- Email: [email protected]
When only one embryo is available, is it better to transfer on Day 3 or to grow on?
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ARTICLE
When only one embryo is available, is it better to transfer on Day 3 or to grow on? BIOGRAPHY
Joyce S Xiao is a final year medical student from Monash University, Australia. She has keen interests in gynaecology and reproductive health and wishes to pursue these through research and innovation. Joyce S Xiao1, Martin Healey2, Alon Talmor3, Beverley Vollenhoven4,*
KEY MESSAGE
In patients with only one viable embryo on Day 3 post-IVF treatment, transferring the embryo into the uterus on Day 3 was found to achieve higher pregnancy and live birth rates compared to Day 4–6 transfer. This challenges current practices and warrants reconsideration of the uterus as the ideal incubator. ABSTRACT
Research question: In patients with only one embryo on Day 3 post-IVF treatment, does transferring the embryo into the uterine environment achieve a higher pregnancy rate than growing the embryo on with a plan to transfer at Day 4–6? Design: This was a retrospective cohort study conducted in patients with only one viable embryo on Day 3 post-IVF treatment. Data were extracted from a standardized IVF database and included 1384 women who fulfilled this study's selection criteria. Outcomes of these embryos were followed up and stratified into two groups: embryos transferred on Day 3 and those grown on to Day 4–6. Pregnancy rate (biochemical and clinical) and live birth rates were analysed with logistic regression and adjusted using a parsimonious model for baseline patient characteristics. Results: Biochemical pregnancy (16.7% versus 9.5%, odds ratio [OR] 1.9, P = 0.001), clinical pregnancy (14.7% versus 6.8%, OR 2.35, P < 0.001) and live birth rates (9.7% versus 4.4%, OR 2.37, P = 0.002) were significantly higher in the Day 3 group than those in the group grown on to Day 4–6. These differences were still significant after adjusting for potential confounders (adjusted OR 2.60, 3.71, 4.08, respectively, P < 0.001). Conclusions: These findings support Day 3 cleavage-stage embryo transfer instead of growing on to Day 4–6 for blastocyst-stage transfer when only a single embryo is available. 1 Monash
University, Clayton Victoria, Australia IVF, 252 Clayton Road, Clayton, Victoria, Australia; Royal Women's Hospital, 20 Flemington Rd, Parkville, Victoria, Australia; Department of Obstetrics and Gynaecology, University of Melbourne, Parkville, Victoria, Australia 3 Department of Obstetrics & Gynaecology, Monash University, Clayton, Victoria, Australia; Womens and Newborn Programme, Monash Health, 246 Clayton Road, Clayton, Victoria, Australia; Monash IVF, 252 Clayton Road, Clayton, Victoria, Australia; Epworth Richmond, 89 Bridge Road, Richmond 3121, Australia 4 Department of Obstetrics & Gynaecology, Monash University, Clayton, Victoria, Australia; Womens and Newborn Programme, Monash Health, 246 Clayton Road, Clayton, Victoria, Australia; Monash IVF, 252 Clayton Road, Clayton, Victoria, Australia 2 Monash
© 2019 Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved. *Corresponding author. E-mail address: [email protected] (B. Vollenhoven). https://doi.org/10.1016/j. rbmo.2019.08.003 1472-6483/© 2019 Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved. Declaration: The authors report no financial or commercial conflicts of interest.
KEYWORDS
Blastocyst Cleavage-stage embryo Embryo transfer IVF Live birth Pregnancy rate
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INTRODUCTION
S
ignificant advances in assisted reproductive technology have been made in recent years. However, the proportion of IVF attempts that succeed in achieving pregnancy remains suboptimal (De Geyter et al., 2018; Penzias, 2012; Zegers-Hochschild et al., 2014). Even in patients with good prognostic factors and high-grade embryos via conventional morphology selection, pregnancy rate remains less than ideal (ranging from 61% to 70%) (Gardner et al., 2004; Yang et al., 2012). Implantation failure accounts for a significant number of unsuccessful IVF attempts (CalhazJorge et al., 2016; Koot et al., 2012; Margalioth et al., 2006). Three broad categories have been proposed to classify causes of implantation failure: inherent developmental issues with the embryo, problems with the uterus and problems with the interaction between the embryo and the uterus (Coughlan et al., 2014; Kliman and Frankfurter, 2019; Macklon, 2017; Ruiz-Alonso et al., 2012). As the precise cellular and mechanical events surrounding implantation have yet to be fully elucidated, research directed towards improving implantation rates is currently focused on improving embryonic quality, as well as on maternal factors affecting embryo receptivity (Das and Holzer, 2012; Mollo et al., 2009; Simon and Laufer, 2012; Sunkara et al., 2010). To reduce the rate of implantation failure associated with the embryo, many studies have focused on finding the optimal day of embryo transfer. Embryo transfer can occur at Day 3 post-fertilization (cleavage stage) or Day 4–6 post-fertilization (blastocyst stage). Traditionally, embryo transfer was carried out on Day 3; however, in recent years there has been a shift in practice to transferring the embryo at Day 4–6. This is due, at least in part, to the development of culture media, which has enabled the support of in-vitro embryo development for longer periods (Gardner et al., 2000). Additional characteristics incorporated into the grading criteria of blastocyst-stage embryos (Gardner et al., 2001) has allowed selection of highgrade embryos that are more likely to be euploid at transfer (Kaur et al., 2014). It has been conventionally postulated that blastocyst-stage embryos have the ability to physiologically synchronize with the host endometrium (Alper et al., 2001);
thus transferring on Day 4–6 would hypothetically increase implantation and overall pregnancy rate (Papanikolaou et al., 2006; Van der Auwera et al., 2002). Despite increasing evidence supporting Day 4–6 blastocyst-stage transfer, the optimal day of embryo transfer remains controversial (Blake et al., 2004; Glujovsky and Farquhar, 2016; Glujovsky et al., 2016; Levron et al., 2002; Martins et al., 2017). In the presence of more than one embryo on Day 3, growing on with the aim to transfer on Day 4–6 allows for selection out of potential non-viable embryos. As Day 4–6 embryos have been selected by surviving, it becomes increasingly likely that their use may lead to a more successful outcome. Previous studies performing a comparison between Day 3 and Day 4–6 embryos focused on outcomes on a per cycle basis rather than per embryo (Aziminekoo et al., 2015; Hatırnaz and Kanat Pektaş, 2017). These studies have not addressed the quandary of how to proceed when only one embryo is available and so the implantation rate and pregnancy outcomes in relation to an individual embryo remain unclear. As such, this study aims to provide additional evidence on whether the implantation and pregnancy rate is higher on Day 3 embryo transfer or with a policy to grow on to Day 4–6 when only a single embryo is available on Day 3.
MATERIALS AND METHODS Study population This retrospectiZve cohort study was carried out using a prospectively completed standardized database of a multi-site private IVF clinic. The criteria for inclusion and exclusion of IVF treatment cycles with suitable embryos were established prior to searching the database (FIGURE 1). All patients who underwent an IVF stimulation cycle between 2008 and 2016 at the multisite IVF clinic were initially included in this study. The exclusion criteria used were: cycles that are not fresh or without ovarian stimulation before fresh embryo transfers, cycles with embryos less than 3 days old, cycles using donor egg/embryo, and cycles with unknown outcome or destination of embryo (FIGURE 1). Only cycles with one viable embryo on Day 3 were included. If a
patient had more than one cycle that met these criteria, only the earliest cycle was included in the study. The decision to transfer an embryo on Day 3 or Day 4–6 was made by the treating clinician in consultation with the patient prior to and during their IVF cycle. Patients were counselled on various embryo transfer options prior to the cycle commencing: Day 3 transfer, growing on the embryo for Day 4–6 transfer and planned Day 4–6 transfer with the option of changing to Day 3 if there was a low embryo number (≤4) or poor embryo grades (C/D) on Day 3. Patients were also counselled regarding embryo progress (embryo number and grade) during their treatment cycle. As per local protocol, cycles with fewer than four viable embryos or embryo grade C and below on Day 3 should have immediate transfer. Occasionally, delayed transfers were performed because of patient convenience, or a decision to undergo embryo biopsy. Despite the range in days for late transfer, all patients who underwent transfers on Day 4–6 were enrolled. This was done to avoid incomplete follow-up of growing on, which would be a potential source of bias. All embryos were graded on Day 3 postfertilization, based on local protocols (Supplementary Table 1). Embryo grading was performed using parameters such as cell count number, compaction and degree of fragmentation. Embryos with grade A to D were included in this study. All embryos were grown in sequential culture media from either Cook Medical or Vitrolife but never both at the same time, and cultured in various types of culture dishes. Embryos were cultured in mini benchtop incubators (Cook Medical MINC™; Bloomington, IN, USA) at 37°C and 6% CO2 (special gas mix: 5% O2, 6% CO2 and 89% N2). Time-lapse incubators were not used. All embryos were treated under the same conditions with the same protocol. A total of 1384 Day 3 embryos were included in this study, separated into two groups based on the day of embryo transfer: 1017 patients (73.5%) who had their embryo transferred on Day 3 and 367 patients (26.5%) whose embryos were grown on with the intent to be transferred on Day 4–6. The outcomes of both fresh and frozen embryo transfers were followed up. The study
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FIGURE 1 Flow chart of the study selection process.
