cycles, particularly from patients with advanced maternal age (AMA) or in low responder patients. For these patients, information on whether or not non-top quality blastocysts result in healthy pregnancies after vitrification is extremely important. The second issue is how to proceed with delayed expanding Day 5 embryos presenting as early blastocysts (ebl) or with delayed blastulation at the stage of morulae or compacting embryos on Day 5. Should embryos delayed in development be transferred in fresh embryo transfer, or should they rather be vitrified with or without prolonged embryo culture to be transferred in a subsequent cryo-cycle for better synchronization between embryo and endometrium, or should they be discarded? We addressed these issues by conducting a retrospective study that investigated the clinical outcomes of fresh embryo transfer versus transfer after vitrification/warming embryo transfer in the same stimulation cycle, taking into account blastocyst grading on Day 5. Additionally, clinical outcomes after fresh embryo transfer or vitrified/warmed embryo transfer after delayed cavitation or blastulation were compared. Possible limitations regarding blastocyst quality for vitrification cycles as well as the benefits of better embryonic-endometrial synchronization should be evaluated. Materials and Methods Study design and patient characteristics We retrospectively compared fresh 1010 fresh embryo transfer and 1270 vitrified/warmed embryo transfer between January 2009 and June 2013 of blastocysts originating from the same stimulation cycles (Fig. 1). In 91.2% of patients, fresh embryo transfer was followed by vitrified/warmed embryo transfer, in 8.8% of patients, no fresh embryo transfer occurred due to OHSS or inadequate endometrial build-up. Clinical outcomes were evaluated according to blastocyst morphology on Day 5 prior to transfer or prior to vitrification. We also analysed whether blastocyst recovery after vitrification/ warming or maternal age had an impact on clinical outcomes. Furthermore, we retrospectively investigated fresh and vitrified/warmed embryo transfer of blastocysts with delayed expansion (ebl) or delayed blastulation (compacted/morula stage) on Day 5 in the same study period (Fig. 2). We included: 636 fresh embryo transfer; 60 vitrified/warmed embryo transfer with slow developing embryos vitrified on Day 5 and warmed 3 h prior embryo transfer (without prolonged culture); 122 vitrified/warmed embryo transfer after vitrification on Day 5 and warming 18 h prior to embryo transfer (prolonged culture after vitrification/ warming) and 122 vitrified/warmed embryo transfer of blastocysts with slow development vitrified on Day 6 and warmed 3 h prior to embryo transfer (prolonged culture before vitrification). All patients in this group were counselled and informed regarding reduced chances of a pregnancy and all firmly decided in favour of embryo transfer. Vitrification after slow development was only performed for patients with no expanding blastocyst on Day 5 and when no better blastocyst development was expected in subsequent stimulation cycles. Main outcomes were SR after vitrification/warming, clinical PR (cpr), IR, miscarriage rate (MR) and LBR. Every patient signed informed consent for analysis of anonymized outcome data. Ovarian stimulation and luteal phase support for fresh embryo transfer and vitrified/ warmed embryo transfer Ovarian stimulation was performed using the GnRHa long protocol, as previously described (Zech et al., 2007). Luteal phase support for fresh embryo transfer was done by i.m. injection of progesterone (Prontogest, IBSA, Austria) until Week 7 of gestation, followed by intravaginal progesterone (Uterogestan, Meda Pharma, Austria) until Week 16. Before vitrified/ warmed embryo transfer, increasing doses of oestradiol valerate (Progynova, Schering, Austria) starting at 4 mg per day were administered. Endometrial build-up was considered to be adequate 8 mm in the fundus. Intramuscular progesterone injection (Prontogest, IBSA, Austria) was started 5 days prior to vitrified/warmed embryo transferand continued until Week 7 ofgestation, followed by intravaginal progesterone until Week 16. Embryo culture and evaluation of blastocyst morphology Retrieved oocytes were maintained in human tubal fluid (HTF) medium (Life- Global, Ontario, Canada) for 2 4 h before denudation. For all patients, the intracytoplasmic morphologically selected sperm injection (IMSI) was applied as a routine procedure (Vanderzwalmen et al., 2008). Embryos were cultured

