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Sequential embryo scoring as a predictor of aneuploidy in poor-prognosis patients

      Abstract

      The delivery rates of 298 patients having preimplantation genetic diagnosis with aneuploidy screening (PGS) were compared with the delivery rates of 144 PGS patients that cancelled the plan for PGS with embryo transfer on day 2 or day 3. The goal of this study was to compare the impact of embryo de-selection with PGS to embryo selection using sequential embryo scoring (SES) on outcome in poor-prognosis patients. Embryos with good sequential scores were more likely to have a normal PGS result than embryos with poor SES scores (34% versus 12%; P < 0.05). Patients proceeding with PGS had an overall delivery rate of 15% per oocyte retrieval. There was a significant difference in delivery rates between patients with less than six embryos and patients with greater than six embryos (6% versus 19%; P < 0.005). The overall delivery rate for patients having transfers without PGS was 23% (P < 0.05 compared with PGS patients) with no difference between low and good responders. It was concluded that PGS neither enhanced nor impaired delivery rates in high responding poor-prognosis patients yet SES may be more accurate than PGS as a means of selection for low-responding poor-prognosis patients.
      Preimplantation genetic diagnosis for aneuploidy screening (PGS) is a technology used to determine the chromosome complement of an embryo prior to its transfer into the uterus. Despite promising initial results, PGS has not drastically increased delivery rates. This study compares the delivery rates of patients having PGS and those canceling the plan for PGS opting for an embryo transfer without it. Embryos were selected by the PGS result (normal or abnormal) for PGS patients or based on embryo morphology for those patients who did not undergo PGS. The results of this study showed that there was no difference in delivery rates between these two groups of patients and, in fact, a lower delivery rate after PGS in patients having less than six embryos.

      Keywords

      Introduction

      Advances in stimulation protocols, embryo culture conditions and methods of embryo selection continue to improve delivery rates following IVF. However, improving success rates in poor-prognosis patients remains challenging especially as more of the patients remaining or entering treatment fall into this category. Preimplantation genetic diagnosis for aneuploidy screening (PGS) has been offered as a means of improving outcomes in repeat failure IVF patients, patients of advanced maternal age and those with recurrent pregnancy loss (
      • Gianaroli L.
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      Will preimplantation genetic diagnosis assist patients with a poor prognosis to achieve pregnancy?.
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      Preimplantation genetic diagnosis for aneuploidies in patients undergoing in vitro fertilization with a poor prognosis: identification of the categories for which it should be proposed.
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      The role of preimplantation genetic diagnosis for aneuploidy.
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      Pronuclear morphology and chromosomal abnormalities as scoring criteria for embryo selection.
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      The beneficial effects of preimplantation genetic diagnosis for aneuploidy support extensive clinical application.
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      The results of aneuploidy screening in 276 couples undergoing assisted reproductive techniques.
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      ). Improved success is based on the assumption that high rates of chromosomal abnormalities in the embryos of these patients account for their lack of a successful pregnancy. Despite reports of improved outcomes (
      • Gianaroli L.
      • Magli M.
      • Munné S.
      Will preimplantation genetic diagnosis assist patients with a poor prognosis to achieve pregnancy?.
      ,
      • Gianaroli L.
      • Magli M.
      • Ferraretti A.
      • Munné S.
      Preimplantation genetic diagnosis for aneuploidies in patients undergoing in vitro fertilization with a poor prognosis: identification of the categories for which it should be proposed.
      ,
      • Gianaroli L.
      • Magli M.
      • Ferraretti A.
      • Fortini D.
      • Grieco N.
      Pronuclear morphology and chromosomal abnormalities as scoring criteria for embryo selection.
      ,
      • Gianaroli L.
      • Magli M.
      • Fiorentino F.
      • Baldi M.
      • Ferraretti A.
      Clinical value of preimplantation genetic diagnosis.
      ,
      • Gianaroli L.
      • Magli M.C.
      • Ferraretti A.
      • et al.
      The beneficial effects of preimplantation genetic diagnosis for aneuploidy support extensive clinical application.
      ,
      • Kahraman S.
      • Benkhalifa M.
      • Donmez E.
      • et al.
      The results of aneuploidy screening in 276 couples undergoing assisted reproductive techniques.
      ,
      • Kuliev A.
      • Verlinsky Y.
      Impact of preimplantation genetic diagnosis for chromosomal disorders on reproductive outcome.
      ,
      • Munné S.
      • Magli C.
      • Cohen J.
      • et al.
      Positive outcome after preimplantation diagnosis of aneuploidy in human embryos.
      ,
      • Munné S.
      • Sandalinas M.
      • Escudero T.
      • et al.
      Improved implantation after preimplantation genetic diagnosis of aneuploidy.
      ,
      • Pehlivan T.
      • Rubio C.
      • Rodrigo L.
      • et al.
      Impact of preimplantation genetic diagnosis on IVF outcome in implantation failure patients.