was approved by the local research and ethics committee on 20 February 2018 (MH15172M). Statistical analysis Data analysis was performed using SPSS Statistics for Windows, Version 24.0 (IBM Corp., Armonk, NY, USA) and
Stata Statistical Software, Release 15 (StataCorp LP, College Station, TX, USA). Descriptive values of the variables were expressed as mean (SD) (SE of the mean) or median [95% confidence interval (CI)]. Continuous variables were compared using the t-test, while the Mann–Whitney U-test was used to compare proportions.
A P-value <0.05 was considered significant. Outcomes were analysed using logistic regression modelling. Crude odds ratios (OR) were provided for study outcomes (biochemical pregnancy rate, clinical pregnancy rate and live birth
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rate). Biochemical pregnancy refers to a transient rise in beta-human chorionic gonadotrophin (beta-HCG), followed by decline without development of an embryo or a gestational sac in the uterus. Clinical pregnancy refers to a pregnancy with detectable fetal heartbeat on ultrasound. Live birth refers to the delivery of a liveborn neonate after 20 weeks of gestation. A parsimonious model was developed by sequentially removing potential confounding variables with P > 0.1, where their removal did not change or reduce the Akaike's information criterion. Body mass index (BMI) was not included as a confounder due to
missing values for 70 cases; however, inclusion of BMI in the models did not substantially alter findings (results not shown). Based on this process, the variables included in the parsimonious model of biochemical pregnancy were: maternal age, gravidity, parity, fibroids on ultrasound, idiopathic infertility, embryo cell number and embryo grade. The variables used to model clinical pregnancy were: maternal age, gravidity, parity, fibroids on ultrasound, embryo cell number and embryo grade. Finally, the variables included in the modelling of live birth were: maternal age, parity, diabetes status (Yes/No), fibroids on ultrasound, endometrioma
on ultrasound, embryo cell number and embryo grade.
RESULTS Outcomes of the selected embryos Destinations (fresh transfer, grow on, freeze then thaw, discard) of all Day 3 and Day 4–6 embryos from the initial IVF treatment cycle are reported in FIGURE 1 . Baseline patient characteristics are documented in TABLE 1. All 1384 Day 3 embryos screened through the selection criteria were included in this study. Of the 1384 Day 3 embryos, 1017 embryos (73.5%) were
TABLE 1 BASELINE CHARACTERISTICS OF PATIENTS, THEIR TREATMENT CYCLE AND EMBRYOS Variables
Day 3 (n = 1017)
Day 4–6 (n = 367)
Mann–Whitney U-test (P); t-test (P)**
Treatment cycle #
3.5 (3.3) (0.1) 2 [1–13]
3.3 (3.5) (0.2) 2 [1–12]
0.006
BMI
25.3 (5.8) (0.2) (n = 967)
24.4 (5.1) (0.3) (n = 347)
0.016**
Follicles
5.0 (4.0) (0.1) 4 [1–14]
6.1 (5.8) (0.3) 5 [1–18]
<0.001**
Total FSH (IU)
3806 (1772) (56)
3848 (1846) (96)
0.70**
ICSI
77.2% (785)
69.2% (254)
0.002
Maternal age
38.7 (4.4) (0.1)
38.5 (4.8) (0.3)
0.53**
Maximum oestradiol
2249 (2063) (65)
2332 (2254) (118)
0.54**
Year
2011 [2008–2015]
2010 [2008–2016]
0.42
Gravidity
1.0 (1.3) (0.04) 1 [0–5]
0.8 (1.6) (0.09) 0 [0–4]
<0.001
Parity
0.4 (0.7) (0.02) 0 [0–2]
0.3 (0.8) (0.04) 0 [0–2]
0.002
Smoker
3.5% (36)
4.1% (15)
0.63
Diabetic
0.9% (9)
0.3% (1)
0.24
Tubal factor
9.3% (95)
9.0% (33)
0.84
Male factor
8.8% (89)
12.5% (46)
0.036
PCOS
2.6% (26)
3.8% (14)
0.22
Endometriosis
12.8% (130)
13.6% (50)
0.68
Idiopathic
35.4% (360)
19.3% (71)
<0.001
U/S endometriomas
0.3% (3)
0.5% (2)
0.49
U/S PCOS
6.2% (63)
4.6% (17)
0.27
U/S fibroids
4.2% (43)
1.9% (7)
0.041
Aetiology
Embryo grade on Day 3
<0.001
A
32.2% (327)
59.3% (217)
B
38.8% (393)
21.3% (78)
CD
18.7% (190)
14.8% (54)
Missing
10.3% (104) n = 3
4.6% (17) n = 1
Day 3 embryo cell number
7.1 (2.0) (0.10) 8 [3–11]
7.0 (2.1) (0.07) 8 [2–11]
0.74**
Values presented as mean (SD) (standard error of the mean) or median [95% confidence interval] or % (n). BMI = body mass index; ICSI = intracytoplasmic sperm injection; PCOS = polycystic ovary syndrome; U/S = ultrasound diagnosis.
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TABLE 2 OUTCOME OF LOGISTIC REGRESSION ANALYSIS Day 3 (n = 1017)
Day 4–6 (n = 367)
Crude OR (P) (univariate)
Adjusted OR (P) [parsimony and no BMI]
Biochemical pregnancy
16.7% (170)
9.5% (35)
1.90 (1.30–2.80) P = 0.001
2.60 (1.66–4.06) P < 0.001
Clinical pregnancy
14.7% (149)
6.8% (25)
2.35 (1.51–3.65) P < 0.001
3.71 (2.22–6.19) P < 0.001
Live birth
9.7% (99)
4.4% (16)
2.37 (1.38–4.07) P = 0.002
4.08 (2.13–7.80) P < 0.001
In the parsimonious model biochemical pregnancy was adjusted for maternal age, gravidity, parity, fibroids on ultrasound, idiopathic infertility, embryo cell number, embryo grade; clinical pregnancy was adjusted for maternal age, gravidity, parity, fibroids on ultrasound, embryo cell number, embryo grade and live birth was adjusted for maternal age, parity, diabetes status (Yes/No), fibroids on ultrasound, endometrioma on ultrasound, embryo cell number, embryo grade. BMI = body mass index; OR = odds ratio.
allocated to undergo Day 3 transfer and 367 embryos (26.5%) for Day 4–6 transfer. The 367 Day 4–6 embryos were either directly grown on to Day 4–6 or frozen on Day 3 and thawed to be grown on to Day 4–6. Within the Day 3 group, 91 embryos (8.9%) were discarded, 29 (2.9%) frozen with the intention to transfer at a later stage and 897 (88.2%) freshly transferred into the maternal uterus. In the Day 4–6 group, 119 embryos (32.4%) were discarded, 19 (5.2%) were frozen and 229 (62.4%) were freshly transferred. Destinations of the frozen embryos were followed up. In the Day 3 group, 29 embryos were frozen, of which five embryos (17.2%) were thawed and discarded, 22 (75.9%) were thawed and transferred, and none were thawed and refrozen. There was one embryo (3.45%) still frozen at the time of this study and one embryo (3.45%) transferred to another IVF facility. In the Day 4–6 group, 6 frozen embryos (31.6%) were thawed and discarded, 13 (68.4%) thawed and transferred and none were refrozen. As 242 embryos in the Day 4–6 group survived blastulation to be freshly transferred or frozen and transferred later, the blastocyst formation rate was 65.9%. Overall, 919 of the 1017 embryos (90.4%) in the Day 3 group underwent embryo transfer (fresh and frozen), and 242 of the 367 embryos (65.9%) underwent embryo transfer in the Day 4–6 group. Pregnancy and live birth rates Cycle outcomes of the two groups are shown in TABLE 2. Biochemical pregnancy rate in the Day 3 group was 16.7% (n = 170) and 9.5% in the Day 4–6 group (n = 35). Crude OR calculated for biochemical pregnancy rate was 1.90 (P = 0.001). Adjusted OR for biochemical pregnancy was 2.60 with parsimony and no BMI (P < 0.001).
