Effect of oxygen concentration on human embryo development evaluated by time-lapse monitoring

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1 Effect of oxygen concentration on human embryo development evaluated by time-lapse monitoring Kirstine Kirkegaard, M.D., Johnny Juhl Hindkjaer, M.Sc., and Hans Jakob Ingerslev, D.M.Sc. The Fertility Clinic, Aarhus University Hospital, Aarhus, Denmark Objective: To evaluate, using time-lapse monitoring, the temporal influence of culture in 5% O 2 or 20% O 2 on human embryonic development. Design: Retrospective cohort study. Setting: University-based fertility clinic. Patient(s): In vitro fertilized embryos from women aged <38 years with no endometriosis and R8 oocytes retrieved. Intervention(s): Culture in 20% O 2 exclusively (group 1), 20% and 5% O 2 combined (group 2), or 5% O 2 exclusively (group 3). Main Outcome Measure(s): Developmental rates and timing of developmental stages. Result(s): The timing of the third cleavage cycle was delayed for embryos cultured in 20% O 2 (group 1) compared with embryos cultured in 5% O 2 (groups 2 and 3). No difference was observed in timing of the early and full blastocyst stages. More embryos in groups 2 and 3 reached the 8-cell, early blastocyst, and full blastocyst stages than in group 1. We found that embryos in group 3 (5% O 2 ) reached the 8-cell stage faster than embryos in group 2 (5% þ 20% O 2 ), but none of the other parameters (i.e., other time points, cumulative development, and embryo score) differed between the two groups. Conclusion(s): Culture in 20% O 2 reduces developmental rates and delays completion of the third cell cycle. The delayed development after culture in atmospheric oxygen was seen in the precompaction embryo only and therefore appears to be stage specific. Clinical Trial Registration Number: NCT (Fertil Steril Ò 2013;99: Ó2013 by American Society for Reproductive Medicine.) Key Words: Oxygen concentration, embryo culture, human, time-lapse, developmental kinetics Discuss: You can discuss this article with its authors and with other ASRM members at fertstertforum.com/kirkegaardk-oxygen-concentration-embryo-culture-time-lapse/ Use your smartphone to scan this QR code and connect to the discussion forum for this article now.* * Download a free QR code scanner by searching for QR scanner in your smartphone s app store or app marketplace. The influence of oxygen concentration on preimplantation embryo development has been a subject of intense research and debate. The introduction of in vitro fertilization (IVF) has led to a dramatic improvement of infertility treatment; yet in vitro culturing though inarguably efficient exposes the preimplantation embryo to a different environment than if fertilized in vivo. Human IVF embryos have traditionally been cultured in atmospheric oxygen (20% O 2 ). Data from animal studies report that cleavage-stage embryos are exposed to an oviductal oxygen concentration of 7%, with a further decrease in the uterine environment that encounters the blastocyst (1). Whether similar conditions exist in humans remains uninvestigated regarding oviductal oxygen concentrations. In one study (2), intrauterine oxygen concentrations in humans were shown Received June 27, 2012; revised November 14, 2012; accepted November 16, 2012; published online December 11, K.K. has nothing to disclose. J.J.H. has nothing to disclose. H.J.I. has nothing to disclose. Supported by Aarhus University, Aase og Einar Danielsen Foundation, Toyota Foundation, and Søster and Verner Lipperts Foundation. Reproductive research at The Fertility Clinic, Aarhus University, is supported by an unrestricted grant from MSD and Ferring. Reprint requests: Kirstine Kirkegaard, M.D., The Fertility Clinic, Aarhus University Hospital, Brendstrupgaardsvej 100, 8200 Aarhus N, Denmark ( kirstine.kirkegaard@ki.au.dk). Fertility and Sterility Vol. 99, No. 3, March 1, /$36.00 Copyright 2013 American Society for Reproductive Medicine, Published by Elsevier Inc. to display considerable variation between different women (range 4% 27% O 2 ) and to be highly dynamic with substantial fluctuations over time. The reported average of 11.8% was, however, well below atmospheric oxygen, which indicates that embryos cultured in vitro under 20% O 2 are exposed to oxygen at nonphysiologic concentrations. Numerous studies of various animal species have documented the detrimental effects of atmospheric oxygen, the primary manifestations being lower blastocyst formation and reduced cell number per blastocyst (3 13). A recent study of mouse embryos by Wale et al. (13) reported that detrimental effects of atmospheric oxygen occur already from the first cleavage cycle, and indicated that the largest impact was 738 VOL. 99 NO. 3 / MARCH 1, 2013