      ,
      • Verlinsky Y.
      • Tur-Kaspa I.
      • Cieslak J.
      • et al.
      Preimplantation testing for chromosomal disorders improves reproductive outcome of poor prognosis patients.
      ), prospective, controlled, randomized studies have not found PGS to significantly improve the chance of having a baby (
      • Blockeel C.
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      Prospectively randomized controlled trial of PGS in IVF/ICSI patients with poor implantation.
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      Preimplantation genetic screening for aneuploidy of embryos after in vitro fertilization in women aged at least 35 years: a prospective randomized trial.
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      Preimplantation genetic screening in women of advanced maternal age caused a decrease in clinical pregnancy rate: a randomized controlled trial.
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      What next for preimplantation genetic screening (PGS)? Experience with blastocyst biopsy and testing for aneuploidy.
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      • et al.
      In vitro fertilization with preimplantation genetic screening.
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      ).
      Within the population of PGS patients there is a subgroup that, due to a paucity of embryos deemed suitable for biopsy or patient choice, converts back to conventional embryo transfer on either day 2 or day 3 following retrieval rather than proceeding with PGS. This group of patients could be considered a control group in a PGS programme since they present with the same conditions for selection to perform PGS but proceed without it.
      Morphological scoring continues to be the primary tool for embryo selection and transfer in patients not having PGS (
      • Claman P.
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      The impact of embryo quality and quantity on implantation and the establishment of viable pregnancies.
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      A formula for scoring human embryo growth rates in in vitro fertilization: its value in predicting pregnancy and in comparison with visual estimates of embryo quality.
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      Implantation: embryo score to predict implantation after in-vitro fertilization: based on 957 single embryo transfers.
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      • Hardarson T.
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      Human embryos with unevenly sized blastomeres have lower pregnancy and implantation rates: indications for aneuploidy and multinucleation.
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      • Nagy Z.
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      • Diamond M.
      • et al.
      Pronuclear morphology evaluation with subsequent evaluation of embryo morphology significantly increases implantation rates.
      ,
      • Puissant F.
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      Embryo scoring as a prognostic tool in IVF treatment.
      ,
      • Salumets A.
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      Early cleavage predicts the viability of human embryos in elective single embryo transfer procedures.
      ,
      • Scott L.
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      The morphology of human pronuclear embryos is positively related to blastocyst development and implantation.
      ,
      • Staessen C.
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      The relationship between embryo quality and the occurrence of multiple pregnancies.
      ,
      • Steer C.V.
      • Mills C.L.
      • Tan S.L.
      • Campbell S.
      • Edwards R.G.
      Short communication: 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.
      ,
      • Van Royen E.
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      • De Neubourg D.
      • Laureys I.
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      Calculating the implantation potential of day 3 embryos in women younger than 38 years of age: a new model.
      ,
      • Ziebe S.
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      • Andersen A.G.
      • Gabrielsen A.
      • Andersen A.N.
      Embryo morphology or cleavage stage: how to select the best embryos for transfer after in-vitro fertilization.
      ). Recently, a prospective trial applying sequential morphometric scoring for embryo selection reported a significant correlation between pronuclear (PN) morphology and day-2 cell size, number and state of nucleation with viable deliveries (
      • Scott L.
      • Finn A.
      • O’Leary T.
      • McClellan S.
      • Hill J.
      Morphologic parameters of early cleavage-stage embryos that correlate with fetal development and delivery: prospective and applied data for increased pregnancy rates.
      ).
      In the study clinic, embryos in the general IVF population are selected for transfer using this system of sequential embryo scoring (SES) (
      • Scott L.
      • Finn A.
      • O’Leary T.
      • McClellan S.
      • Hill J.
      Morphologic parameters of early cleavage-stage embryos that correlate with fetal development and delivery: prospective and applied data for increased pregnancy rates.
      ). Embryos consisting of 6–8 cells on day 3 are preferentially selected based on morphological scores on day 1 and day 2 with day-2 parameters having the highest weighting on selection criteria. In contrast, embryos in the IVF–PGS population are de-selected based on the results of aneuploidy screening. The embryos deemed ‘normal’ or euploid after aneuploidy screening are only further selected based on morphological score when there is a surplus of good-quality euploid blastocysts.
      This study postulated that euploidy and aneuploidy were strongly related to both the morphological scores and the biological basis behind SES (
      • Scott L.
      • Finn A.
      • O’Leary T.
      • McClellan S.
      • Hill J.
      Morphologic parameters of early cleavage-stage embryos that correlate with fetal development and delivery: prospective and applied data for increased pregnancy rates.
      ). This hypothesis was tested by comparing PGS results to embryo morphology scores independently and to cumulative SES scores. The delivery rates of PGS patients having PGS and those who cancelled PGS were also compared in order to reveal the efficacy of PGS versus strict SES for poor-prognosis patients.