3 LBR of expanded and delayed developing blastocysts 1687 Figure 1 Flow chart diagram of study design, patient cohort and comparison of clinical outcomes after fresh embryo transfer or vitrified/warmed embryo transfer of blastocysts with expansion grades 2 5 on Day 5. Clinical outcome results are shown in Tables I III. Standard deviations are provided in italics, and ranges are shown in parenthesis. BMI, body mass index; COC, cumulus oocyte complex; OPU, ovum pick-up. in a single-step medium (LifeGlobal, Ontario, Canada) supplemented with human serum albumin (LifeGlobal, Ontario, Canada) in Nunc 4-well dishes for IVF (Roskilde, Denmark). For fresh embryo transfer, blastocyst scoring was performed just before transfer. For vitrified/warmed embryo transfer, we took into account blastocyst quality just before vitrification, as aim of this study was to evaluate which qualities can safely be cryopreserved. All blastocyst scorings were done according to the Gardner score as follows: Expansion score 0 ¼ no cavity (Fig. 3B), score 1 ¼ blastocoel cavity less than half volume of the embryo (ebl), score 2 ¼ blastocoel cavity more than half volume of the embryo (Fig. 3A), Score 3 ¼ cavity completely filling the embryo, Score 4 ¼ cavity larger than the embryo and thinning zona, Score 5 ¼ hatching blastocyst; for ICM, Grade A ¼ formed by many tightly packed cells, Grade B ¼ several loosely packed cells, Grade C ¼ few cells; for TE, Grade A ¼ many cells forming a cohesive layer, Grade B ¼ few cells and loose layer, Grade C ¼ very few large cells (Supplementary data, Fig. S1A and S1B; Gardner et al., 2000). The following groups were established based on the scoring (Fig. 3A): (i) Top quality blastocysts expansion grades 4 5 or 2 3, and ICM and TE with AA, AB or BA classifications. (ii) Intermediate quality blastocysts expansion grades 4 5 or 2 3, and ICM and TE with a BB classification. (iii) Non-top quality blastocysts expansion grades 4 5 or 2 3, and ICM and TE with AC, CA, BC, CB classifications.

4 1688 Wirleitner et al. Figure 2 Flow chart diagram of study design, patient cohort and comparison of clinical outcomes after fresh or vitrified/warmed embryo transfer of delayed expanding and blastulating Day 5 embryos. Clinical outcome results are shown in Table IV. Standard deviations are provided in italics, and ranges are shown in parenthesis. BMI, body mass index; COC, cumulus oocyte complex; OPU, ovum pick-up. Scoring embryos with delayed expansion or blastulation was performed on Day 5 before fresh embryo transfer, before vitrification on Day 5, or before prolonged culture following vitrification on Day 6 (Fig. 3B): (1) Top quality ebl with many cells forming the blastocyst and no blastomeres excluded from blastocyst formation. (2) Non-top quality ebl with few cells forming the blastocyst and/or blastomeres excluded from blastocyst formation. (3) Delayed blastulation (morulae or compacting embryos). The best embryo(s) according to the morphological grading (one or two per embryo transfer) were selected for fresh embryo transfer and surplus embryos were vitrified. Exclusion criteria for vitrification on Day 5 were ICM and TE classified as CC (Supplemental data, Fig. S2). Exclusion criteria for blastocysts vitrified on Day 6 after prolonged culture included expansion grades,2 and/or ICM and TE with CC classifications. Aseptic vitrification and warming Blastocysts and embryos were aseptically vitrified as previously described and according to the protocol mentioned by the manufacturer (FertiPro, Belgium) (Vanderzwalmen et al., 2009). Blastocyst survival after warming was defined as re-expansion and further development 3 h after warming. Blastocysts with slow and incomplete re-expansion within 3 h and with severely lower morphology grading in relation to pre-freeze grades were defined as blastocysts with delayed recovery. Cancellation of vitrified/ warmed embryo transfer occurred when all blastocysts warmed did not re-expand after 3 h and cells showed signs of degeneration or lyses. Embryo transfer Fresh embryo transfer or vitrified/warmed embryo transfer was performed in a Day 5 endometrium as previously described (Spitzer et al., 2012). Embryos were placed 0.5 cm below the fundus by a Wallace embryo replacement catheter (Smiths Medical International, Kent, UK). Clinical outcomes The cpr was confirmed by fetal heartbeat 8 12 weeks after embryo transfer. IR was determined from the number of fetal heartbeats per number of blastocysts that were transferred. MR was defined by pregnancy loss after detection of fetal heartbeat. LBR was determined from the numberof livebirths per embryo transfer.