2 Fertility and Sterility found on the precompaction stages. Their finding supports other animal studies suggesting that the postcompaction embryo is less susceptible to the effects of a high oxygen concentration (5, 14 16). Because a comprehensive assessment of human embryos has been unachievable with traditional static evaluation without compromising development, it remains to be investigated whether this stage-specific temporal effect of different oxygen concentrations exists in human embryos. The recent development of clinical time-lapse incubators has enabled continuous monitoring of human embryos and safe noninvasive studies of early embryogenesis (17, 18). The objective of the present retrospective descriptive study was to evaluate the influence of oxygen tension on human preimplantation development with the use of time-lapse monitoring. METHODS Design and Participants The present study was conducted as a retrospective analysis of dynamic development in embryos recruited for two consecutive studies at The Fertility Clinic, Aarhus University Hospital, from June 2010 to February 2011 (study A) and from February 2011 to November 2011 (study B). Eligible for both studies were patients aged <38 years without endometriosis in whom R8 oocytes had been retrieved. Patients were excluded if their prior treatments had failed to result in a normal fertilization (R50%). Written informed consent was obtained from each couple before inclusion. The Central Denmark Region Committees on Biomedical Research Ethics and the Danish Data Protection Agency approved both studies. The studies were registered at ClinicalTrial.gov, with accession numbers and NCT In both studies, human embryos were cultured in a timelapse incubator (EmbryoScope; Unisense Fertilitech) to the blastocyst stage. Based on oxygen exposure, they were retrospectively divided into three groups: group 1 (20% O 2 exclusively), group 2 (20% and 5% O 2 combined), and group 3 (5% O 2 exclusively). Study A was originally conducted to evaluate the safety of the time-lapse incubator by randomizing embryos from each patient 1:1 to culture in the time-lapse incubator or a conventional incubator (17). All embryos from study A cultured at 20% O 2 in a time-lapse incubator formed the basis of group 1. Study B evaluated time-lapse parameters for embryo selection (unpublished data). In study B, all IVF embryos were cultured for 18 hours in 20% O 2 in a conventional incubator, and after inspection for pronuclei (PN) they were transferred to 5% O 2 in the time-lapse incubator. These embryos formed the basis for group 2. Microinseminated (intracytoplasmic sperm injection [ICSI]) embryos in study B were cultured in the time-lapse incubator in 5% O 2 only, forming the basis for group 3. Group 1 consisted of 120 IVF/ICSI embryos from 26 patients recruited to study A. Because embryos from study A comprised a random half of all embryos from each patient, embryos from study B used to create groups 2 and 3 were therefore selected in a manner that mimicked the randomization procedure used in study A. A statistical program (Stata) randomly chose half of the embryos from each patient, thereby generating group 2. Group 2 thus consisted of 123 IVF embryos from 28 patients. Group 3 consisted of 120 ICSI embryos from 30 patients. Baseline and cycle characteristics for the three groups are presented in Table 1. The distinctive difference in fertilization method between the three groups originates from the described construction of the groups determined by the different principles of embryo culture for IVF and ICSI embryos. In Vitro Fertilization, Embryo Culture, and Embryo Score Following retrieval, oocytes were fertilized with the use of conventional IVF or ICSI procedures. ICSI-fertilized embryos were placed in the time-lapse incubator immediately after injection, whereas IVF embryos were cultured 18 hours in a conventional incubator after insemination before they were transferred for further culturing in the time-lapse incubator. The 18-hour delay in transfer to the time-lapse incubator was introduced to allow for optimal fertilization before the necessary denudation of IVF embryos performed to achieve optimal image acquisition in the time-lapse incubator. Culture conditions were identical in the three groups, apart from the differences in O 2 concentrations. All embryos were TABLE 1 Baseline and cycle characteristics. Group 1 Group 2 Group 3 P value Oxygen exposure 20% 20% for 24 h, followed by 5% 5% No. of treatment cycles Maternal age (y) Maternal BMI (kg/m 2 ) 22.1 ( ) 22.5 ( ) 23.1 ( ).46 Cumulative FSH dose (IU) 1, , , No. of retrieved oocytes/cycle No. of ICSI procedures No. of IVF procedures Note: Where continuous data did not approximate to a normal distribution with equal standard deviations, data are displayed as medians with range and Kruskal-Wallis test was applied (BMI); otherwise, one-way analysis of variance was used and estimates are given as mean SD (age, oocytes retrieved). FSH data were normal distributed after log transformation, and estimates are therefore given as median SD. Categoric data (IVF/ICSI procedures) are presented as number of cases. P value tests the hypothesis of no difference between the groups. BMI ¼ body mass index; ICSI ¼ intracytoplasmic sperm injection; IVF ¼ in vitro fertilization. VOL. 99 NO. 3 / MARCH 1,