      Materials and methods

      Study design

      This study was a retrospective cohort analysis of embryo and cycle outcome of all poor-prognosis patients requesting PGS between January 2005 and December 2008. PGS was suggested for patients with advanced maternal age (>37 years), repeat failure IVF (more than two failed IVF attempts; all diagnoses) and recurrent pregnancy loss (more than five spontaneous abortions) (Table 1). Patients electing PGS were further categorized into two groups: those proceeding with PGS (group 1) and those converting back to conventional embryo transfer on day 2 or day 3 without PGS (group 2). The groups were further identified as either low responders (between one and five fertilized oocytes on day 1) or good responders (six or more fertilized oocytes on day 1) (Figure 1). Embryo morphology scores on day 1 and day 3 were related with PGS results from all patients having PGS. Delivery rate was also compared between the two groups (PGS versus no PGS; Figure 1).
      Table 1Patient diagnosis.
      DiagnosisPGS patients (n=298)PGS converted to IVF (n=144)
      Advanced maternal age12.822.9
      Recurrent pregnancy loss24.620.1
      Male19.522.2
      Unexplained15.011.1
      Tubal8.34.9
      Ovulatory6.14.2
      Endometriosis2.95.5
      Uterine2.94.9
      Other8.04.2
      Values are percentages.
      Figure thumbnail gr1
      Figure 1Prenatal genetic screening patients 2005–2008. D2/D3 = day 2/day 3; ET = embryo transfer; OR = oocyte retrieval; PGS = prenatal genetic screening.
      All patients had an agonist suppression, agonist flare or antagonist suppression as the ovarian stimulation protocol (Table 2). Agonist suppression protocols were typically prescribed for higher-responding, better-prognosis patients while antagonist and agonist flare protocols were usually reserved for patients with a poorer prognosis based on age, prior response and cycle day-3 FSH concentrations. Oocyte retrievals were performed 36 h after human chorionic gonadotrophin (HCG) administration and intracytoplasmic sperm injection or standard insemination occurred 39–41 h post HCG injection. Oocytes were cultured in individual, numbered drops and all resulting embryos were assigned an individual tracking number. All cultures were in standard media micro-drops under an oil overlay (Sage Complete Cleavage media and Sage Complete Blastocyst media; Cooper Surgical, NJ, USA). Embryos for transfer were selected based on PGS results and/or on embryo scoring where biopsy was performed and by SES when PGS was not performed.
      Table 2Patient demographics and cycle characteristics.
      Mean age (years)Mean cycle numberMean no. of embryos transferredImplantation rate (%)
      Implantation rate defined as number of gestational sacs with fetal heart beat per number of embryos transferred.
      Agonist suppression cycles (%)Antagonist cycles (%)Agonist flare cycles (%)
      PGS patients38.32.61.859/364 (16)127 (43)59 (20)112 (38)
      PGS converted to IVF39.32.42.442/346 (12)39 (27)
      P<0.005.
      50 (35)
      P<0.001.
      55 (38)
      a Implantation rate defined as number of gestational sacs with fetal heart beat per number of embryos transferred.
      b P < 0.005.
      c P < 0.001.
      All oocytes were scored for fertilization 16–18 h post insemination or intracytoplasmic sperm injection (55–57 h after HCG administration). Fertilized oocytes were assigned Z-scores based on PN morphology and nucleolar precursor body (NPB) alignment (
      • Scott L.
      • Alvero R.
      • Leondires M.
      • Miller B.
      The morphology of human pronuclear embryos is positively related to blastocyst development and implantation.
      ). Zygotes with aligned pronuclei and equal numbered, aligned NPB were designated as Z1, those with equal numbered and scattered NPB in both pronuclei were scored as Z2. Zygotes with 2PN containing unequal and or misaligned NPB were scored as Z3. Zygotes designated as Z4 had 2PN that were more than 20% unequal in size or 2PN that were not touching each other. Day-2 scoring was performed at 42–44 h post insemination (81–83 h post HCG). Embryos were scored for cell number, presence or absence of mono or multinucleated cells and cell size. Scoring on day 3 was performed between 64 and 65 h post insemination (103–105 h post HCG). The score on day 3 included number of blastomeres, percentage fragmentation and cell size (
      • Scott L.
      • Finn A.
      • O’Leary T.
      • McClellan S.
      • Hill J.
      Morphologic parameters of early cleavage-stage embryos that correlate with fetal development and delivery: prospective and applied data for increased pregnancy rates.
      ).
      Patients in group 1 (undergoing PGS) had embryo biopsy and blastomere fixation on the morning of culture day 3 approximately 68 h post insemination and 108 h post HCG. Embryos containing five cells or more with less than 30% fragmentation were biopsied. Embryo biopsy was performed with assisted hatching using acidified medium and biopsy in calcium- and magnesium-free media (Sage Modified HTF, Cooper Surgical). A second cell was biopsied only in the event that a nucleus could not be detected at the time of fixation. After biopsy, embryos were returned to individual culture drops and transferred to blastocyst culture media later in the day. Blastomeres removed at biopsy were fixed to glass slides using the Tarkowski method modified by Munné for single blastomeres (
      • Munné S.
