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A deep dive into the morphokinetics and ploidy of low-quality blastocysts

Open AccessPublished:June 17, 2022DOI:https://doi.org/10.1016/j.xfre.2022.06.004

      Objective

      To describe morphokinetic parameters and ploidy among low-quality blastocysts not meeting the criteria for clinical use.

      Design

      Prospective cohort study.

      Setting

      Academic medical center.

      Patient(s)

      Two hundred patients undergoing in vitro fertilization between February 2018 and November 2019.

      Intervention(s)

      All embryos were cultured in a time-lapse incubator. All expanded blastocysts underwent preimplantation genetic testing for aneuploidy using next-generation sequencing.

      Main Outcome Measure(s)

      Static blastocyst morphology grading; morphokinetic parameters, including time to each cell division (2-cell formation to 8-cell formation); time to morula formation; time to the start of blastulation; time to blastocyst formation; and preimplantation genetic testing for aneuploidy results.

      Result(s)

      A total of 1,306 embryos progressed to the expanded blastocyst stage; of these, 935 embryos met the criteria for clinical use and were designated as high quality, whereas 371 embryos were graded as low quality and did not meet the criteria for use. In morphokinetic evaluation, low-quality embryos developed more quickly to 5-cell formation (t5) 48.4 [42.4–48.7) vs 50.2 [46.3–50.1] hours, but progressed more slowly thereafter with tM 91.5 [85.9–92.3] vs 88.3 [82.1–88.3] and tB 114.0 [106.4–113.9] vs 106.9 [101.3–107.4] hours. Among the low-quality embryos, 75.5% were aneuploid, 22.4% were euploid, and 2.2% had undetermined chromosome copy number results. Morphokinetic parameters did not differ between the euploid and aneuploid low-quality embryos.

      Conclusion(s)

      Morphokinetic analysis did not distinguish between euploid and aneuploid low-quality embryos.

      Key Words

      Discuss: You can discuss this article with its authors and other readers at https://www.fertstertdialog.com/posts/xfre-d-22-00075
      In vitro fertilization has progressed to a stage at which the focus is no longer on the ability to achieve a pregnancy but on the time to singleton pregnancy and live birth. Improvements in the embryology laboratory have allowed for blastocyst culture and other methods aimed at selecting the single best embryo for transfer. One of these methods is trophectoderm (TE) biopsy at the blastocyst stage for preimplantation genetic testing for aneuploidy (PGT-A). A downside of PGT-A is the need for a “freeze all” cycle with an attendant delay in transfer and a requirement for vitrification and subsequent warming of a selected embryo. Time-lapse imaging (TLI) has been explored as a noninvasive mechanism for identifying an embryo that is most likely to result in successful implantation via continuous image acquisition and study of individual embryo morphokinetics. Multiple algorithms have been designed to use embryo morphokinetics to predict embryos with a higher probability of live birth upon transfer; however, none of these have demonstrated an association with ploidy that is strong enough to supplant the clinical application of PGT-A (
      • Mumusoglu S.
      • Yarali I.
      • Bozdag G.
      • Ozdemir P.
      • Polat M.
      • Sokmensuer L.K.
      • et al.
      Time-lapse morphokinetic assessment has low to moderate ability to predict euploidy when patient- and ovarian stimulation-related factors are taken into account with the use of clustered data analysis.
      ).
      Historically, standard static embryo morphology gradings have been used to select embryos for transfer. In practices performing a high volume of PGT-A, it is common to define a criterion for biopsy that excludes embryos with poor-quality TE or inner cell mass (ICM) grading from undergoing TE biopsy for PGT-A. In this setting, low-quality embryos are discarded (
      • Knudtson J.F.
      • Robinson R.D.
      • Sparks A.E.
      • Hill M.J.
      • Chang T.A.
      • Van Voorhis B.J.
      Common practices among consistently high-performing in vitro fertilization programs in the United States: 10-year update.
      ). However, studies have shown that low-quality embryos can result in successful pregnancies (
      • Capalbo A.
      • Rienzi L.
      • Cimadomo D.
      • Maggiulli R.
      • Elliott T.
      • Wright G.
      • et al.
      Correlation between standard blastocyst morphology, euploidy and implantation: an observational study in two centers involving 956 screened blastocysts.
      ). We sought to describe the morphokinetics of low-quality embryos and any relationship to embryonic ploidy in an attempt to elucidate features that could predict euploid status from a low-quality embryo. Our hypothesis is that morphokinetic parameters predict blastocyst quality as measured by static morphology; however, we predict that morphokinetic parameters will have a more limited impact on embryonic ploidy.