5 1689 LBR of expanded and delayed developing blastocysts Figure 3 Morphological grading. (A) Classification of blastocyst morphology according to the Gardner score (Gardner et al., 2000). (B) Classification of delayed expanding and cavitating Day 5 embryos. Statistical analysis Differences in IR, cpr and LBR were evaluated using Pearson s x 2 test. One-way ANOVA was applied to test statistical differences in patient s ages at the time of fresh embryo transfer or vitrified/warmed embryo transfer between groups. A two-tailed t-test was used to analyse differences in SR and top blastocyst rate. Differences between the groups were considered statistically significant when the P-value was,0.05. Statistical analysis was performed using the Statistical Package for Social Sciences (SPSS) software version 22.0 for Windows (SPSS, Inc., Chicago, IL, USA). Results Clinical outcomes after fresh embryo transfer or vitrified/warmed embryo transfer according to blastocyst quality on Day 5 Fresh embryo transfer of blastocysts expansion scores 2 5 on Day 5 A total of 1010 women received fresh embryo transfer (Fig. 1), and clinical outcomes were analysed with regard to blastocyst quality.

6 1690 Wirleitner et al. Table I Clinical outcome after fresh embryo transfer versus vitrified/warmed embryo transfer of blastocysts expansion 2 5 in the same patient cohort. Fresh embryo transfer Vitrified/warmed embryo transfer... Expansion grades 4-5 Top quality Embryo transfer (Warming cycles) (397) SR 88.6% Maternal age Embryos/embryo transfer cpr 41.5% 36.5% IR 29.8% 27.4% MR 8.1% 14.6% LBR 33.4% 31.2% Intermediate Embryo transfer (Warming cycles) (283) SR 90.7% Maternal age Embryos/embryo transfer cpr 34.1% 33.9% IR 22.1% 23.8% MR 4.1% 13.8% LBR 30.0% 29.2% Non-top Embryo transfer (Warming cycles) (115) SR 92.9% Maternal age Embryos/embryo transfer cpr 37.5% 33.8% IR 23.1% 20.8% MR 10.7% 5.3%* LBR 26.8% 31.9% Expansion grades 2 3 Top quality Embryo transfer (Warming cycles) (152) SR 92.7% Maternal age Embryos/embryo transfer cpr 40.2% 35.6% IR 24.8% 45.0%*** MR 10.7% 22.6% LBR 29.5% 27.5% Intermediate Embryo transfer (Warming cycles) (250) SR 93.7% Maternal age Embryos/embryo transfer cpr 28.3% 32.7% IR 20.4% 36.5% MR 4.3% 6.2% LBR 23.9% 30.6% Non-top Embryo transfer (Warming cycles) (91) SR 95.4% Maternal age Embryos/embryo transfer cpr 10.5% 28.4%* IR 6.7% 22.6%* MR 2.6% 20.0% LBR 7.9% 22.5% Numbers of warming cycles are given in italics and parenthesis. Standard deviation is given in italics. Top quality blastocysts: (AA, AB, BA), intermediate quality blastocysts: (BB), non-top quality blastocysts: (AC, CA, BC, CB). cpr, clinical pregnancy rate; IR, implantation rate; LBR, live birth rate; MR, miscarriage rate; SR, survival rate. Maternal age and embryos/embryo transfer are given as mean + standard deviation. *P, 0.05; **P, 0.01; ***P, Results are shown in Table I. After embryo transfer of blastocysts with expansion grades of 4 5 and ICM and TE grades of (AA, AB, BA), (BB) and (AC, CA, BC, CB), IRs of 29.8, 22.1 and 23.1%; and LBRs of 33.4, 30.0 and 26.8% were observed, respectively. Blastocysts with expansion grades of 2 3 and ICM and TE grades of (AA, AB, BA), (BB) and (AC, CA, BC, CB) showed IRs of 24.8, 20.4 and 6.7%; and LBRs of 29.5, 23.9 and 7.9%, respectively.