      • Cohen J.
      Chromosome abnormalities in human embryos.
      ,
      • Tarkowski A.
      An air-drying method for chromosome preparation mouse eggs.
      ). The slides were then sent for fluorescence in-situ hybridization (FISH) analysis of chromosomes X, Y, 13, 15, 16, 17, 18, 21 and 22. PGS results were received the afternoon of culture day 4 and transfer of euploid embryos was performed either the afternoon of day 4 (2005 only) or the morning of culture day 5 (2005–2008).
      Patients in group 2 elected to cancel PGS after oocyte retrieval. The reasons for cancelling PGS included low embryo number, poor embryo quality and patient preference. These patients had embryo transfers on culture day 2 or culture day 3. Sequential embryo scoring (
      • Scott L.
      • Finn A.
      • O’Leary T.
      • McClellan S.
      • Hill J.
      Morphologic parameters of early cleavage-stage embryos that correlate with fetal development and delivery: prospective and applied data for increased pregnancy rates.
      ) was used to select embryos for transfer in these patients. Morphology reflecting embryo biology with the highest implantation rates determined the embryos’ qualifications for transfer. Day-2 scores were previously shown to have the highest impact on implantation (
      • Scott L.
      • Finn A.
      • O’Leary T.
      • McClellan S.
      • Hill J.
      Morphologic parameters of early cleavage-stage embryos that correlate with fetal development and delivery: prospective and applied data for increased pregnancy rates.
      ) and therefore were weighted the most heavily during selection, followed by PN score. Embryos consisting of 6–8 cells were selected on day 3 for transfer based on day-2 and PN morphology.
      A serum HCG test was performed on all patients 12–14 days after embryo transfer. A positive test was considered to be any value above 8 IU/ml that continued to rise after 48 h. This value was used to designate a positive pregnancy test, as it is the lowest value from which an intrauterine sac has been detected at 6 weeks of gestation in the clinic’s practice. Ultrasound was performed at 6 weeks of gestation. Spontaneous abortions (SAB) in this study were defined as any positive pregnancy test that did not result in a delivery. All deliveries were confirmed with patients either verbally or in writing. Results from the data analysed were evaluated using Fisher’s exact test.

      Results

      PGS was recommended for 442 IVF patients having oocyte retrievals between January 2005 and December 2008; 298 of which proceeded with PGS (group 1). Of these, 86 were defined as low responders (less than 6 2PN, 29%) and 212 as good responders (6 or more 2PN, 71%), and 127 (43%) had agonist suppression stimulation protocols, 59 (20%) had antagonist cycles and 112 (38%) had agonist flare stimulation cycles (Table 2). In 20 of the PGS cycles (7%), all embryos were cryopreserved at the 2PN stage for future PGS, resulting in no fresh transfer. Previously cryopreserved embryos were thawed and combined with fresh embryos in 24 of the total PGS cycles. PGS results showed no euploid embryos and therefore no embryo transfers in 44 of the low responders (51%) and 42 of the good responders (20%) in group 1. Deliveries occurred in 45 patients, resulting in a 15% delivery rate per oocyte retrieval and a 23% delivery rate per embryo transfer in group 1 (Table 3). There were five deliveries from low responders having PGS (6% delivery rate per oocyte retrieval) and 40 deliveries from good responders having PGS (19% delivery rate per oocyte retrieval; P < 0.005.
      Table 3Poor-prognosis patient outcomes based on number of two pronucleate oocytes in prenatal genetic screening (PGS) versus cancelled PGS groups.
      ParameterPGSPGS patients converting back to day-2 or day-3 ETLow responders
      Low responders=poor-prognosis patients with less than six 2PN oocytes.
      Good responders
      Good responders=poor-prognosis patients with less than six 2PN oocytes.
      Group 1 (PGS)Group 2 (no PGS)Group 1 (PGS)Group 2 (no PGS)
      Patients2981448611721227
      Cycles with ET1921382611216626
      Positive βHCG (% per ET)71 (37)52 (38)7 (27)41 (37)64 (49)11 (42)
      SAB26 (37)18 (35)2 (29)13 (32)24 (38)5 (45)
      Delivery (% per OR)45 (15)33 (23)
      P<0.05 versus PGS patients.
      5 (6)28 (24)
      P<0.005 versus low responders having PGS.
      40 (19)
      P<0.005 versus low responders having PGS.
      6 (22)
      Delivery (% per ET)45 (23)33 (24)5 (19)28 (25)40 (24)6 (23)
      ET = embryo transfer; OR = oocyte retrieval; SAB = spontaneous abortion.
      a Low responders = poor-prognosis patients with less than six 2PN oocytes.
      b Good responders = poor-prognosis patients with less than six 2PN oocytes.
      c P < 0.05 versus PGS patients.
      d P < 0.005 versus low responders having PGS.