3 ORIGINAL ARTICLE: ASSISTED REPRODUCTION cultured in sequential culture medium (Sydney IVF Fertilization/Cleavage/Blastocyst Medium; Cook) under oil at 37 C and 6% CO 2. Day 3 embryo assessment was performed 68 hours after oocyte retrieval. Embryos were grouped as high, medium, or low quality according to number of cells, fragmentation, and evenness (high: R6 cells, <20% fragmentation, even or uneven cells; medium: R6 cells, 20% 50% fragmentation, uneven cells; low: <6 cells, >50% fragmentation, uneven cells). Blastocyst assessment was performed 115 hours after fertilization according to the criteria described by Gardner et al. (7). In brief, blastocysts with blastoceles that were less than half the volume of the embryo were graded 1, and hatched blastocysts were graded 6. For blastocysts with grades 3 6, the inner cell mass (ICM) and trophectoderm (TE) were graded A C based on number and cohesiveness of cells. Embryos failing to develop a blastocele received a score of 0. Based on this assessment, subsequent grouping into three major categories was performed: 1) high: blastocysts with excellent/good ICM and TE; 2) medium: early blastocysts and other blastocysts with poor ICM and TE; 3) low: no blastocele. Because the three groups of embryos forming the basis of this study were constructed by randomly selecting half of the embryos from each patient without distinguishing between which embryos were transferred, a meaningful evaluation of pregnancy rates was not possible. Time-Lapse Monitoring The time-lapse system consists of a commercially available incubator (EmbryoScope) with a built-in microscope and camera connected to a computer. Images were recorded automatically every 20 minutes in seven planes (15-mm intervals). Key events, such as time points for each cell-division and blastocyst stage, were registered until 115 hours after fertilization. Only 2PN embryos completing the first cleavage were evaluated. Time points refer to the point of time when an image is recorded and are reported as hours after fertilization (t ¼ 0). For ICSI embryos, fertilization was registered as time of injection; for IVF embryos, fertilization was registered as the time of adding sperm cells to the wells. Statistical Analysis The hypothesis of no difference in time points of embryonic stages between the three groups was tested by one-way analysis of variance, assuming that data followed a normal distribution with equal standard deviations. The assumption of normality and equal standard deviations was checked with histograms, QQ-plots, and Bartlett test for equal variances. If data did not fulfill the assumption of normality and equal standard deviations, the hypothesis of no difference between the groups was tested with the Kruskall-Wallis test. The hypothesis of no association between the proportion of embryos reaching an embryonic stage and group was tested with a logrank test. Categoric data were compared with the c 2 test. Estimates are reported as medians with 95% confidence intervals. Two-sided P values of <.05 were considered to be significant. All statistical analyses were performed in the statistical package Stata for Mac, version 11.0 (Statacorp). RESULTS The three groups did not statistically significantly differ in the timing of the first and second cleavage cycles resulting in 2- and 4-cell embryos, respectively. The timing of the third cleavage cycle, i.e., division from 4 to 8 cells, was increasingly delayed for embryos cultured in 20% O 2 (group 1) compared with embryos cultured both partly and exclusively in 5% O 2 (groups 2 and 3, respectively; Table 2). The largest difference was found at the 8-cell stage: group 1: 61.2 h ( ); group 2: 57.5 h ( ); and group 3: 54.5 h ( ). This finding remained unchanged by exclusion of embryos arresting development before the blastocyst stage (data not shown). No difference was observed among the three groups in timing of the early and full blastocyst stages (Table 2). Comparing culture in 5% O 2 exclusively (group 3) and culture in 20% O 2 for 18 hours followed by culture in 5% O 2 (group 2), we found that embryos in group 3 reached the 8-cell stage faster than embryos in group 2 (P¼.03; Table 2), but none of the other parameters