7 LBR of expanded and delayed developing blastocysts 1691 Table II Impact of blastocyst recovery after vitrification/warming on implantation rates (IR) and live birth rates (LBR). Blastocyst recovery after warming... Blastocyst grading before vitrification Good recovery Delayed recovery Expansion grades 4 5 Top quality vitet (n) IR 30.3% 18.1%** LBR 33.7% 21.5%* Intermediate vitet (n) IR 26.8% 5.8%*** LBR 33.1% 7.3%*** Non-top vitet (n) IR 21.5% 15.0% LBR 33.0% 23.1% Expansion grades 2 3 Top quality vitet (n) IR 27.8% 9.1% LBR 29.4% 7.7% Intermediate vitet (n) IR 24.7% 11.4%* LBR 33.2% 16.2%* Non-top vitet (n) 80 9 IR 19.7% 6.3% LBR 23.8% 11.1% Top quality blastocysts: (AA, AB, BA), intermediate quality blastocysts: (BB), non-top quality blastocysts: (AC, CA, BC, CB). vitet, vitrified/warmed embryo transfer. *P, 0.05; **P, 0.01; ***P, Vitrified/warmed embryo transfer of blastocysts with expansion scores 2 5 on Day 5 Of 1288 warming cycles, only 18 vitrified/warmed embryo transfer (1.4%) had to be cancelled because none of the warmed blastocysts survived. The cancellation rate did not increase with decreasing blastocyst morphology quality scores. SR ranged between 88.6 and 95.4% and did not associate with blastocyst morphology (Table I). There were no significant differences between the SR of the different blastocyst groups. The clinical outcomes obtained after vitrified/warmed embryo transfer of blastocysts with expansion grades 4 5 and ICM and TE grades of (AA,AB,BA), (BB), and (AC,CA,BC,CB) were as follows: IRs of 27.4, 23.8 and 20.8%; and LBRs of 31.2, 29.2 and 31.9%, respectively. Blastocysts with expansion grades of 4 5 and ICM and TE grades of (AC,AC,BC,CB) showed a significantly higher MR rate after fresh embryo transfer than after vitrified/warmed embryo transfer (10.7 versus 5.3%, respectively). After vitrification/warming embryo transfer of blastocysts expansion grades of 2 3, ICM and TE grades of (AA,AB,BA), (BB) or (AC,CA,BC,CB), IRs of 45.0, 36.5 and 22.6%, and LBRs of 27.5, 30.6 and 22.5% were observed, respectively. Compared with fresh embryo transfer in the same patient cohort, a statistically significant differences were observed in IRs with blastocyst grades (AA,AB,BA) (24.8 versus 45.0%) (Table I) and for blastocyst grades (AC,CA,BC,CB) in cpr (10.5 versus 28.5%) and in IR (6.7 versus 22.6%). Clinical outcomes after vitrified/warmed embryo transfer according to blastocyst scoring performed before and after vitrification/warming Blastocysts with good recovery after warming provided better clinical outcomes than blastocysts with delayed recovery. Significantly different outcomes were observed in three out of the six morphological groups (Table II). Clinical outcomes after vitrified/warmed embryo transfer according to blastocyst scores and maternal age Patients were allocated to three groups according to female age at OPU: 18 35, and years (Table III). Lower LBRs were observed in women between the ages of 40 and 44 years, with statistical significance for blastocysts with expansion grades 4 5 and ICM/TE grades of (AA,AB,BA) or (AC,CA,BC,CB) (Table III). However, despite AMA, non-top quality blastocysts also resulted in live births after vitrified/warmed embryo transfer with a LBR of 5.4% with top quality blastocysts expansion grades 4 5 grades (AA,AB,BA) versus 22.7% with top quality blastocysts expansions 2 3. Clinical outcomes after vitrified/warmed embryo transfer following delayed expansion and blastulation After fresh embryo transfer on Day 5, LBRs of 25.4, 6.6 and 2.8% were observed with top quality ebl, non-top quality ebl and morulae/ compacting embryos, respectively (Table IV). After vitrified/warmed embryo transfer without prolonged embryo culture, LBRs of 31.3 or 21.4% with top or non-top quality ebl were noted. When warming was performed 18 h before vitrified/warmed embryo transfer (prolonged culture after warming), which gives the embryos more time to develop, LBRs of 23.9 and 13.0% were observed with top or non-top quality ebl. However, none of the five patients with delayed blastulating embryos in this group became pregnant. As another strategy, non-top ebl or delayed blastulating embryos were cultured 1 additional day and vitrified on Day 6. Warming was performed 3 h prior to vitrified/ warmed embryo transfer. We observed a LBR of 28.3% with non-top