Clinical pregnancy rate in the Day 3 group was 14.7% (n = 149) and 6.8% in the Day 4–6 group (n = 25). Crude OR was 2.35 (P < 0.001), adjusted OR was 3.71 with parsimony and no BMI (P < 0.001). Live birth rate was 9.7% in the Day 3 group (n = 99) and 4.4% in the Day 4–6 group (n = 16). Crude OR was 2.37 (P = 0.002), adjusted OR was 4.08 with parsimony and no BMI (P < 0.001). To account for the concerns about heterogeneity within the Day 4–6 group, two subgroup analyses were carried out: Day 3 versus Day 5 embryo transfers (Supplementary Table 2) and Day 3 versus Day 5 fresh embryo transfers only (Supplementary Table 3).
DISCUSSION This retrospective cohort study evaluated pregnancy and live birth rates in patients with only one embryo on Day 3, who underwent either Day 3 embryo transfer or were grown on for planned Day 4–6 embryo transfer. Higher pregnancy (biochemical and clinical) and live birth rates were observed with the Day 3 embryo transfer group as opposed to the planned Day 4–6 embryo transfer group. The difference in pregnancy and live birth rates between these groups persisted and increased with adjustment for potential confounders, as seen from the higher adjusted OR across all three outcomes measured. The increase in adjusted compared with crude OR indicates that women who were planned for Day 3 embryo transfers were more likely to have confounding characteristics at baseline associated with poorer outcome when compared with the Day 4–6 group. Conventional practice today is based, at least in part, on previous studies that
have examined patients with multiple embryos and compared pregnancy rate per cycle of IVF treatment (Aziminekoo et al., 2015; Glujovsky, et al., 2016; Hatırnaz and Kanat Pektaş, 2017). The outcomes of such studies would favour Day 4–6 embryo transfer, because embryos undergo self-selection and poorquality embryos would be eliminated by Day 4–6 during this natural selection process. By limiting the patient sample to those with one surviving embryo only, this study uniquely examines the pregnancy rate per embryo in an IVF cycle, hence accounting for the natural attrition rate of embryos and allowing us to study the best time for embryo transfer, with the aim of perfecting current clinical practice when only one embryo is available on Day 3. There may be concern that the range of days in the group grown on to Day 4–6 are too wide, resulting in heterogeneous characteristics and outcomes within this group. However, there is increasing evidence showing that Day 4 and Day 6 transfers are comparable to Day 5 (Simopoulou et al., 2019; Taylor et al., 2014; Yee and Tian, 2019). As discussed above (Materials and methods), the authors decided that exclusion of any data would introduce a source of bias, but note the validity of these concerns. As such, a subgroup analysis of Day 3 versus Day 5 embryo transfer and Day 3 versus Day 5 fresh transfers only were carried out. The results remained unchanged – Day 3 embryo transfer leads to higher pregnancy and live birth rates than Day 5 transfer, in patients with only one viable embryo on Day 3. The aetiology of infertility could influence IVF success and potentially confound results. These include factors such as male factor infertility, maternal age and primary versus secondary infertility. A
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study conducted by Gaafar et al. (2015) found that in couples who utilized IVF due to male factor infertility, transferring blastocyst-stage embryos compared with cleavage-stage embryos significantly increased clinical pregnancy rates (60% versus 35%, P < 0.01). In the present study, Day 3 cleavage-stage embryo transfer resulted in increased pregnancy rates, despite the rate of male factor infertility being significantly higher in the Day 4–6 than the Day 3 group (12.5% versus 8.8%, P = 0.036). Male factor infertility as a potential confounding factor was adjusted for in the initial modelling. It did not contribute to the model (P > 0.1) due to interactions with other confounders and so was not included in the final parsimonious model. The mean maternal age in this study did not differ significantly between the Day 3 and Day 4–6 groups (38.7 versus 38.5 years). It has previously been found that patients aged 35 or older benefit from Day 5 as opposed to Day 3 embryo transfer, leading to a higher cumulative pregnancy rate (Fernandez-Shaw et al., 2015), but this study did not examine the pregnancy rate per embryo transfer, and its findings regarding maternal age cannot be extrapolated to patients with only one embryo on Day 3. Primary versus secondary infertility did not influence the outcomes of the current study, because patients in the Day 3 group had significantly higher gravidity and parity than the Day 4–6 group (P < 0.001 and P = 0.002, respectively); however, the outcomes remain unchanged following statistical adjustment for these factors. For poor responders to IVF treatment, some studies have suggested early embryo transfer at the cleavage stage instead of blastocyst stage, as prolonged in-vitro embryonic culture has been associated with cleavage arrest (Laverge et al., 2001). Various aetiologies and contributing factors have been proposed for this – poor metabolism, DNA damage and culture media used, all of which could lead to disruptions in early embryonic development (Jurisicova and Acton, 2004). This is supported by the findings from this study, which demonstrated early embryo transfer leading to improved IVF outcomes when only one embryo was available. In the subgroup of poor responders, a study by Dayal et al. (2011) also found no
difference in pregnancy rate between Day 2 or 3 transfer. Further studies need to explore the differences between Day 2 and 3 transfer in patients with only one embryo post-fertilization, as well as determining the exact cellular mechanisms contributing to the uterus being the optimal incubator. In addition to finding the optimal day of embryo transfer, the policies used for embryo transfer should also be considered in order to maximise IVF success. It has been suggested that FET policy should be employed over fresh embryo transfer, as it leads to higher rates of implantation. The frozen– thawed approach allows the uterine endometrium to recover after ovarian stimulation and for synchronicity to resume. Doing so increases endometrial receptivity to the embryo, increasing the likelihood of implantation (Roque et al., 2015). In the present study, both fresh and frozen–thawed embryos were included to avoid selection bias. One of the main reasons behind transferring a blastocyst embryo over a cleavage embryo is the ability for self-selection. It is well known that the morphological criteria used to grade cleavage embryos are limited and many studies have debated the correlation between morphological features and pregnancy rates (Scott et al., 2000; Sjoblom et al., 2006; Steer et al., 1992). In conjunction with evidence showing cleavage embryos having a higher incidence of chromosomal abnormalities than blastocysts, many authors subsequently concluded that blastocyst embryo transfer is better than cleavage embryo transfer when multiple embryos are available. In this present study, all cleavage-stage embryos were graded on Day 3 regardless of day of transfer, using a local protocol that is based on cell number and degree of compaction or fragmentation. Embryos with all grades (A–D) were transferred. Despite the Day 3 group having higher percentages of grade C and D embryos than the Day 4–6 group (18.7% versus 14.8% and 10.3% versus 4.6%, respectively), Day 3 embryo transfer resulted in higher pregnancy and live birth rates, when both unadjusted and adjusted for embryo grading. This raises the possibility that current morphological criteria used to grade cleavage embryos do not correlate well with pregnancy outcome. Therefore, refinement of the grading
system for cleavage embryos is necessary in order to better distinguish between good- and poor-quality embryos. At present, time-lapse morphokinetics is a non-invasive alternative that studies the developmental kinetics between a normal euploid and aneuploid embryo (Basile et al., 2014). A recent study by Ezoe et al. (2019) has demonstrated evidence for using blastomere movement to predict pregnancy outcome after fresh embryo transfer of Day 2 embryos. Further development in time-lapse morphokinetics will therefore allow better identification of embryonic characteristics to support early embryo transfer. A limitation of this study is its duration of 8 years (2008–2016), during which there have been some changes to the local laboratory protocols in IVF techniques and culture media. This possible heterogeneity has been broadly accounted and adjusted for by the inclusion of ‘year of treatment’ in the parsimonious model adjustment. Overall, the adjusted OR still favours the transfer of the single embryo on Day 3. While the findings of this study are valuable in contributing towards current understanding of the relationship between day of embryo transfer and pregnancy outcome, further research needs to be undertaken before the results can be extrapolated to the general population. In particular, a prospective study with an intention-totreat protocol that randomly compares Day 3 transfer and a policy to grow on to blastocyst stage is necessary. Furthermore, the results from the current study are only applicable to the unique subgroup of women with one surviving embryo during Day 3. There is therefore a need to assess, on a pregnancy per embryo basis, the outcomes of Day 3 versus grown on blastocyst policies when patients have multiple available embryos. In conclusion, this study's findings have demonstrated that in a woman with one embryo available, transferring Day 3 cleavage-stage embryos into the uterus leads to higher rates of pregnancy and live birth, compared with growing the embryo on and aiming for Day 4–6 embryo transfer. Further studies are needed to elucidate the interaction between the host uterus and cleavagestage embryo to understand why the
uterus could be a better incubator than the laboratory.