      A total of 2244 embryos were biopsied for patients in group 1 with 2177 (97%) having interpretable FISH results. Of these, 566 (26%) were screened euploid for chromosomes X, Y, 13, 15, 16, 17, 18, 21 and 22, 409 of which were transferred. A second cell was removed in 151 (7%) embryos and 26 (6%) of these embryos were transferred. A second cell was only removed when a nucleus could not be found at the time of fixation. Therefore, no results on mosaicism could be obtained from these embryos, as there was typically only one cell that gave results.
      The PN, day-2 and day-3 morphology was compared with the euploidy results. The percentage of euploid in each morphological category was calculated as well as the percentage of each morphological category in the total euploid embryo population. Although Z1/Z2 embryos had the same rates of euploidy as Z3/Z4 embryos, the embryos with Z1 or Z2 PN scores comprised 72% of the total euploid embryos compared with Z3 or Z4 embryos which only accounted for 28% of the euploid embryos (P < 0.001; Table 4). Therefore, the selection of a Z1 or Z2 embryo was more likely to lead to the selection of a euploid embryo. Embryos with even cell sizes and no multinucleation on day 2 had a 31% euploidy rate and comprised 73% of the total euploid embryos after PGS. This was significantly different compared with embryos with even cell sizes and multinucleation, which were 20% euploid (P < 0.005) and made up only 7% of the euploid population (P < 0.0001; Table 5). Similarly, embryos with uneven cell sizes but no multinucleation had a euploidy rate of 19% (P < 0.0001) and accounted for 18% of the total euploid population (P < 0.0001; Table 5). Embryos with both uneven cell sizes and multinucleation were only 11% euploid (P < 0.0001), which accounted for only 3% of the euploid embryos after PGS (P < 0.0001; Table 5). Embryos with 7–8 cells on day 3 had a euploidy rate of 31% and comprised 66% of the total euploid embryos compared with embryos with 6 cells or less that showed a euploidy rate of 17% (P < 0.0001) and made up 27% of the total euploid embryos (P < 0.0001; Table 6). Embryos with more than 8 cells had a 23% euploidy rate (P < 0.05) and accounted for only 7% of the total euploid embryos (P < 0.0001; Table 6).
      Table 4Zygote morphology and percentage of euploid embryos.
      TypeNo.Euploid (%)% of total euploid (n=495)
      Total embryos Z–scored2015495 (25)
      Z1/Z21447358 (25)72
      Z3/Z4568137 (24)28
      P<0.001.
      a P < 0.001.
      Table 5Day-2 morphology and percentage of euploid embryos.
      Day-2 morphologyNo.Euploid (%)% of total euploid (n=520)
      Even cell size, no MN1225378 (31)73
      Even cell size, MN17835 (20)
      P<0.0001 compared to embryos with even cell size and no multinucleation.
      7
      P<0.005 compared to embryos with even cell size and no multinucleation.
      Uneven cell size, no MN49292 (19)
      P<0.005 compared to embryos with even cell size and no multinucleation.
      18
      P<0.005 compared to embryos with even cell size and no multinucleation.
      Uneven cell size, MN13915 (11)
      P<0.005 compared to embryos with even cell size and no multinucleation.
      3
      P<0.005 compared to embryos with even cell size and no multinucleation.
      MN = multinucleation.
      a P < 0.0001 compared to embryos with even cell size and no multinucleation.
      b P < 0.005 compared to embryos with even cell size and no multinucleation.
      Table 6Day-3 cell number and percentage of euploid embryos.
      Day-3 cell numberNo.Euploid (%)% of total euploid (n=542)
      7–81172359 (31)66
      ⩽6831145 (17)
      P<0.0001 compared to 7–8 cell embryos.
      27
      P<0.0001 compared to 7–8 cell embryos.
      >816638 (23)
      P<0.05 compared to 7–8 cell embryos.
      7
      P<0.0001 compared to 7–8 cell embryos.
      a P < 0.0001 compared to 7–8 cell embryos.
      b P < 0.05 compared to 7–8 cell embryos.