8 1692 Wirleitner et al. Table III Impact of maternal age on clinical outcome after vitrified/warmed embryo transfer. Maternal Age... Blastocyst Grading years years years (mean 31.9) (mean 37.9) (mean 41.5) Expansion grades 4 5 Top quality vitet (n) IR 30.2% 29.5% 4.5% *** LBR 33.7% 34.3% 5.4% *** Intermediate vitet (n) IR 27.4% 21.6% 9.3%* LBR 32.9% 27.2% 12.5%* Non-top vitet (n) IR 23.3% 23.3% 0%** LBR 35.9% 39.4% 0%* Expansion grades 2 3 Top quality vitet (n) IR 36.4% 23.6% 16.7% LBR 32.5% 20.5% 22.7% Intermediate vitet (n) IR 25.6% 24.5% 12.9%* LBR 34.2% 33.3% 17.0%* Non-top vitet (n) IR 23.3% 12.5% 19.2% LBR 34.1% 11.8%* 14.3% Top quality blastocysts: (AA, AB, BA), intermediate quality blastocysts: (BB), non-top quality blastocysts: (AC, CA, BC, CB). Mean maternal age is given in parenthesis. LBR, live birth rate; IR, implantation rate; vitet, vitrified/warmed embryo transfer. *P, 0.05; **P, 0.01; ***P, ebl and a LBR of 21.7% with delayed blastulation, resulting in a clinical outcome that was statistically significantly better compared with fresh embryo transfer with embryos of the same quality. Discussion A pivotal issue for IVF is identifying embryos that have the capacity to implant and result in a healthy pregnancy. The identification of embryos (blastocysts) that have the capacity to implant and result in a healthy pregnancy is a crucial part of IVF and more information is needed to determine which embryos should be transferred in fresh embryo transfer, which should be vitrified, or which should be discarded. Our results demonstrate that the use of a well-established vitrification protocol allowed cryopreservation of all types of blastocysts. Non-top quality blastocysts did not lead to reduced SRs after warming. Furthermore, no statistically significant differences between LBRs after fresh embryo transfer or vitrified/warmed embryo transfer of blastocysts originating from the same stimulation cycle were observed, but interestingly, there was a trend towards higher LBRs after vitrified/warmed embryo transfer with non-top quality blastocysts. The efficiency of the vitrification/warming procedure played an important role, as statistically significantly reduced LBRs were found after vitrified/warmed embryo transfer of blastocysts that showed poor recovery after warming. Another important factor that significantly affected clinical outcomes was maternal age. However, this was true for top quality as well as non-top quality blastocysts. These results are important as vitrification of non-top quality blastocysts becomes more relevant in patients with AMA. With increasing female age, the rate and quality of blastocysts obtained declines, as does the probability of producing better quality blastocysts in a subsequent stimulation cycle. In this study, we further observed that embryos delayed in blastulation and/or expansion can result in live birth after vitrified/warmed embryo transfer and prolonged embryo culture. Thereby a combination of two main factors, namely a better synchronization between embryo development and endometrial receptivity, as well as an improved embryo selection might play important roles. The morphology of blastocysts transferred in elective single fresh or frozen embryo transfer is known to be closely related to pregnancy and LBR. However, in contrast to top quality blastocysts, little is known about outcomes after vitrified/warmed embryo transfer of non-top quality blastocysts (Gardner et al., 2000; Ahlström et al., 2011; Van den Abbeel et al., 2013). By applying an aseptic vitrification protocol, we found that non-top quality blastocysts did not show reduced SRs after vitrification/warming, which suggests that the freezing process is not a limitation in deciding which type of embryo can be vitrified. Clinical outcomes after fresh embryo transfer and vitrified/warmed embryo transfer were compared. In terms of IRs, no difference was noticed between fresh embryo transfer and vitrified/warmed embryo transfer with blastocyst