      Materials and methods

      Trial Design and Study Population

      This is a secondary analysis of a sibling oocyte study of 2 different culture media systems designed to study early embryonic development within a time-lapse incubator (
      • Quinn M.M.
      • Marsh P.
      • Ribeiro S.
      • Simbulan R.K.
      • Hickman C.
      • Berntsen J.
      • et al.
      Aneuploidy rates and morphokinetic parameters of embryos cultured in distinct culture media: a sibling oocyte study.
      ). In the primary study, embryo quality, morphokinetic parameters, and aneuploidy rates from TE biopsy were similar between sibling embryos cultured in distinct media systems from the time of gamete isolation. For this study, we focused on the static morphology, morphokinetics, and ploidy of high-quality embryos vs. those of poor-quality embryos that were graded insufficient for clinical use. Individuals planning in vitro fertilization with the intent of blastocyst culture and PGT-A were offered enrollment in the study before their treatment cycle between February 2018 and November 2019. During this time frame, 631 patients were eligible to participate and 200 patients consented to participation. The inclusion and exclusion criteria have been described previously (
      • Quinn M.M.
      • Marsh P.
      • Ribeiro S.
      • Simbulan R.K.
      • Hickman C.
      • Berntsen J.
      • et al.
      Aneuploidy rates and morphokinetic parameters of embryos cultured in distinct culture media: a sibling oocyte study.
      ). One hundred seventy-six individuals completed the study.
      The study was approved by the institutional review board of the University of California San Francisco (IRB #17-22331) and registered on clinicaltrials.gov (NCT 03503877). Written informed consent was obtained from all study subjects before participation.

      Ovarian Hyperstimulation and Laboratory Procedures

      Ovarian stimulation was performed as described previously (
      • Quinn M.M.
      • Marsh P.
      • Ribeiro S.
      • Simbulan R.K.
      • Hickman C.
      • Berntsen J.
      • et al.
      Aneuploidy rates and morphokinetic parameters of embryos cultured in distinct culture media: a sibling oocyte study.
      ). Oocyte retrieval was performed according to clinic standard 36 hours after ovulatory trigger. A semen sample was obtained by masturbation within 1 hour of oocyte retrieval. The method of fertilization—via conventional insemination vs. intracytoplasmic sperm injection—was determined by the patient’s primary physician.
      At 16–18 hours, fertilization was evaluated by the existence of 2 pronuclei. All embryos were cultured in the EmbryoScope+ time-lapse incubator (Vitrolife A/S, Viby J, Denmark). Once placed in the EmbryoScope+ time-lapse incubator, embryos were cultured at 37 °C with 6.5% CO2 and 5.0% O2 for up to 6 days without media exchange.
      The EmbryoScope+ incubation chamber contains a built-in microscope and camera, allowing for continuous monitoring of embryonic development. An image acquisition software was used to obtain high-contrast images every 10 minutes from several focal planes to create time-lapse videos. Conventional embryonic assessment was made through observations at prespecified time points. Cleavage-stage embryos were assessed for cell number, symmetry, percentage fragmentation, evidence of multinucleation, and progression of compaction. Blastocysts were evaluated to assess for blastocele volume and expansion, ICM development, and TE organization.
      Additional morphokinetic parameters were assessed with time-lapse videos, including time to pronuclear fading or syngamy, time to 2–8 cells, time to morula, time to start of blastulation, time to blastocyst, and time to expanded blastocyst. Cleavage anomalies were recorded.
      Embryonic biopsy for preimplantation genetic testing was performed at the blastocyst stage in all embryos reaching full blastocyst. On the day of the biopsy, 5–10 TE cells were gently aspirated. Biopsied cells were washed and cryopreserved before being sent for testing. Biopsied TE cells were analyzed for all 24 chromosomes by the testing laboratory (PacGenomics, Agoura Hills, CA) using a next-generation sequencing–based assay.

      Predictors

      The primary predictors or exposure variables were morphokinetic parameters assessing time to specific developmental endpoints from TLI (as delineated earlier). The age of the oocyte from which an embryo was derived was dichotomized to <35 years or ≥35 years for stratified analysis.