measured (i.e., other time points, cumulative development, and embryo score) differed between the two groups. TABLE 2 Time points of embryonic stages. Stage n Group 1 n Group 2 n Group 3 P value 2-cell ( ) ( ) ( ).24 a 3-cell ( ) ( ) ( ).12 b 4-cell ( ) ( ) ( ).41 b 5-cell ( ) ( ) ( ).09 b 6-cell ( ) ( ) ( ).03 b 7-cell ( ) ( ) ( ) <.001 b 8-cell ( ) ( ) ( ) <.001 a Early blastocyst ( ) ( ) ( ).84 a Full blastocyst ( ) ( ) ( ).08 a Note: All data followed a normal distribution after log transformation and are displayed as medians with 95% confidence intervals. P value tests the hypothesis of no difference between the groups. The number of 8-cell embryos is smaller than the number of early blastocysts, because some embryos compacted earlier than the 8-cell stage and those embryos were therefore not included in the number of 8-cell embryos. a Analysis of variance. b Kruskal-Wallis test. 740 VOL. 99 NO. 3 / MARCH 1, 2013

4 Fertility and Sterility FIGURE 1 A B The cumulative development of embryos progressing through (A) third cleavage cycle and (B) early blastocyst stage. Cumulative analysis of key developmental stages showed that the total percentage of embryos reaching the 8-cell and early blastocyst stages was less at any given time point for embryos cultured in 20% O 2 (group 1) than for embryos cultured partly or exclusively in 5% O 2 (groups 2 and 3, respectively; Fig. 1). Consequently, significantly more embryos in groups 2 and 3 reached the 8-cell and early and full blastocyst stages compared with group 1 (Table 3). However, the proportion of embryos that developed into full blastocysts after completing the third cell cycle did not differ between the groups (P¼.28; c 2 test). To address the influence of fertilization method on the above findings, we performed a subanalysis of IVF and ICSI embryos separately. Overall, the results were unchanged, though there was a tendency toward the differences found between the groups being more pronounced for the IVF than the ICSI embryos (Supplemental Tables 1 and 2, available online at Evaluating day 3 embryo quality, we found significantly more embryos of low quality (n ¼ 52) in the high-oxygen group (Supplemental Table 3, available online at than in the combined (n ¼ 27) and reduced (n ¼ 26) oxygen groups (P¼.001) on day 3. We also found significantly more low-quality embryos (n ¼ 63) on day 5 after fertilization (score 0) in the high-oxygen group than in the combined (n ¼ 39) and reduced (n ¼ 33) oxygen groups (P<.001), which reflects the finding that fewer embryos reached the blastocyst stage in group 1 (Supplemental Table 3). Excluding grade 3 embryos, thereby comparing only embryos actually reaching the blastocyst stage, no difference in embryo quality was found, however, among the three groups (P¼.37). In particular, no difference in scoring of ICM was observed (P¼.45; Supplemental Table 4, available online at DISCUSSION We describe for the first time the influence of oxygen on dynamic development in human embryos with the use of timelapse monitoring. In this observational study we found that culture in 20% O 2 reduced the cumulative development at the 8-cell and early and full blastocyst stages. Furthermore, the timing of the third cleavage cycle, i.e., division to 8 cells, was delayed for embryos cultured in 20% O 2 compared with embryos cultured partly or exclusively in 5% O 2. We were unable to demonstrate a delay in the timing of the postcompaction stages for embryos cultured in 20% O 2. Because the reduced blastocyst development was based on a reduced number of embryos completing the third cleavage cycle in group 1, no difference between the groups in the proportion of embryos arresting development after completion of the third cleavage cycle stage was found. Together, these two observations indicate that the precompaction human embryo is VOL. 99 NO. 3 / MARCH 1,

5 ORIGINAL ARTICLE: ASSISTED REPRODUCTION TABLE 3 Rates of embryo development. Embryonic stage Group 1 Group 2 Group 3 P value First cell cycle, n start Second cell cycle 110 (92%) 114 (93%) 116 (97%).30 Third cell cycle 69 (58%) 96 (78%) 86 (72%).004 Early blastocyst 57 (48%) 85 (69%) 87 (73%) <.001 Full blastocyst 46 (38%) 70 (57%) 61 (51%).01 Note: Data are displayed as number and proportion of embryos reaching a developmental stage. P value tests the hypothesis of no association between group and percentage of embryos reaching the embryonic stage, with the use of a log-rank test. more susceptible to the detrimental effects of a high oxygen concentration than later stages. Similar findings have recently been reported for mice (10, 13). Although the effects of atmospheric oxygen on the timing of the developmental stages in the present study seem to be less profound in human embryos than the