ACKNOWLEDGEMENTS The authors would like to thank Ms Vivien MacLachlan, Donor, Data and Research Manager of Monash IVF, for her assistance with data collection.
SUPPLEMENTARY MATERIALS Supplementary material associated with this article can be found in the online version at doi:10.1016/j.rbmo.2019.08.003.
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REFERENCES Alper, M.M., Brinsden, P., Fischer, R., Wikland, M. To blastocyst or not to blastocyst? That is the question. Hum. Reprod. 2001; 16: 617–619. https://doi.org/10.1093/humrep/16.4.617 Aziminekoo, E., Mohseni Salehi, M.S., Kalantari, V., Shahrokh Tehraninejad, E., Haghollahi, F., Hossein Rashidi, B., Zandieh, Z. Pregnancy outcome after blastocyst stage transfer comparing to early cleavage stage embryo transfer. Gynecol. Endocrinol. 2015; 31: 880–884. https://doi.org/10.3109/09513590.20 15.1056141 Basile, N., Nogales Mdel, C., Bronet, F., Florensa, M., Riqueiros, M., Rodrigo, L., García-Velasco, J., Meseguer, M. Increasing the probability of selecting chromosomally normal embryos by time-lapse morphokinetics analysis. Fertil. Steril. 2014; 101: 699–704. https://doi. org/10.1016/j.fertnstert.2013.12.005 Blake, D.A., Proctor, M., Johnson, N.P. The merits of blastocyst versus cleavage stage embryo transfer: a Cochrane review. Hum. Reprod. 2004; 19: 2174. https://doi.org/10.1093/humrep/ deh104 Calhaz-Jorge, C., de Geyter, C., Kupka, M.S., de Mouzon, J., Erb, K., Mocanu, E., Motrenko, T., Scaravelli, G., Wyns, C., Goossens, V. Assisted reproductive technology in Europe, 2012: results generated from European registers by ESHRE. Hum. Reprod. 2016; 31: 1638–1652. https://doi.org/10.1093/humrep/dew151 Coughlan, C., Ledger, W., Wang, Q., Liu, F., Demirole, A., Gurgan, T., Cutting, R., Ong, K., Sallam, H., Li, T.C. Recurrent implantation failure: definition and management. Reprod. Biomed. Online. 2014; 28: 14–38. https://doi. org/10.1016/j.rbmo.2013.08.011 Das, M., Holzer, H.E. Recurrent implantation failure: gamete and embryo factors. Fertil. Steril. 2012; 97: 1021–1027. https://doi. org/10.1016/j.fertnstert.2012.02.029 Dayal, M.B., Frankfurter, D., Athanasiadis, I., Peak, D., Dubey, A., Gindoff, P.R. Day 2 embryo transfer (ET) and day 3 ET afford similar reproductive outcomes in the poor responder. Fertil. Steril. 2011; 95: 1130–1132. https://doi.org/10.1016/j.fertnstert.2010.10.010 de Geyter, C., Calhaz-Jorge, C., Kupka, M.S., Wyns, C., Mocanu, E., Motrenko, T., Scaravelli, G., Smeenk, J., Vidakovic, S., Goossens, V. ART in Europe, 2014: results generated from European registries by ESHRE: The European IVF-monitoring Consortium (EIM) for the European Society of Human Reproduction and Embryology (ESHRE). Hum. Reprod. 2018; 33: 1586–1601. https://doi.org/10.1093/humrep/ dey242 Ezoe, K., Ohata1, K., Morita, H., Ueno, S., Miki, T., Okimura, T., Uchiyama, K., Yabuuchi, A., Kobayashi, T., Montag, M., Kato, K. Prolonged blastomere movement induced by the delay of pronuclear fading and first cell division adversely affects pregnancy outcomes after fresh embryo transfer on Day 2: a time-lapse study. Reprod. Biomed. Online. 2019; 38: 659– 668. https://doi.org/10.1016/j.rbmo.2018.12.014 Fernandez-Shaw, S., Cercas, R., Brana, C., Villas, C., Pons, I. Ongoing and cumulative pregnancy rate after cleavage-stage versus blastocyst-stage embryo transfer using vitrification for cryopreservation: Impact of age on the results. J. Assist. Reprod. Genet.
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2015; 32: 177–184. https://doi.org/10.1007/ s10815–014–0387–9 Gaafar, S.H., El Fourtia, I., El Tawil, S., El Guiziry, D., El Maghraby, H. Blastocyst versus day 2–3 transfer in ICSI cycles for male factor infertility: a multicentre randomized controlled trial. Hum. Reprod. 2015; 30 Gardner, D.K., Lane, M., Stevens, J., Schlenker, T., Schoolcraft, W.B. Blastocyst score affects implantation and pregnancy outcome: towards a single blastocyst transfer. Fertil. Steril. 2000; 73: 1155–1158. https://doi. org/10.1016/S0015–0282(00)00518–5 Gardner, D.K., Surrey, E., Minjarez, D., Leitz, A., Stevens, J., Schoolcraft, W.B. Single blastocyst transfer: a prospective randomized trial. Fertil. Steril. 2004; 81: 551–555. https://doi. org/10.1016/j.fertnstert.2003.07.023 Gardner, D.K., Weissman, A., Howles, C., Zeev, S. 2001 Textbook of Assisted Reproductive Techniques, first ed. CRC press, London. https://doi.org/10.1201/9781439813942 Glujovsky, D., Farquhar, C. Cleavage-stage or blastocyst transfer: what are the benefits and harms? Fertil. Steril. 2016; 106: 244–250. https://doi.org/10.1016/j.fertnstert.2016.06.029 Glujovsky, D., Farquhar, C., Quinteiro Retamar, A.M., Alvarez Sedo, C.R., Blake, D. Cleavage stage versus blastocyst stage embryo transfer in assisted reproductive technology. Cochrane. Database. Syst. Rev. 2016CD002118. https://doi.org/10.1002/14651858.CD002118. pub5 Hatırnaz, Ş., Kanat Pektaş, M. Day 3 embryo transfer versus day 5 blastocyst transfers: A prospective randomized controlled trial. Turk. J. Obstet. Gynecol. 2017; 14: 82–88. https://doi. org/10.4274/tjod.99076 Jurisicova, A., Acton, B.M. Deadly decisions: the role of genes regulating programmed cell death in human preimplantation embryo development. Reprod. 2004; 128: 281–291. https://doi.org/10.1530/rep.1.00241 Kaur, P., Swarankar, M.L., Maheshwari, M., Acharya, V. A comparative study between cleavage stage embryo transfer at day 3 and blastocyst stage transfer at day 5 in in-vitro fertilization/ intra-cytoplasmic sperm injection on clinical pregnancy rates. J. Hum. Reprod. Sci. 2014; 7: 194. https://doi.org/10.4103/0974–1208.142481 Kliman, H.J., Frankfurter, D. Clinical approach to recurrent implantation failure: evidencebased evaluation of the endometrium. Fertil. Steril. 