      Comparing the cumulative SES scoring with aneuploidy revealed embryos with a perfect SES score (day-3 embryos with 6–8 cells, good PN scores, even cell sizes and no multinucleation on day 2) had a euploidy rate of 34% and comprised 55% of the total euploid embryos (Table 7). Day-3 embryos with 6–8 cells, good PN scores, no multinucleation but uneven cell sizes on day 2 had a 17% euploidy rate (P < 0.0001 compared with perfect SES-scored embryos; Table 7) and made up 10% of the total euploid embryos (P < 0.0001 compared with perfect SES-scored embryos; Table 7). Day-3 embryos with 6–8 cells, good PN scores, even cell sizes yet multinucleation on day 2 were 22% euploid (P < 0.05 compared with perfect SES-scored embryos; Table 7) and accounted for 4% of the total euploid embryos (P < 0.0001 compared with perfect SES-scored embryos; Table 7). Day-3 embryos with 6–8 cells, good PN scores but with both uneven cell sizes and multinucleation on day 2 were 10% euploid (P < 0.0005 compared with perfect SES-scored embryos; Table 7) and comprised only 1% of the total euploid embryos (P < 0.0001 compared with perfect SES-scored embryos; Table 7). Day-3 embryos with 6–8 cells, poor PN scores but even cell sizes and no multinucleation on day 2 had a 30% euploidy rate (not significant when compared with perfect SES-scored embryos) and accounted for 21% of the total euploid embryos (P < 0.0001 compared with perfect SES-scored embryos; Table 7). Day-3 embryos with 6–8 cells, poor PN scores, no multinucleation yet uneven cell sizes on day 2 were 26% euploid (not significant when compared with perfect SES-scored embryos) and made up 7% of the total euploid embryos (P < 0.0001 compared with perfect SES-scored embryos; Table 7). Day-3 embryos with 6–8 cells, poor PN scores, even cell sizes but multinucleation on day 2 had a euploidy rate of 26% (not significant compared with perfect SES-scored embryos) and comprised only 2% of the total euploid embryos (P < 0.0001 compared with perfect SES-scored embryos; Table 7). Embryos with the poorest SES score (6–8 cells on day 3 with poor PN scores, uneven cell sizes and multinucleation on day 2) had a 12% euploidy rate (P < 0.05 compared with perfect SES-scored embryos; Table 6) and only accounted for 1% of the total euploid population (P < 0.0001 compared with perfect SES-scored embryos; Table 7).
      Table 7Sequential embryo scoring (SES) of pronuclear and day-2 morphology of day-3 embryos with 6–8 cells and percentage of euploid embryos.
      DescriptionNo. with SES score (n=1421)Euploid (%)% of total euploid (n=397)
      Z1/Z2, even cell sizes, no MN on day 2644219 (34)55
      Z3/Z4 even cell sizes, no MN on day 227983 (30)21
      P<0.0001 compared to embryos that were Z1/Z2, even cell sizes and no multinucleation on day 2.
      Z1/Z2, uneven cell sizes, no MN on day 223741 (17)
      P<0.0001 compared to embryos that were Z1/Z2, even cell sizes and no multinucleation on day 2.
      10
      P<0.0001 compared to embryos that were Z1/Z2, even cell sizes and no multinucleation on day 2.
      Z3/Z4 uneven cell sizes, no MN on day 210126 (26)7
      P<0.0001 compared to embryos that were Z1/Z2, even cell sizes and no multinucleation on day 2.
      Z1/Z2 even cell sizes but MN on day 26414 (22)
      P<0.05 compared to embryos that were Z1/Z2, even cell sizes and no multinucleation on day 2.
      4
      P<0.0001 compared to embryos that were Z1/Z2, even cell sizes and no multinucleation on day 2.
      Z3/Z4 even cell sizes and MN on day 2236 (26)2
      P<0.0001 compared to embryos that were Z1/Z2, even cell sizes and no multinucleation on day 2.
      Z1/Z2, uneven cell sizes and MN on day 2485 (10)
      P<0.0005 compared to embryos that were Z1/Z2, even cell sizes and no multinucleation on day 2.MN=multinucleation.
      1
      P<0.0001 compared to embryos that were Z1/Z2, even cell sizes and no multinucleation on day 2.
      Z3/Z4 uneven cell sizes and MN on day 2253 (12)
      P<0.05 compared to embryos that were Z1/Z2, even cell sizes and no multinucleation on day 2.
      1
      P<0.0001 compared to embryos that were Z1/Z2, even cell sizes and no multinucleation on day 2.
      a P < 0.0001 compared to embryos that were Z1/Z2, even cell sizes and no multinucleation on day 2.
      b P < 0.05 compared to embryos that were Z1/Z2, even cell sizes and no multinucleation on day 2.
      c P < 0.0005 compared to embryos that were Z1/Z2, even cell sizes and no multinucleation on day 2.MN = multinucleation.