      Outcomes

      The primary outcome was static blastocyst embryo morphology. High-quality embryos meeting the criteria for clinical use were defined as blastocysts with expansion grade 3–6 according to Gardner criteria and at least a B grading for ICM and TE (
      • Gardner D.K.
      • Lane M.
      • Stevens J.
      • Schlenker T.
      • Schoolcraft W.B.
      Blastocyst score affects implantation and pregnancy outcome: towards a single blastocyst transfer.
      ). Expanded blastocysts with C grading for either t TE or ICM were defined as low-quality embryos and were deemed unsuitable for clinical use. A secondary outcome was embryonic ploidy determined by TE biopsy with PGT-A. A subanalysis included the type of aneuploidy (simple, segmental, or complex).

      Statistical Analysis

      Outcomes were assessed for normality of distribution. Mean, standard deviation, medians, and interquartile ranges are reported. Chi-square was used as appropriate. Morphokinetic evaluations were compared using Wilcoxon rank sum testing with Bonferroni correction to adjust for multiple comparisons. All analyses were performed in the statistical software package R version 3.6.3 (R Core Team, 2020).

      Results

      Baseline characteristics and stimulation parameters of the enrolled patients were reported previously (
      • Quinn M.M.
      • Marsh P.
      • Ribeiro S.
      • Simbulan R.K.
      • Hickman C.
      • Berntsen J.
      • et al.
      Aneuploidy rates and morphokinetic parameters of embryos cultured in distinct culture media: a sibling oocyte study.
      ). One hundred seventy patients contributed embryos to the study. The median number of oocytes collected was 13.5 (10.0–19.75). The median number of normally fertilized oocytes (2PN) per patient was 10.5 (7–15) and that of blastocysts formed was 7 (4–11). Blastulation rate (blastocysts/2PN) per patient was 71% (55%–85%).
      There were 935 high-quality blastocysts and 371 low-quality blastocysts that did not meet the criteria for clinical use. The distribution of static morphology grading at the cleavage stage and the final blastocyst grading is depicted in Figure 1. Full automatic annotations were possible for 864 high-quality and 328 low-quality blastocysts. Time-lapse imaging revealed a shorter time to 5 cells in embryos that subsequently became low-quality blastocysts: 48.4 (42.4–48.7) hours vs. 50.2 (46.3–50.1) hours; P = .02 (Table 1). Embryos that would become low-quality blastocysts reached all subsequent morphokinetic milestones at a slower pace. The time to blastocyst formation was 114.0 (106.4–113.9) hours vs 106.9 (101.3–107.4) hours, P<.0001 (Table 1). A similar pattern was observed when restricting the morphokinetic analysis to euploid embryos graded low vs. high quality (Supplemental Table 1, available online).
      Figure thumbnail gr1
      Figure 1(A) Cleavage-stage embryo grading among high- and low-quality embryos. (B) Blastocyst stage final embryo grading among high- and low-quality embryos (Gardner criteria). ICM = inner cell mass. TE = trophectoderm.
      Table 1Morphokinetic parameters for high-quality and low-quality embryos in time-lapse imaging.
      ParameterHigh qualityLow qualityP value (Wilcoxon rank sum)P value (Bonferroni corrected)
      nMean ± SDMedian (IQR)nMean ± SDMedian (IQR)
      tPNf
      From insemination to pronuclear fading.
      86424.0 ± 3.623.6 (21.8–24.0)32824.5 ± 3.924.3 (22.0–24.5).028.396
      t2
      Two-cell formation.
      86426.5 ± 3.726.1 (24.3–26.5)32827.1 ± 4.126.9 (24.6–27.1).011.158
      t3
      Three-cell formation.
      86437.5 ± 4.737.5 (34.9–37.5)32837.2 ± 5.737.4 (34.2–37.2).4771
      t4
      Four-cell formation.
      86438.5 ± 4.638.2 (35.8–38.5)32839.3 ± 5.539.0 (35.6–39.3).048.668
      t5
      Five-cell formation.
      86450.1 ± 7.050.2 (46.3–50.1)32848.7 ± 8.548.4 (42.4–48.7).001.020
      t6
      Six-cell formation.
      86452.2 ± 6.551.9 (48.5–52.2)32852.8 ± 8.652.4 (47.4–52.8).5631
      t7
      Seven-cell formation.
      86454.2 ± 7.453.3 (49.6–54.2)32856.0 ± 9.054.7 (49.9–56.0).003.0370
      t8
      Eight-cell formation.
      86457.7 ± 8.956.1 (51.6–57.7)32860.3 ± 10.159.0 (53.0–60.3)<.001<.001
      tM
      Morula formation.
      86488.3 ± 9.588.3 (82.1–88.3)32892.3 ± 10.091.5 (85.9–92.3)<.001<.001
      tSB
      Appearance of blastocele/start of blastulation.