2019; 111: 618–628. https://doi. org/10.1016/j.fertnstert.2019.02.011 Koot, Y.E.M., Teklenburg, G., Salker, M.S., Brosens, J.J., Macklon, N.S. Molecular aspects of implantation failure. Biochim. Biophys. Acta. Mol. Basis. Dis. 2012; 1822: 1943–1950. https:// doi.org/10.1016/j.bbadis.2012.05.017 Laverge, H., De Sutter, P., Van der Elst, J., Dhont, M. A prospective, randomized study comparing day 2 versus day 3 embryo transfer in human IVF. Hum. Reprod. 2001; 16: 476– 480. https://doi.org/10.1093/humrep/16.3.476 Levron, J., Shulman, A., Bider, D., Seidman, D., Levin, T., Dor, J. A prospective randomized study comparing day 3 with blastocyst-stage embryo transfer. Fertil. Steril. 2002; 77: 1300–1301. https://doi.org/10.1016/S0015– 0282(02)03090-X Macklon, N. Recurrent implantation failure is a pathology with a specific transcriptomic signature. Fertil. Steril. 2017; 108: 9–14. https:// doi.org/10.1016/j.fertnstert.2017.05.028
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Margalioth, E.J., Ben-Chetrit, A., Gal, M., EldarGeva, T. Investigation and treatment of repeated implantation failure following IVF-ET. Hum. Reprod. 2006; 21: 3036–3043. https:// doi.org/10.1093/humrep/del305 Martins, W.P., Nastri, C.O., Rienzi, L., van der Poel, S.Z., Gracia, C., Racowsky, C. Blastocyst versus cleavage-stage embryo transfer: systematic review and meta-analysis of reproductive outcomes. Ultrasound. Obstet. Gynecol. 2017; 49: 583–591. https://doi. org/10.1002/uog.17327 Mollo, A., De Franciscis, P., Colacurci, N., Cobellis, L., Perino, A., Venezia, R., Alviggi, C., De Placido, G. Hysteroscopic resection of the septum improves the pregnancy rate of women with unexplained infertility: a prospective controlled trial. Fertil. Steril. 2009; 91. https://doi.org/10.1016/j. fertnstert.2008.04.011 Papanikolaou, E.G., Camus, M., Kolibianakis, E.M., Van Landuyt, L., Van Steirteghem, A., Devroey, P. In vitro Fertilization with Single BlastocystStage versus Single Cleavage-Stage Embryos. N. Engl. J. Med. 2006; 354: 1139–1146. https:// doi.org/10.1056/NEJMoa053524 Penzias, A.S. Recurrent IVF failure: other factors. Fertil. Steril. 2012; 97: 1033–1038. https://doi. org/10.1016/j.fertnstert.2012.03.017 Roque, M., Valle, M., Guimarães, F., Sampaio, M., Geber, S. Freeze-all policy: fresh versus frozen-thawed embryo transfer. Fertil. Steril. 2015; 103: 1190–1193. https://doi.org/10.1016/j. fertnstert.2015.01.045 Ruiz-Alonso, M., Blesa, D., Simon, C. The genomics of the human endometrium. Biochim. Biophys. Acta. 2012; 1822: 1931–1942. https://doi.org/10.1016/j.bbadis.2012.05.004 Scott, L., Alvero, R., Leondires, M., Miller, B. The morphology of human pronuclear
embryos is positively related to blastocyst development and implantation. Hum. Reprod. 2000; 15: 2394–2403. https://doi.org/10.1093/ humrep/15.11.2394 Simon, A., Laufer, N. Repeated implantation failure: clinical approach. Fertil. Steril. 2012; 97: 1039–1043. https://doi.org/10.1016/j. fertnstert.2012.03.010 Simopoulou, M., Sfakianoudis, K., Tsioulou, P., Rapani, A., Maziotis, E., Giannelou, P., Grigoriadis, S., Pantou, A., Nikolettos, K., Vlahos, N., Pantos, K., Koutsilieris, M. Should the flexibility enabled by performing a day-4 embryo transfer remain as a valid option in the IVF laboratory? A systematic review and network meta-analysis. J. Assist. Reprod. Genet. 2019; 36: 1049–1061. https://doi. org/10.1007/s10815–019–01475–0 Sjoblom, P., Menezes, J., Cummins, L., Mathiyalagan, B., Costello, M.F. Prediction of embryo developmental potential and pregnancy based on early stage morphological characteristics. Fertil. Steril. 2006; 86: 848–861. https://doi.org/10.1016/j. fertnstert.2006.03.040 Steer, C.V., Mills, C.L., Tan, S.L., Campbell, S., Edwards, R.G. The cumulative embryo score: a predictive embryo scoring technique to select the optimal number of embryos to transfer in an in-vitro fertilization and embryo transfer programme. Hum. Reprod. 1992; 7: 117–119. https://doi.org/10.1093/oxfordjournals.humrep. a137542 Sunkara, S.K., Khairy, M., El-Toukhy, T., Khalaf, Y., Coomarasamy, A. The effect of intramural fibroids without uterine cavity involvement on the outcome of IVF treatment: a systematic review and meta-analysis. Hum. Reprod. 2010; 25: 418–429. https://doi.org/10.1093/humrep/ dep396
Taylor, T., Patrick, J., Gitlin, S., Wilson, J., Crain, J., Griffin, D. Comparison of aneuploidy, pregnancy and live birth rates between day 5 and day 6 blastocysts. Reprod. Biomed. Online 2014; 29: 305–310. https://doi.org/10.1016/J. RBMO.2014.06.001 Van der Auwera, I., Debrock, S., Spiessens, C., Afschrift, H., Bakelants, E., Meuleman, C., Meeuwis, L., D'Hooghe, T.M. A prospective randomized study: day 2 versus day 5 embryo transfer. Hum. Reprod. 2002; 17: 1507–1512. https://doi.org/10.1093/ humrep/17.6.1507 Yang, Z., Liu, J., Collins, G.S., Salem, S.A., Liu, X., Lyle, S.S., Peck, A.C., Sills, E.S., Salem, R.D. Selection of single blastocysts for fresh transfer via standard morphology assessment alone and with array CGH for good prognosis IVF patients: results from a randomized pilot study. Mol. Cytogenet. 2012; 5: 24. https://doi. org/10.1186/1755–8166–5-24 Yee, C.S., Tian, T.S. Choosing between Day 5 and Day 6 blastocyst: Comparison of aneuploidy rates and pregnancy rates. Reprod. Biomed. Online 2019; 38: e31. https://doi.org/10.1016/j. rbmo.2019.03.051 Zegers-Hochschild, F., Mansour, R., Ishihara, O., Adamson, G.D., de Mouzon, J., Nygren, K.G., Sullivan, E.A. International Committee for Monitoring Assisted Reproductive Technology: world report on assisted reproductive technology, 2005. Fertil. Steril. 2014; 101: 366–378. https://doi.org/10.1016/j. fertnstert.2013.10.005 Received 13 March 2019; received in revised form 26 July 2019; accepted 14 August 2019.