      A total of 144 patients (group 2), initially set up for PGS, cancelled PGS. Thirty-nine (27%) of these patients had agonist suppression stimulation cycles, 50 (35%) of these patients had antagonist cycles and 55 (38%) had agonist flare stimulation cycles (Table 2). Embryo transfers occurred on day 2 (n = 88) or day 3 (n = 50). Six patients in this group did not have an embryo transfer after the decision to cancel PGS due to poor or arrested embryo development. The decision for day-2 or day-3 transfer was based on number of embryos, embryo quality and timing of decision to cancel PGS. Reasons for cancelling PGS included low embryo number (n = 118, 82%), poor embryo quality (n = 20, 14%) and patient preference (n = 6, 4%). Within this group, there were 117 low responders (less than 6 2PN, 81%) and 27 good responders (6 or more 2PN, 19%). There were no statistically significant differences in mean patient age, cycle number or implantation rate between these patients (group 2) and patients having PGS (group 1) (Table 2). Patients in group 2 had an average of 2.4 embryos transferred compared with an average of 1.8 in group 1. Significantly fewer patients in group 2 underwent agonist suppression cycles (27% versus 43%; P < 0.005) whereas a higher proportion underwent antagonist cycles (35% versus 20%; P < 0.001) compared with the patients in group 1 (Table 2). Deliveries occurred in 34 cycles, resulting in a delivery rate of 23% per oocyte retrieval and 24% per embryo transfer with no significant difference found between low and good responders (Table 3). The delivery rates per oocyte retrieval of the patients in group 2 were significantly different from those in group 1 (23% versus 15%; P < 0.05; Table 3) with no difference in SAB rate between group 2 and group 1 (35% versus 37%; Table 3). The low responders in group 2 had significantly higher delivery rates per oocyte retrieval compared with the low responders in group 1 (24% versus 6%; P < 0.005; Table 3) while the good responders did not show significantly different delivery rates between those having PGS and those converting to conventional transfer on day 2 or 3.

      Discussion

      The data presented here show that there is no advantage to aneuploidy screening when SES is employed in a routine manner. The results further support previous studies showing no significant increase in ongoing pregnancy rates using PGS (
      • Blockeel C.
      • Schutyser V.
      • Vos A.D.
      • et al.
      Prospectively randomized controlled trial of PGS in IVF/ICSI patients with poor implantation.
      ,
      • Hardarson T.
      • Hanson C.
      • Lundin K.
      • et al.
      Preimplantation genetic screening in women of advanced maternal age caused a decrease in clinical pregnancy rate: a randomized controlled trial.
      ,
      • Jansen R.P.S.
      • Bowman M.C.
      • De Boer K.A.
      • Leigh D.A.
      • Lieberman D.B.
      • Mcarthur S.J.
      What next for preimplantation genetic screening (PGS)? Experience with blastocyst biopsy and testing for aneuploidy.
      ,
      • Mastenbroek S.
      • Twisk M.
      • Van Echten-Arends J.
      • et al.
      In vitro fertilization with preimplantation genetic screening.
      ,
      • Staessen C.
      • Platteau P.
      • Van Assche E.
      Comparison of blastocyst transfer with or without preimplantation genetic diagnosis for aneuploidy screening in couples with advanced maternal age: a prospective randomized trial.
      ,
      • Staessen C.
      • Verpoest W.
      • Donoso P.
      • et al.
      Preimplantation genetic screening does not improve delivery rate in women under the age of 36 following single-embryo transfer.
      ,
      • Twisk M.
      • Mastenbroek S.
      • Wely M.V.
      • Maas J.H.
      • Veen F.V.D.
      • Repping S.
      Preimplantation genetic screening for abnormal number of chromosomes (aneuploidies) in in vitro fertilisation or intracytoplasmic sperm injection.
      ,
      • Twisk M.
      • Mastenbroek S.
      • Hoek A.
      • et al.
      No beneficial effect of preimplantation genetic screening in women of advanced maternal age with a high risk for embryonic aneuploidy.
      ). However the current data is a unique retrospective analysis in that it compares the outcomes of PGS patients that elected or qualified for a PGS option, but did not proceed with it, to outcomes when the same group did proceed with PGS. Therefore, the patient groups are well matched making this study comparable with randomized controlled trials (
      • Blockeel C.
      • Schutyser V.
      • Vos A.D.
      • et al.
      Prospectively randomized controlled trial of PGS in IVF/ICSI patients with poor implantation.
      ,
      • Debrock S.
      • Melotte C.
      • Splessens C.
      • et al.
      Preimplantation genetic screening for aneuploidy of embryos after in vitro fertilization in women aged at least 35 years: a prospective randomized trial.
      ,
      • Hardarson T.
      • Hanson C.
      • Lundin K.
      • et al.
      Preimplantation genetic screening in women of advanced maternal age caused a decrease in clinical pregnancy rate: a randomized controlled trial.
      ,
      • Jansen R.P.S.
      • Bowman M.C.
      • De Boer K.A.
      • Leigh D.A.
      • Lieberman D.B.
      • Mcarthur S.J.
      What next for preimplantation genetic screening (PGS)? Experience with blastocyst biopsy and testing for aneuploidy.
      ,
      • Mastenbroek S.
      • Twisk M.
      • Van Echten-Arends J.
      • et al.
      In vitro fertilization with preimplantation genetic screening.
      ,
      • Staessen C.
      • Platteau P.
      • Van Assche E.
      Comparison of blastocyst transfer with or without preimplantation genetic diagnosis for aneuploidy screening in couples with advanced maternal age: a prospective randomized trial.