      86499.0 ± 8.498.4 (93.4–99.0)328103.3 ± 9.1102.7 (97.2–103.3)<.001<.001
      tB
      Formation of blastocyst.
      847107.4 ± 9.0106.9 (101.3–107.4)312113.9 ± 10.5114.0 (106.4–113.9)<.001<.001
      cc2
      Duration of second cell cycle.
      86411.0 ± 2.311.3 (10.6–11.0)32810.1 ± 4.311.2 (10.0–10.1).0971
      cc3
      Duration of third cell cycle.
      86411.6 ± 4.912.2 (10.9–11.6)3289.4 ± 6.611.3 (1.3–9.4)<.001<.001
      db
      Duration of blastulation (tb–tSB).
      8478.5 ± 3.67.8 (6.1–8.5)31211.0 ± 5.69.8 (7.0–11.0)<.001<.001
      Note: All times are presented in hours; tSB notation required for inclusion. IQR = interquartile range.
      a From insemination to pronuclear fading.
      b Two-cell formation.
      c Three-cell formation.
      d Four-cell formation.
      e Five-cell formation.
      f Six-cell formation.
      g Seven-cell formation.
      h Eight-cell formation.
      i Morula formation.
      j Appearance of blastocele/start of blastulation.
      k Formation of blastocyst.
      l Duration of second cell cycle.
      m Duration of third cell cycle.
      n Duration of blastulation (tb–tSB).
      Among low-quality blastocysts, 280 were aneuploid, 83 were euploid, and 8 had an indeterminate result. Morphokinetic parameters did not differ between euploid and aneuploid low-quality blastocysts (Table 2). High-quality embryos were more likely to be euploid (41.5% vs. 22.4%, P<.001). This was true in a subgroup analysis of embryos derived from women aged ≤ 35 years and >35 years (Table 3). Complex aneuploidy was more frequently identified in poor-quality embryos, particularly in women aged >35 years (Supplemental Table 2).
      Table 2Morphokinetic parameters for euploid and aneuploid low-quality embryos in time-lapse imaging.
      ParameterEuploidAneuploidP value (Wilcoxon rank sum)P value (Bonferroni corrected)
      nMean ± SDMedian (IQR)nMean ± SDMedian (IQR)
      tPNf
      From insemination to pronuclear fading.
      7324.7 ± 3.524.7 (22.2–24.7)24724.4 ± 4.124.2 (21.9–24.4).5711
      t2
      Two-cell formation.
      7327.3 ± 3.627.4 (24.9–27.3)24727.0 ± 4.326.8 (24.4–27.0).4631
      t3
      Three-cell formation.
      7337.5 ± 5.137.8 (34.4–37.5)24737.0 ± 5.837.0 (34.1–37.0).4531
      t4
      Four-cell formation.
      7339.5 ± 5.339.3 (36.8–39.5)24739.1 ± 5.538.9 (35.5–39.1).6541
      t5
      Five-cell formation.
      7348.2 ± 7.749.0 (41.9–48.2)24748.7 ± 8.648.1 (42.7–48.7).8451
      t6
      Six-cell formation.
      7352.5 ± 7.552.6 (47.5–52.5)24752.6 ± 8.751.8 (47.4–52.6).6301
      t7
      Seven-cell formation.
      7355.9 ± 8.655.5 (51.2–55.9)24755.7 ± 9.054.5 (49.7–55.7).7211
      t8
      Eight-cell formation.
      7360.6 ± 10.058.6 (53.3–60.6)24760.1 ± 10.259.1 (52.4–60.1).8131
      tM
      Morula formation.
      7393.5 ± 10.294.1 (87.8–93.5)24791.6 ± 9.791.1 (85.8–91.6).1321
      tSB
      Appearance of blastocele/start of blastulation.
      73103.6 ± 9.5102.6 (97.9–103.6)247102.9 ± 8.8102.6 (96.8–102.9).6561
      tB
      Formation of blastocyst.
      71113.7 ± 9.8113.5 (106.9–113.7)233113.7 ± 10.6113.9 (106.2–113.7).9591
      cc2
      Duration of second cell cycle.
      7310.2 ± 4.711.3 (10.0–10.2)24710.1 ± 4.311.2 (9.9–10.1).5591
      cc3
      Duration of third cell cycle.
      738.7 ± 6.211.1 (1.0–8.7)2479.5 ± 6.611.4 (1.4–9.5).3491
      db
      Duration of blastulation (tb–tSB).
      7110.6 ± 5.49.6 (6.6–10.6)23311.1 ± 5.710.0 (7.2–11.1).3801
      Noe: All times are presented in hours, tSB notation required for inclusion. IQR = interquartile range.
      a From insemination to pronuclear fading.
      b Two-cell formation.
      c Three-cell formation.
      d Four-cell formation.
      e Five-cell formation.
      f Six-cell formation.
      g Seven-cell formation.
      h Eight-cell formation.
      i Morula formation.
      j Appearance of blastocele/start of blastulation.
      k Formation of blastocyst.
      l Duration of second cell cycle.
      m Duration of third cell cycle.
      n Duration of blastulation (tb–tSB).
      Table 3Ploidy status of high-quality and low-quality embryos by age ≤35 years or >35 years.
      GroupCharacteristicsHigh qualityLow qualityP value (chi-square)
      n%n%
      AllEuploid38841.58322.4<.001
      Aneuploid (all)53056.728075.5
      Undetermined171.882.2
      Age (y)