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ARTICLE
When only one embryo is available, is it better to transfer on Day 3 or to grow on? BIOGRAPHY
Joyce S Xiao is a final year medical student from Monash University, Australia. She has keen interests in gynaecology and reproductive health and wishes to pursue these through research and innovation. Joyce S Xiao1, Martin Healey2, Alon Talmor3, Beverley Vollenhoven4,*
KEY MESSAGE
In patients with only one viable embryo on Day 3 post-IVF treatment, transferring the embryo into the uterus on Day 3 was found to achieve higher pregnancy and live birth rates compared to Day 4–6 transfer. This challenges current practices and warrants reconsideration of the uterus as the ideal incubator. ABSTRACT
Research question: In patients with only one embryo on Day 3 post-IVF treatment, does transferring the embryo into the uterine environment achieve a higher pregnancy rate than growing the embryo on with a plan to transfer at Day 4–6? Design: This was a retrospective cohort study conducted in patients with only one viable embryo on Day 3 post-IVF treatment. Data were extracted from a standardized IVF database and included 1384 women who fulfilled this study's selection criteria. Outcomes of these embryos were followed up and stratified into two groups: embryos transferred on Day 3 and those grown on to Day 4–6. Pregnancy rate (biochemical and clinical) and live birth rates were analysed with logistic regression and adjusted using a parsimonious model for baseline patient characteristics. Results: Biochemical pregnancy (16.7% versus 9.5%, odds ratio [OR] 1.9, P = 0.001), clinical pregnancy (14.7% versus 6.8%, OR 2.35, P < 0.001) and live birth rates (9.7% versus 4.4%, OR 2.37, P = 0.002) were significantly higher in the Day 3 group than those in the group grown on to Day 4–6. These differences were still significant after adjusting for potential confounders (adjusted OR 2.60, 3.71, 4.08, respectively, P < 0.001). Conclusions: These findings support Day 3 cleavage-stage embryo transfer instead of growing on to Day 4–6 for blastocyst-stage transfer when only a single embryo is available. 1 Monash
University, Clayton Victoria, Australia IVF, 252 Clayton Road, Clayton, Victoria, Australia; Royal Women's Hospital, 20 Flemington Rd, Parkville, Victoria, Australia; Department of Obstetrics and Gynaecology, University of Melbourne, Parkville, Victoria, Australia 3 Department of Obstetrics & Gynaecology, Monash University, Clayton, Victoria, Australia; Womens and Newborn Programme, Monash Health, 246 Clayton Road, Clayton, Victoria, Australia; Monash IVF, 252 Clayton Road, Clayton, Victoria, Australia; Epworth Richmond, 89 Bridge Road, Richmond 3121, Australia 4 Department of Obstetrics & Gynaecology, Monash University, Clayton, Victoria, Australia; Womens and Newborn Programme, Monash Health, 246 Clayton Road, Clayton, Victoria, Australia; Monash IVF, 252 Clayton Road, Clayton, Victoria, Australia 2 Monash
© 2019 Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved. *Corresponding author. E-mail address: [email protected] (B. Vollenhoven). https://doi.org/10.1016/j. rbmo.2019.08.003 1472-6483/© 2019 Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved. Declaration: The authors report no financial or commercial conflicts of interest.
KEYWORDS
Blastocyst Cleavage-stage embryo Embryo transfer IVF Live birth Pregnancy rate
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INTRODUCTION
S
ignificant advances in assisted reproductive technology have been made in recent years. However, the proportion of IVF attempts that succeed in achieving pregnancy remains suboptimal (De Geyter et al., 2018; Penzias, 2012; Zegers-Hochschild et al., 2014). Even in patients with good prognostic factors and high-grade embryos via conventional morphology selection, pregnancy rate remains less than ideal (ranging from 61% to 70%) (Gardner et al., 2004; Yang et al., 2012). Implantation failure accounts for a significant number of unsuccessful IVF attempts (CalhazJorge et al., 2016; Koot et al., 2012; Margalioth et al., 2006). Three broad categories have been proposed to classify causes of implantation failure: inherent developmental issues with the embryo, problems with the uterus and problems with the interaction between the embryo and the uterus (Coughlan et al., 2014; Kliman and Frankfurter, 2019; Macklon, 2017; Ruiz-Alonso et al., 2012). As the precise cellular and mechanical events surrounding implantation have yet to be fully elucidated, research directed towards improving implantation rates is currently focused on improving embryonic quality, as well as on maternal factors affecting embryo receptivity (Das and Holzer, 2012; Mollo et al., 2009; Simon and Laufer, 2012; Sunkara et al., 2010). To reduce the rate of implantation failure associated with the embryo, many studies have focused on finding the optimal day of embryo transfer. Embryo transfer can occur at Day 3 post-fertilization (cleavage stage) or Day 4–6 post-fertilization (blastocyst stage). Traditionally, embryo transfer was carried out on Day 3; however, in recent years there has been a shift in practice to transferring the embryo at Day 4–6. This is due, at least in part, to the development of culture media, which has enabled the support of in-vitro embryo development for longer periods (Gardner et al., 2000). Additional characteristics incorporated into the grading criteria of blastocyst-stage embryos (Gardner et al., 2001) has allowed selection of highgrade embryos that are more likely to be euploid at transfer (Kaur et al., 2014). It has been conventionally postulated that blastocyst-stage embryos have the ability to physiologically synchronize with the host endometrium (Alper et al., 2001);
thus transferring on Day 4–6 would hypothetically increase implantation and overall pregnancy rate (Papanikolaou et al., 2006; Van der Auwera et al., 2002). Despite increasing evidence supporting Day 4–6 blastocyst-stage transfer, the optimal day of embryo transfer remains controversial (Blake et al., 2004; Glujovsky and Farquhar, 2016; Glujovsky et al., 2016; Levron et al., 2002; Martins et al., 2017). In the presence of more than one embryo on Day 3, growing on with the aim to transfer on Day 4–6 allows for selection out of potential non-viable embryos. As Day 4–6 embryos have been selected by surviving, it becomes increasingly likely that their use may lead to a more successful outcome. Previous studies performing a comparison between Day 3 and Day 4–6 embryos focused on outcomes on a per cycle basis rather than per embryo (Aziminekoo et al., 2015; Hatırnaz and Kanat Pektaş, 2017). These studies have not addressed the quandary of how to proceed when only one embryo is available and so the implantation rate and pregnancy outcomes in relation to an individual embryo remain unclear. As such, this study aims to provide additional evidence on whether the implantation and pregnancy rate is higher on Day 3 embryo transfer or with a policy to grow on to Day 4–6 when only a single embryo is available on Day 3.
MATERIALS AND METHODS Study population This retrospectiZve cohort study was carried out using a prospectively completed standardized database of a multi-site private IVF clinic. The criteria for inclusion and exclusion of IVF treatment cycles with suitable embryos were established prior to searching the database (FIGURE 1). All patients who underwent an IVF stimulation cycle between 2008 and 2016 at the multisite IVF clinic were initially included in this study. The exclusion criteria used were: cycles that are not fresh or without ovarian stimulation before fresh embryo transfers, cycles with embryos less than 3 days old, cycles using donor egg/embryo, and cycles with unknown outcome or destination of embryo (FIGURE 1). Only cycles with one viable embryo on Day 3 were included. If a
patient had more than one cycle that met these criteria, only the earliest cycle was included in the study. The decision to transfer an embryo on Day 3 or Day 4–6 was made by the treating clinician in consultation with the patient prior to and during their IVF cycle. Patients were counselled on various embryo transfer options prior to the cycle commencing: Day 3 transfer, growing on the embryo for Day 4–6 transfer and planned Day 4–6 transfer with the option of changing to Day 3 if there was a low embryo number (≤4) or poor embryo grades (C/D) on Day 3. Patients were also counselled regarding embryo progress (embryo number and grade) during their treatment cycle. As per local protocol, cycles with fewer than four viable embryos or embryo grade C and below on Day 3 should have immediate transfer. Occasionally, delayed transfers were performed because of patient convenience, or a decision to undergo embryo biopsy. Despite the range in days for late transfer, all patients who underwent transfers on Day 4–6 were enrolled. This was done to avoid incomplete follow-up of growing on, which would be a potential source of bias. All embryos were graded on Day 3 postfertilization, based on local protocols (Supplementary Table 1). Embryo grading was performed using parameters such as cell count number, compaction and degree of fragmentation. Embryos with grade A to D were included in this study. All embryos were grown in sequential culture media from either Cook Medical or Vitrolife but never both at the same time, and cultured in various types of culture dishes. Embryos were cultured in mini benchtop incubators (Cook Medical MINC™; Bloomington, IN, USA) at 37°C and 6% CO2 (special gas mix: 5% O2, 6% CO2 and 89% N2). Time-lapse incubators were not used. All embryos were treated under the same conditions with the same protocol. A total of 1384 Day 3 embryos were included in this study, separated into two groups based on the day of embryo transfer: 1017 patients (73.5%) who had their embryo transferred on Day 3 and 367 patients (26.5%) whose embryos were grown on with the intent to be transferred on Day 4–6. The outcomes of both fresh and frozen embryo transfers were followed up. The study
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FIGURE 1 Flow chart of the study selection process.
was approved by the local research and ethics committee on 20 February 2018 (MH15172M). Statistical analysis Data analysis was performed using SPSS Statistics for Windows, Version 24.0 (IBM Corp., Armonk, NY, USA) and
Stata Statistical Software, Release 15 (StataCorp LP, College Station, TX, USA). Descriptive values of the variables were expressed as mean (SD) (SE of the mean) or median [95% confidence interval (CI)]. Continuous variables were compared using the t-test, while the Mann–Whitney U-test was used to compare proportions.