expansion grades 4 5. A 2-fold increase in IRs after vitrified/warmed embryo transfer was even observed with lower expansion blastocysts (Grades 2 3). A trend towards higher LBRs after vitrified/warmed embryo transfer of non-top quality blastocysts compared with fresh embryo transfer was noted. These findings strongly support the hypothesis of improved endometrial receptiveness in vitrified/warmed embryo transfer cycles (Shapiro et al., 2014). To date, only a few studies have directly analysed clinical outcomes after fresh and vitrified/warmed embryo transfer of blastocysts during a single IVF stimulation cycle. Doherty et al. evaluated 247 frozen embryo transfer and found that the cpr and LBR were significantly higher after vitrified/warmed embryo transfer following an unsuccessful

9 LBR of expanded and delayed developing blastocysts 1693 Table IV Clinical outcome after fresh or vitrified/warmed embryo transfer (vitet) following slow blastulation and/or delayed cavitation. Fresh embryo transfer Vitrification... On Day 5 On Day 6... vitet 3h post-warming vitet 18 h post-warming vitet 3h post-warming No prolonged Prolonged culture Prolonged culture culture after warming before vitrification... Expansion 1 Top Quality Embryo transfer (Warming cycles) (33) 71(71) (early SR 90.1% 92.9% blastocysts) Maternal age Embryos/embryo transfer cpr 29.3% 34.4% 32.4% IR 21.2% 20.7% 20.7% MR 13.2% 9.1% 26.1% LBR 25.4% 31.3% 23.9% Expansion 0 Non-top Quality Morulae, compactions Embryo transfer (Warming cycles) (28) 46(48) 99(101) SR 88.4% 89.5% 91.6% Maternal age Embryos/embryo transfer cpr 10.0% 25.0%* 17.4% 33.3%*** IR 8.0% 16.7% 12.7% 24.7%*** MR 14.3% 14.3% 25.0% 15.2% LBR 6.6% 21.4%* 13.0% 28.3%*** Embryo transfer (Warming cycles) (5) 23(23) SR 76.9% 94.1% Maternal age Embryos/embryo transfer cpr 3.7% 21.7%*** IR 3.1% 21.9%*** MR 22.2% LBR 2.8% 21.7%*** Early blastocysts (expansion grade: 1) of top and non-top qualities and morulae and compactions (expansion grade: 0) Numbers of warming cycles are given in italics and parenthesis. Embryos/embryo transfer and maternal age are given as mean + standard deviation. cpr, clinical pregnancy rate; IR, implantation rate; LBR, live birth rate; MR, miscarriage rate; SR, survival rate. *P, 0.05; **P, 0.01; ***P, fresh embryo transfer than after vitrified/warmed embryo transfer following successful fresh embryo transfer (Doherty et al., 2014). They proposed that only a small proportion of embryos retrieved during a single cycle were capable of developing into a viable pregnancy. However, the authors did not report the morphological grades of the blastocysts that were frozen and transferred. Statistically significant lower LBRs were observed in all blastocyst quality groups as evaluated before vitrification when poor recovery after warming was observed. This finding is in line with earlier observations and highlights the importance of a well-established vitrification protocol (Ebner et al., 2009; Maezawa et al., 2014). Analysis of the impact of maternal age on LBR in relation to blastocyst quality showed that clinical outcomes after vitrified/warmed embryo transfer were significantly reduced in AMA patients aged years, which is well known (Moragianni and Penzias, 2010). However, no statistically significant reduction in LBRs with vitrified non-top blastocysts within the same age group was observed. Similar to top quality blastocysts, vitrified/warmed embryo transfer of blastocysts of non-top quality resulted in live births in women with AMA. This finding becomes even more important with the known reduced blastocyst rates and reduced blastocyst qualities in AMA. In addition to investigating which blastocysts can be vitrified on Day 5, we further evaluated whether outcomes of Day 5 embryos with delayed expansion and blastulation could be improved by using vitrified/warmed embryo transfer with or without prolonged culture. Embryos frequently reach the blastocyst stage only on Day 6. Reasons for this phenomenon might involve cytoplasmic immaturity of oocytes, chromosomal abnormalities in blastomeres and suboptimal culture conditions that might result in prolonged intermitotic periods and therefore delayed development of the embryo (Campbell et al., 2013). Ivec et al. showed in their setting that 84.4% of Day 5 morulae can reach the blastocyst stage; however, only 25.8% of these embryos grew to top quality blastocysts (Ivec et al., 2011). Similar SRs were found after vitrified/warmed of top or non-top quality ebl, morulae, compacting embryos or blastocysts vitrified on Day 6. Clinical outcomes after fresh embryo transfer or vitrified/ warmed embryo transfer of top quality ebl with or without prolonged culture did not differ. However, an improvement in LBR was obtained