      ≤35 years
      Euploid14648.84232.6.008
      Aneuploid14749.28465.1
      Undetermined62.032.3
      Age (y)

      >35 years
      Euploid24238.14116.9<.001
      Aneuploid38360.219681.0
      Undetermined111.752.1

      Discussion

      In this secondary analysis of a large sibling oocytes study using TLI and TE biopsy for PGT-A to evaluate the developmental competence of blastocysts, we demonstrated that nearly a quarter of low-quality blastocysts were euploid; however, TLI was unable to distinguish euploid vs. aneuploid low-quality embryos.
      Multiple prior publications have explored the use of morphokinetic timings gleamed from TLI to predict ploidy status among blastocysts. A recent review on the topic concluded that although morphokinetic parameters from TLI may relate to ploidy status, the predictive value was inadequate to replace PGT-A for aneuploidy screening (
      • Zaninovic N.
      • Irani M.
      • Meseguer M.
      Assessment of embryo morphology and developmental dynamics by time-lapse microscopy: is there a relation to implantation and ploidy?.
      ). This was especially true when kinetic risk models developed at different centers were adopted before internal validation (
      • Kramer Y.G.
      • Kofinas J.D.
      • Melzer K.
      • Noyes N.
      • McCaffrey C.
      • Buldo-Licciardi J.
      • et al.
      Assessing morphokinetic parameters via time lapse microscopy (TLM) to predict euploidy: are aneuploidy risk classification models universal?.
      ). Minasi et al. (
      • Minasi M.G.
      • Colasante A.
      • Riccio T.
      • Ruberti A.
      • Casciani V.
      • Scarselli F.
      • et al.
      Correlation between aneuploidy, standard morphology evaluation and morphokinetic development in 1730 biopsied blastocysts: a consecutive case series study.
      ) explored the relationship among standard morphology, morphokinetic development, and embryonic ploidy as determined by TE biopsy with PGT-A via array comparative genomic hybridization in 928 blastocysts. Euploid embryos demonstrated a shorter time to start blastulation, expansion, and hatching than that demonstrated by aneuploid embryos. Notably, standard morphology was poorly predictive of ploidy with C gradings for ICM among 17.1% of euploid blastocysts and C gradings for TE among 26.6% of euploid blastocysts (
      • Minasi M.G.
      • Colasante A.
      • Riccio T.
      • Ruberti A.
      • Casciani V.
      • Scarselli F.
      • et al.
      Correlation between aneuploidy, standard morphology evaluation and morphokinetic development in 1730 biopsied blastocysts: a consecutive case series study.
      ). Similarly, Capalbo et al (
      • Capalbo A.
      • Rienzi L.
      • Cimadomo D.
      • Maggiulli R.
      • Elliott T.
      • Wright G.
      • et al.
      Correlation between standard blastocyst morphology, euploidy and implantation: an observational study in two centers involving 956 screened blastocysts.
      ) found a 25.5% euploidy rate among poor-quality blastocysts, a rate of 30.1% when ICM was “C,” and a rate of 23.4% with a TE “C” score. Of note, this study included only 153 total poor-quality blastocysts from 2 centers. This finding that poor-quality blastocysts have potential for euploid status is important when many clinics exclude embryos for biopsy on the basis of static morphology grading (
      • Knudtson J.F.
      • Robinson R.D.
      • Sparks A.E.
      • Hill M.J.
      • Chang T.A.
      • Van Voorhis B.J.
      Common practices among consistently high-performing in vitro fertilization programs in the United States: 10-year update.
      ).