A P-value <0.05 was considered significant. Outcomes were analysed using logistic regression modelling. Crude odds ratios (OR) were provided for study outcomes (biochemical pregnancy rate, clinical pregnancy rate and live birth
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rate). Biochemical pregnancy refers to a transient rise in beta-human chorionic gonadotrophin (beta-HCG), followed by decline without development of an embryo or a gestational sac in the uterus. Clinical pregnancy refers to a pregnancy with detectable fetal heartbeat on ultrasound. Live birth refers to the delivery of a liveborn neonate after 20 weeks of gestation. A parsimonious model was developed by sequentially removing potential confounding variables with P > 0.1, where their removal did not change or reduce the Akaike's information criterion. Body mass index (BMI) was not included as a confounder due to
missing values for 70 cases; however, inclusion of BMI in the models did not substantially alter findings (results not shown). Based on this process, the variables included in the parsimonious model of biochemical pregnancy were: maternal age, gravidity, parity, fibroids on ultrasound, idiopathic infertility, embryo cell number and embryo grade. The variables used to model clinical pregnancy were: maternal age, gravidity, parity, fibroids on ultrasound, embryo cell number and embryo grade. Finally, the variables included in the modelling of live birth were: maternal age, parity, diabetes status (Yes/No), fibroids on ultrasound, endometrioma
on ultrasound, embryo cell number and embryo grade.
RESULTS Outcomes of the selected embryos Destinations (fresh transfer, grow on, freeze then thaw, discard) of all Day 3 and Day 4–6 embryos from the initial IVF treatment cycle are reported in FIGURE 1 . Baseline patient characteristics are documented in TABLE 1. All 1384 Day 3 embryos screened through the selection criteria were included in this study. Of the 1384 Day 3 embryos, 1017 embryos (73.5%) were
TABLE 1 BASELINE CHARACTERISTICS OF PATIENTS, THEIR TREATMENT CYCLE AND EMBRYOS Variables
Day 3 (n = 1017)
Day 4–6 (n = 367)
Mann–Whitney U-test (P); t-test (P)**
Treatment cycle #
3.5 (3.3) (0.1) 2 [1–13]
3.3 (3.5) (0.2) 2 [1–12]
0.006
BMI
25.3 (5.8) (0.2) (n = 967)
24.4 (5.1) (0.3) (n = 347)
0.016**
Follicles
5.0 (4.0) (0.1) 4 [1–14]
6.1 (5.8) (0.3) 5 [1–18]
<0.001**
Total FSH (IU)
3806 (1772) (56)
3848 (1846) (96)
0.70**
ICSI
77.2% (785)
69.2% (254)
0.002
Maternal age
38.7 (4.4) (0.1)
38.5 (4.8) (0.3)
0.53**
Maximum oestradiol
2249 (2063) (65)
2332 (2254) (118)
0.54**
Year
2011 [2008–2015]
2010 [2008–2016]
0.42
Gravidity
1.0 (1.3) (0.04) 1 [0–5]
0.8 (1.6) (0.09) 0 [0–4]
<0.001
Parity
0.4 (0.7) (0.02) 0 [0–2]
0.3 (0.8) (0.04) 0 [0–2]
0.002
Smoker
3.5% (36)
4.1% (15)
0.63
Diabetic
0.9% (9)
0.3% (1)
0.24
Tubal factor
9.3% (95)
9.0% (33)
0.84
Male factor
8.8% (89)
12.5% (46)
0.036
PCOS
2.6% (26)
3.8% (14)
0.22
Endometriosis
12.8% (130)
13.6% (50)
0.68
Idiopathic
35.4% (360)
19.3% (71)
<0.001
U/S endometriomas
0.3% (3)
0.5% (2)
0.49
U/S PCOS
6.2% (63)
4.6% (17)
0.27
U/S fibroids
4.2% (43)
1.9% (7)
0.041
Aetiology
Embryo grade on Day 3
<0.001
A
32.2% (327)
59.3% (217)
B
38.8% (393)
21.3% (78)
CD
18.7% (190)
14.8% (54)
Missing
10.3% (104) n = 3
4.6% (17) n = 1
Day 3 embryo cell number
7.1 (2.0) (0.10) 8 [3–11]
7.0 (2.1) (0.07) 8 [2–11]
0.74**
Values presented as mean (SD) (standard error of the mean) or median [95% confidence interval] or % (n). BMI = body mass index; ICSI = intracytoplasmic sperm injection; PCOS = polycystic ovary syndrome; U/S = ultrasound diagnosis.
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TABLE 2 OUTCOME OF LOGISTIC REGRESSION ANALYSIS Day 3 (n = 1017)
Day 4–6 (n = 367)
Crude OR (P) (univariate)
Adjusted OR (P) [parsimony and no BMI]
Biochemical pregnancy
16.7% (170)
9.5% (35)
1.90 (1.30–2.80) P = 0.001
2.60 (1.66–4.06) P < 0.001
Clinical pregnancy
14.7% (149)
6.8% (25)
2.35 (1.51–3.65) P < 0.001
3.71 (2.22–6.19) P < 0.001
Live birth
9.7% (99)
4.4% (16)
2.37 (1.38–4.07) P = 0.002
4.08 (2.13–7.80) P < 0.001
In the parsimonious model biochemical pregnancy was adjusted for maternal age, gravidity, parity, fibroids on ultrasound, idiopathic infertility, embryo cell number, embryo grade; clinical pregnancy was adjusted for maternal age, gravidity, parity, fibroids on ultrasound, embryo cell number, embryo grade and live birth was adjusted for maternal age, parity, diabetes status (Yes/No), fibroids on ultrasound, endometrioma on ultrasound, embryo cell number, embryo grade. BMI = body mass index; OR = odds ratio.
allocated to undergo Day 3 transfer and 367 embryos (26.5%) for Day 4–6 transfer. The 367 Day 4–6 embryos were either directly grown on to Day 4–6 or frozen on Day 3 and thawed to be grown on to Day 4–6. Within the Day 3 group, 91 embryos (8.9%) were discarded, 29 (2.9%) frozen with the intention to transfer at a later stage and 897 (88.2%) freshly transferred into the maternal uterus. In the Day 4–6 group, 119 embryos (32.4%) were discarded, 19 (5.2%) were frozen and 229 (62.4%) were freshly transferred. Destinations of the frozen embryos were followed up. In the Day 3 group, 29 embryos were frozen, of which five embryos (17.2%) were thawed and discarded, 22 (75.9%) were thawed and transferred, and none were thawed and refrozen. There was one embryo (3.45%) still frozen at the time of this study and one embryo (3.45%) transferred to another IVF facility. In the Day 4–6 group, 6 frozen embryos (31.6%) were thawed and discarded, 13 (68.4%) thawed and transferred and none were refrozen. As 242 embryos in the Day 4–6 group survived blastulation to be freshly transferred or frozen and transferred later, the blastocyst formation rate was 65.9%. Overall, 919 of the 1017 embryos (90.4%) in the Day 3 group underwent embryo transfer (fresh and frozen), and 242 of the 367 embryos (65.9%) underwent embryo transfer in the Day 4–6 group. Pregnancy and live birth rates Cycle outcomes of the two groups are shown in TABLE 2. Biochemical pregnancy rate in the Day 3 group was 16.7% (n = 170) and 9.5% in the Day 4–6 group (n = 35). Crude OR calculated for biochemical pregnancy rate was 1.90 (P = 0.001). Adjusted OR for biochemical pregnancy was 2.60 with parsimony and no BMI (P < 0.001).
Clinical pregnancy rate in the Day 3 group was 14.7% (n = 149) and 6.8% in the Day 4–6 group (n = 25). Crude OR was 2.35 (P < 0.001), adjusted OR was 3.71 with parsimony and no BMI (P < 0.001). Live birth rate was 9.7% in the Day 3 group (n = 99) and 4.4% in the Day 4–6 group (n = 16). Crude OR was 2.37 (P = 0.002), adjusted OR was 4.08 with parsimony and no BMI (P < 0.001). To account for the concerns about heterogeneity within the Day 4–6 group, two subgroup analyses were carried out: Day 3 versus Day 5 embryo transfers (Supplementary Table 2) and Day 3 versus Day 5 fresh embryo transfers only (Supplementary Table 3).