effects reported in mice, our findings do support the conclusion that the embryo's response to oxygen appears to be stage dependent, with the largest effect on timing seen in the precompaction embryo. Pre- and postcompaction embryos differ in many ways. Mammalian embryonic gene expression has been demonstrated to follow a stage-specific pattern, with a shift from translation of the maternal to the embryonic genome during the first days of development (19 23). This shift occurs in human embryos approximately on day 3 after fertilization during the third cleavage cycle (19, 23 28). This shift may offer an explanation to the proposed differences in response to high oxygen concentrations between pre- and postcompaction embryos, because embryos may not be able to provide protection to oxidative stress before genomic activation. The described effect was most pronounced for embryos cultured exclusively in 20% O 2 compared with embryos cultured exclusively in 5% O 2 (group 3). Because embryos in these two groups were subjected to constant levels of either high or low oxygen throughout their entire culture period, the comparison between these two groups is the most relevant to consider. We were, however, able to detect a possible effect of a transient exposure to atmospheric oxygen by comparing embryos cultured under 5% O 2 (group 3) with embryos cultured 18 hours under 20% O 2 followed by culture under 5% O 2 (group 2). We found that embryos in group 3 reached the 8-cell stage faster than embryos in group 2, indicating an adverse effect of even transient exposure to 20% O 2, thus supporting similar findings in mice (10). Although the impact of a transient exposure to atmospheric oxygen would be expected to be less than if cultured entirely at high oxygen, this interpretation must, however, be subject to caution, because a considerable number of embryos initiated compaction at the 7-cell stage, leaving only few embryos for analysis at the 8-cell stage. Bovine studies have demonstrated a significant prolongation of the embryonic stage just before embryonic arrest (29). We reperformed the analysis without embryos that had not reached the blastocyst stage to affirm that the observed delay of the third cleavage cycle after culture in 20% O 2 was not caused by a prolonged cell stage before developmental arrest. This exclusion did not change the result, which indicates that the prolonged third cleavage cycle is not associated with subsequent developmental arrest. The overall detrimental effect of atmospheric oxygen has been studied extensively in mouse models, where retarded growth, reduced blastocyst rates, and total cell number have been convincingly demonstrated (5 12). Studies in various other animal species report similar findings (3, 4, 30 37). The corresponding beneficial effects of culture at reduced oxygen concentrations are reported in most human studies regarding embryo development and quality (38 46). Our study confirmed the reduced blastocyst rates and retarded growth found in animal studies, although the retarded growth was detected at the precompaction stages only. We were unable to confirm the reduction in blastocyst cell number found in several animal studies (5) after culture in atmospheric oxygen, evaluated by ICM grading, and the overall blastocyst score of surviving embryos did not differ among the groups, suggesting that the embryos available for transfer were of similar quality. It should be noted, however, that embryos with normal morphology are not necessarily without potential underlying abnormalities, such as altered metabolism and gene expression, as demonstrated in mouse studies (13, 47), and the lack of difference between the groups may well be a result of the limitations of the evaluation with the use of present scoring systems. Because the interpretation of the results is based on an assumption of three comparable groups, the retrospective study design presents an important limitation, particularly regarding the differences in fertilization methods between the groups. The embryos included in the present analysis originated from patients participating in two prospective studies with similar inclusion and exclusion criteria conducted at the same clinic within a relatively short time span, which implicates a minimal risk of bias. However, the fertilization method has been suggested to influence the timing of development, with ICSI-fertilized embryos displaying an earlier first cleavage than IVF embryos (48 50). The influence of the fertilization method on the findings was addressed by performing a subanalysis of IVF and ICSI embryos separately. Although the number of embryos thus analyzed was smaller, the analysis supports our interpretation of the observed difference in timing and development being