      Although we demonstrate a shorter time to 5-cell formation in embryos that subsequently became low-quality embryos, the significance of this finding is uncertain. Notably, 5-cell formation is before embryonic genome activation. Prior research limited by the assessment of embryonic ploidy at the cleavage stage by array comparative genomic hybridization reported some ability to distinguish between aneuploid and euploid embryos on the basis of morphokinetic development to the 4-cell stage (
      • Basile N.
      • del Carmen Nogales M.
      • Bronet F.
      • Florensa M.
      • Riqueiros M.
      • Rodrigo L.
      • et al.
      Increasing the probability of selecting chromosomally normal embryos by time-lapse morphokinetics analysis.
      ). Specifically, the investigators demonstrated a greater standard deviation in time to early cell divisions among embryos with meiotic errors (
      • Chavez S.L.
      • Loewke K.E.
      • Han J.
      • Moussavi F.
      • Colls P.
      • Munne S.
      • et al.
      Dynamic blastomere behaviour reflects human embryo ploidy by the four-cell stage.
      ) and an increased risk for falling outside optimal ranges proposed for 5-cell formation to 2-cell formation and duration of third cell cycle (
      • Basile N.
      • del Carmen Nogales M.
      • Bronet F.
      • Florensa M.
      • Riqueiros M.
      • Rodrigo L.
      • et al.
      Increasing the probability of selecting chromosomally normal embryos by time-lapse morphokinetics analysis.
      ). Although this differential was demonstrated in the time to 5-cell formation between high and low-quality embryos within our study, it was not seen when comparing the time to 5-cell formation between low-quality euploid and aneuploid blastocysts.
      A significant limitation of our study is that low-quality euploid embryos were not transferred. As a result, we are unable to describe the reproductive potential of these low-quality blastocysts. In the study by Capalbo et al (
      • Capalbo A.
      • Rienzi L.
      • Cimadomo D.
      • Maggiulli R.
      • Elliott T.
      • Wright G.
      • et al.
      Correlation between standard blastocyst morphology, euploidy and implantation: an observational study in two centers involving 956 screened blastocysts.
      ), poor-quality euploid embryos were eligible for transfer, and 7 of 13 (53.8%) of these resulted in ongoing implantation. This outcome was not different from euploid embryos that were graded as average quality (
      • Capalbo A.
      • Rienzi L.
      • Cimadomo D.
      • Maggiulli R.
      • Elliott T.
      • Wright G.
      • et al.
      Correlation between standard blastocyst morphology, euploidy and implantation: an observational study in two centers involving 956 screened blastocysts.
      ). These data raise the question of whether embryology laboratories should consider poor-quality embryos suitable for clinical use either for transfer untested or when PGT-A is planned. This question reveals the tension between an approach that favors high levels of embryo selection designed to minimize time to pregnancy and an alternative that maximizes cumulative live birth per retrieval cycle. Future investigations should evaluate the reproductive potential of low-quality euploid blastocysts in larger cohorts.

      Acknowledgments

      The authors acknowledge the work of the embryology team at the University of California San Francisco’s Center for Reproductive Health, without which this study would not be possible. Furthermore, they thank PacGenomics for providing preimplantation genetic testing for aneuploidy and CooperSurgical for supplying embryo culture media. Additionally, the authors appreciate the assistance of Jørgen Berntsen, M.Sc. and Mark Larman, Ph.D. with data analysis.

      Supplementary data

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