DISCUSSION This retrospective cohort study evaluated pregnancy and live birth rates in patients with only one embryo on Day 3, who underwent either Day 3 embryo transfer or were grown on for planned Day 4–6 embryo transfer. Higher pregnancy (biochemical and clinical) and live birth rates were observed with the Day 3 embryo transfer group as opposed to the planned Day 4–6 embryo transfer group. The difference in pregnancy and live birth rates between these groups persisted and increased with adjustment for potential confounders, as seen from the higher adjusted OR across all three outcomes measured. The increase in adjusted compared with crude OR indicates that women who were planned for Day 3 embryo transfers were more likely to have confounding characteristics at baseline associated with poorer outcome when compared with the Day 4–6 group. Conventional practice today is based, at least in part, on previous studies that
have examined patients with multiple embryos and compared pregnancy rate per cycle of IVF treatment (Aziminekoo et al., 2015; Glujovsky, et al., 2016; Hatırnaz and Kanat Pektaş, 2017). The outcomes of such studies would favour Day 4–6 embryo transfer, because embryos undergo self-selection and poorquality embryos would be eliminated by Day 4–6 during this natural selection process. By limiting the patient sample to those with one surviving embryo only, this study uniquely examines the pregnancy rate per embryo in an IVF cycle, hence accounting for the natural attrition rate of embryos and allowing us to study the best time for embryo transfer, with the aim of perfecting current clinical practice when only one embryo is available on Day 3. There may be concern that the range of days in the group grown on to Day 4–6 are too wide, resulting in heterogeneous characteristics and outcomes within this group. However, there is increasing evidence showing that Day 4 and Day 6 transfers are comparable to Day 5 (Simopoulou et al., 2019; Taylor et al., 2014; Yee and Tian, 2019). As discussed above (Materials and methods), the authors decided that exclusion of any data would introduce a source of bias, but note the validity of these concerns. As such, a subgroup analysis of Day 3 versus Day 5 embryo transfer and Day 3 versus Day 5 fresh transfers only were carried out. The results remained unchanged – Day 3 embryo transfer leads to higher pregnancy and live birth rates than Day 5 transfer, in patients with only one viable embryo on Day 3. The aetiology of infertility could influence IVF success and potentially confound results. These include factors such as male factor infertility, maternal age and primary versus secondary infertility. A
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study conducted by Gaafar et al. (2015) found that in couples who utilized IVF due to male factor infertility, transferring blastocyst-stage embryos compared with cleavage-stage embryos significantly increased clinical pregnancy rates (60% versus 35%, P < 0.01). In the present study, Day 3 cleavage-stage embryo transfer resulted in increased pregnancy rates, despite the rate of male factor infertility being significantly higher in the Day 4–6 than the Day 3 group (12.5% versus 8.8%, P = 0.036). Male factor infertility as a potential confounding factor was adjusted for in the initial modelling. It did not contribute to the model (P > 0.1) due to interactions with other confounders and so was not included in the final parsimonious model. The mean maternal age in this study did not differ significantly between the Day 3 and Day 4–6 groups (38.7 versus 38.5 years). It has previously been found that patients aged 35 or older benefit from Day 5 as opposed to Day 3 embryo transfer, leading to a higher cumulative pregnancy rate (Fernandez-Shaw et al., 2015), but this study did not examine the pregnancy rate per embryo transfer, and its findings regarding maternal age cannot be extrapolated to patients with only one embryo on Day 3. Primary versus secondary infertility did not influence the outcomes of the current study, because patients in the Day 3 group had significantly higher gravidity and parity than the Day 4–6 group (P < 0.001 and P = 0.002, respectively); however, the outcomes remain unchanged following statistical adjustment for these factors. For poor responders to IVF treatment, some studies have suggested early embryo transfer at the cleavage stage instead of blastocyst stage, as prolonged in-vitro embryonic culture has been associated with cleavage arrest (Laverge et al., 2001). Various aetiologies and contributing factors have been proposed for this – poor metabolism, DNA damage and culture media used, all of which could lead to disruptions in early embryonic development (Jurisicova and Acton, 2004). This is supported by the findings from this study, which demonstrated early embryo transfer leading to improved IVF outcomes when only one embryo was available. In the subgroup of poor responders, a study by Dayal et al. (2011) also found no
difference in pregnancy rate between Day 2 or 3 transfer. Further studies need to explore the differences between Day 2 and 3 transfer in patients with only one embryo post-fertilization, as well as determining the exact cellular mechanisms contributing to the uterus being the optimal incubator. In addition to finding the optimal day of embryo transfer, the policies used for embryo transfer should also be considered in order to maximise IVF success. It has been suggested that FET policy should be employed over fresh embryo transfer, as it leads to higher rates of implantation. The frozen– thawed approach allows the uterine endometrium to recover after ovarian stimulation and for synchronicity to resume. Doing so increases endometrial receptivity to the embryo, increasing the likelihood of implantation (Roque et al., 2015). In the present study, both fresh and frozen–thawed embryos were included to avoid selection bias. One of the main reasons behind transferring a blastocyst embryo over a cleavage embryo is the ability for self-selection. It is well known that the morphological criteria used to grade cleavage embryos are limited and many studies have debated the correlation between morphological features and pregnancy rates (Scott et al., 2000; Sjoblom et al., 2006; Steer et al., 1992). In conjunction with evidence showing cleavage embryos having a higher incidence of chromosomal abnormalities than blastocysts, many authors subsequently concluded that blastocyst embryo transfer is better than cleavage embryo transfer when multiple embryos are available. In this present study, all cleavage-stage embryos were graded on Day 3 regardless of day of transfer, using a local protocol that is based on cell number and degree of compaction or fragmentation. Embryos with all grades (A–D) were transferred. Despite the Day 3 group having higher percentages of grade C and D embryos than the Day 4–6 group (18.7% versus 14.8% and 10.3% versus 4.6%, respectively), Day 3 embryo transfer resulted in higher pregnancy and live birth rates, when both unadjusted and adjusted for embryo grading. This raises the possibility that current morphological criteria used to grade cleavage embryos do not correlate well with pregnancy outcome. Therefore, refinement of the grading
system for cleavage embryos is necessary in order to better distinguish between good- and poor-quality embryos. At present, time-lapse morphokinetics is a non-invasive alternative that studies the developmental kinetics between a normal euploid and aneuploid embryo (Basile et al., 2014). A recent study by Ezoe et al. (2019) has demonstrated evidence for using blastomere movement to predict pregnancy outcome after fresh embryo transfer of Day 2 embryos. Further development in time-lapse morphokinetics will therefore allow better identification of embryonic characteristics to support early embryo transfer. A limitation of this study is its duration of 8 years (2008–2016), during which there have been some changes to the local laboratory protocols in IVF techniques and culture media. This possible heterogeneity has been broadly accounted and adjusted for by the inclusion of ‘year of treatment’ in the parsimonious model adjustment. Overall, the adjusted OR still favours the transfer of the single embryo on Day 3. While the findings of this study are valuable in contributing towards current understanding of the relationship between day of embryo transfer and pregnancy outcome, further research needs to be undertaken before the results can be extrapolated to the general population. In particular, a prospective study with an intention-totreat protocol that randomly compares Day 3 transfer and a policy to grow on to blastocyst stage is necessary. Furthermore, the results from the current study are only applicable to the unique subgroup of women with one surviving embryo during Day 3. There is therefore a need to assess, on a pregnancy per embryo basis, the outcomes of Day 3 versus grown on blastocyst policies when patients have multiple available embryos. In conclusion, this study's findings have demonstrated that in a woman with one embryo available, transferring Day 3 cleavage-stage embryos into the uterus leads to higher rates of pregnancy and live birth, compared with growing the embryo on and aiming for Day 4–6 embryo transfer. Further studies are needed to elucidate the interaction between the host uterus and cleavagestage embryo to understand why the
uterus could be a better incubator than the laboratory.
ACKNOWLEDGEMENTS The authors would like to thank Ms Vivien MacLachlan, Donor, Data and Research Manager of Monash IVF, for her assistance with data collection.
SUPPLEMENTARY MATERIALS Supplementary material associated with this article can be found in the online version at doi:10.1016/j.rbmo.2019.08.003.
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