10 1694 Wirleitner et al. with vitrified/warmed embryo transfer using non-top quality ebl and the improvement was even more pronounced when using morulae or compacting embryos. Data showed that the best strategy for non-top quality ebl or embryos delayed in blastulation was prolonged culture before vitrification on Day 6, as the LBR was significantly improved compared with fresh embryo transfer on Day 5. The data suggest that endometrium receptivity is increased in vitrified/warmed embryo transfer, partly due to avoidance of additional hormonal stimulation to induce follicular growth. Prolonged embryo culture before vitrification could also lead to improved LBRs because of better synchronization between embryo development and endometrial receptivity (Shapiro et al., 2001). This hypothesis is in line with earlier publications showing that fresh Day 5 embryo transfer usually produces better IRs than fresh Day 6 embryo transfer (37.4 versus 20.6%, respectively; Shapiro et al., 2001 and 22.1 versus 3.6%, respectively; Barrenetxea et al., 2005), which could be improved when Day 6 blastocysts are frozen and transferred in a subsequent cryo-cycles (Shapiro et al., 2008). However, our findings contradict those of a previous study in which blastocysts with delayed development were shown to be at increased risk for chromosomal abnormalities, resulting in a reduced IR, increased MR and very low LBR (Campbell et al., 2013). One of the current challenges is to analyse whether time-lapse imaging and specific morphokinetic parameters could help us to foresee whether an embryo will be specifically sensitive to vitrification or will have the capacity to implant. In conclusion, in our retrospective study, blastocysts with expansion grades of 4 5 on Day 5 produce similar IRs and LBRs after fresh embryo transfer or vitrified/warmed embryo transfer. For non-top quality Day-5 blastocysts with expansion grades of 2 3, vitrified/ warmed embryo transfer resulted in higher IRs and LBRs compared with fresh embryo transfer. Based on our data, we report that non-top quality blastocysts should be vitrified not only for, but also for, AMA patients. Further, clinical outcomes of non-top quality ebl and cases of delayed blastulation were improved by prolonged embryo culture and vitrified/warmed embryo transfer, suggesting a freeze-all strategy for these qualities. As final question remains about how we consider the quality of an embryo: based on matching morphological and morphokinetic criteria or based on the potential to implant (Stecher et al., 2014). Our data suggest that too many embryos that might have the capacity for successful implantation after vitrified/warmed embryo transfer are currently discarded instead of being cryopreserved. Supplementary data Supplementary data are available at Authors roles B.W. collected the data, B.W. and P.V. designed the study and interpreted the data, and B.W., P.V. and M.S. wrote the manuscript. B.W. performed statistical analysis of the data. A.S. and M.M. reviewed the manuscript. Funding No external funding was either sought or obtained for this study. Conflict of interest None declared. References Ahlström A, Westin C, Reismer E, Wikland M, Hardarson T. Trophectoderm morphology: an important parameter for predicting live birth after single blastocyst transfer. Hum Reprod 2011;26: Barnhart K. Are we ready to eliminate the transfer of fresh embryos in in vitro fertilization? Fertil Steril 2014;102:1 2. Barrenetxea G, López de Larruzea A, Ganzabal T, Jiménez R, Carbonero K, Mandiola M. 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