caused by the differences in oxygen concentration, because the overall results were unchanged. We do, however, suspect that the ICSI embryos in group 3 were of slightly lower oocyte quality than the embryos in groups 1 and 2. This concern arises from the observation that more patients were needed to produce similarly sized groups of 2PN embryos completing first cleavage in group 3 than in groups 1 and 2. This suggests a lower rate of successful fertilization in group 3. Moreover, based on the difference in fertilization method only, embryos in group 3 were expected to cleave more quickly than embryos in group 2, a difference that was not observed in the present study. Finally, we found a nonsignificant tendency of the differences in timing and development between the 742 VOL. 99 NO. 3 / MARCH 1, 2013

6 Fertility and Sterility groups to be more pronounced for IVF than for ICSI embryos, which could be explained by a lower quality of ICSI embryos in group 3. The influence, if any, on the results would work toward the hypothesis of no difference between the groups. Therefore, the effect of culture in 20% O 2 on the timing of development is potentially larger than the effect observed in this study. Acknowledging the superiority of a prospective randomized study in terms of providing comparable groups, we therefore consider the observed differences to be explained by the differences in oxygen concentration during culture rather than by the fertilization method. The increasing evidence of the detrimental effects of atmospheric oxygen concentration on human embryo quality and development impedes the possibility of performing a similar prospective study, thus encouraging the interpretation of results from the present study, though performed retrospectively. 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8 Fertility and Sterility SUPPLEMENTAL TABLE 1 Time points of embryonic stages ICSI embryos only. Embryonic stage n Group 1 (n start [ 58) n Group 3 (n start [ 120) P value (time point/% developed) First cell cycle 58 (100%) 26.9 ( ) 120 (100%) 27.9 ( ).18 Second cell cycle 55 (95%) 39.4 ( ) 116 (97%) 40.5 ( ).36/.68 Third cell cycle 36 (62%) 61.9 ( ) 86 (72%) 55.4 ( ).001/.20 Early blastocyst 28 (48%) 95.5 ( ) 87 (73%) 97.2 ( ).33/.002 Full blastocyst 23 (40%) ( ) 61 (51%) ( ).08/.16 Note: Continuous data are displayed as median with 95% confidence interval and compared using Student t test. Categoric data are presented as total number and percentage of embryos reaching a developmental stage and the hypothesis of no difference tested with the use of the c 2 test. The number of 8-cell embryos is smaller than the number of early blastocysts, because some embryos compacted earlier than the 8-cell stage and those embryos were therefore not included in the number of 8-cell embryos. VOL. 99 NO. 3 / MARCH 1, e1

9 ORIGINAL ARTICLE: ASSISTED REPRODUCTION SUPPLEMENTAL TABLE 2 Time points of embryonic stages IVF embryos only. Embryonic stage n Group 1 (n start [ 62) n Group 2 (n start [ 123) P value (time point/% developed) First cell cycle ( ) ( ).76 Second cell cycle 55 (89%) 42.6 ( ) 114 (93%) 41.0 ( ).18/.41 Third cell cycle 33 (53%) 61.5 ( ) 96 (78%) 57.5 ( ).02/.001 Early blastocyst 29 (47%) 97.4 ( ) 85 (69%) 96.7 ( ).63/.003 Full blastocyst 23 (37%) ( ) 70 (57%) ( ).16/.01 Note: Continuous data are displayed as median with 95% confidence interval and compared with the use of Student t test. Categoric data are presented as total number and percentage of embryos reaching a developmental stage and the hypothesis of no difference tested with the use of the c 2 test. 744.e2 VOL. 99 NO. 3 / MARCH 1, 2013

10 Fertility and Sterility SUPPLEMENTAL TABLE 3 Day 3 grades and blastocyst (day 5) grades. Day of score Grade Group 1 (n [ 120) Group 2 (n [ 123) Group 3 (n [ 120) Day 3 a 1 (high) (medium) (low) Day 5 b 1 (high) (medium) (low) a 1 (high): R6 cells, < 20% fragmentation, even or uneven cells; 2 (medium): R6 cells, 20% 50% fragmentation, uneven cells; 3 (low): <6 cells, >50% fragmentation, uneven. Fisher exact test of the hypothesis of no difference in scoring among the three groups: P¼.001. b 1 (high): blastocysts with excellent/good inner cell mass (ICM) and trophectoderm (TE); 2 (medium): early blastocysts and other blastocysts with poor ICM and TE; 3 (low): no blastocele. Fisher exact test of the hypothesis of no difference in scoring among the groups: P<.001; P¼.37 comparing only embryos developing to the blastocyst stage. VOL. 99 NO. 3 / MARCH 1, e3

11 ORIGINAL ARTICLE: ASSISTED REPRODUCTION SUPPLEMENTAL TABLE 4 Scoring of inner cell mass. Score Group 1 (n [ 46) Group 2 (n [ 70) Group 3 (n [ 61) A B C Note: P¼.45; c 2 test. Kirkegaard. Temporal effect of O2 on human embryos. Fertil Steril e4 VOL. 99 NO. 3 / MARCH 1, 2013