      ,
      • Yakin K.
      • Ata B.
      • Ercelen N.
      • Balaban B.
      • Urman B.
      The effect of preimplantation genetic screening on the probability of live birth in young women with recurrent implantation failure; a nonrandomized parallel group trial.
      ). In addition, the morphological characteristics of all biopsied embryos were related with the PGS results in order to support these findings of improved or similar outcomes using SES to select embryos without PGS. Comparisons of embryo morphology to aneuploidy are not unique (
      • Magli M.
      • Gianaroli L.
      • Ferraretti A.
      Chromosomal abnormalities in embryos.
      ,
      • Munné S.
      Chromosome abnormalities and their relationship to morphology and development of human embryos.
      ,
      • Munné S.
      • Chen S.
      • Colls P.
      • et al.
      Maternal age, morphology, development and chromosome abnormalities in over 6000 cleavage stage embryos.
      ); however, this is the first study to relate cumulative, sequential morphology scores at multiple stages of pre-embryo development with aneuploidy.
      These data indicate no difference in ongoing delivery rates per transfer between poor-prognosis patients having PGS and those electing no PGS. The results demonstrate that ongoing delivery rates per embryo transfer were neither enhanced nor impaired with PGS. The low responders (less than six embryos) having PGS in this study had the same ongoing pregnancy rate per transfer as those having either day-2 or day-3 embryo transfer without PGS. However, delivery rates were four times higher per retrieval if PGS was not performed in low responders. These results are similar to those reported in other studies (
      • Hardarson T.
      • Hanson C.
      • Lundin K.
      • et al.
      Preimplantation genetic screening in women of advanced maternal age caused a decrease in clinical pregnancy rate: a randomized controlled trial.
      ,
      • Jansen R.P.S.
      • Bowman M.C.
      • De Boer K.A.
      • Leigh D.A.
      • Lieberman D.B.
      • Mcarthur S.J.
      What next for preimplantation genetic screening (PGS)? Experience with blastocyst biopsy and testing for aneuploidy.
      ,
      • Mastenbroek S.
      • Twisk M.
      • Van Echten-Arends J.
      • et al.
      In vitro fertilization with preimplantation genetic screening.
      ), which found lower clinical pregnancy rates per retrieval after PGS. The current study is different in that it only found a difference in the poor-prognosis patients with less than six embryos (low responders). The rates per retrieval for the poor-prognosis patients that had six or more embryos (good responders) were the same with and without PGS in this study.
      Theoretically, if a patient is at high risk of producing aneuploid embryos, the number of embryos available for PGS should not change the overall outcome (i.e. delivery rate) of the cycle. However, these data suggest that at least six embryos are required for a successful PGS cycle in poor-prognosis patients. Other studies have reported similar findings regarding oocyte and embryo number and PGS success (
      • Munné S.
      • Sandalinas M.
      • Escudero T.
      • et al.
      Improved implantation after preimplantation genetic diagnosis of aneuploidy.
      ,
      • Vandervorst M.
      • Liebars I.
      • Sermon K.
      • et al.
      Successful preimplantation genetic diagnosis is related to the number of available cumulus–oocyte complexes.
      ). The current data suggest that patients with a low number of embryos are at a significantly higher risk of having no euploid embryos after PGS (51% compared with 20% in the poor-prognosis good responders; Figure 1) and therefore no embryo transfer resulting in significantly lower delivery rates. On the other hand, when these patients have embryos transferred on day 2 or day 3 (PGS patients electing transfer without biopsy) using SES, there was no significant difference in delivery rates or SAB rates per retrieval or transfer compared with the good responders and significantly higher delivery rates compared with the low responders having PGS. The reasons for this discrepancy are unclear. Based on these data, it is unlikely that the biopsy technique has a negative effect on outcome since the delivery rates per embryo transfer were not impaired with PGS in any group.
      Misdiagnosis of the PGS/FISH procedure itself appears to be the most likely explanation for a lack of significantly improved delivery rates per retrieval after aneuploidy screening in the study clinic but the mechanism or reason behind this misdiagnosis is still unknown. Low confirmation rates of aneuploidy have been reported after day-5 re-analysis of embryos biopsied on day 3 (
      • Baart E.B.
      • Van Opstal D.
      • Los F.J.
      • Fauser B.C.J.M.
      • Martini E.
      Fluorescence in situ hybridization analysis of two blastomeres from day 3 frozen-thawed embryos followed by analysis of the remaining embryo on day 5.
      ,
      • Staessen C.
      • Platteau P.
      • Van Assche E.
      Comparison of blastocyst transfer with or without preimplantation genetic diagnosis for aneuploidy screening in couples with advanced maternal age: a prospective randomized trial.
      ). One study reported an error rate as high as 50% after re-analysis (
      • Baart E.B.
      • Van Opstal D.
      • Los F.J.
      • Fauser B.C.J.M.
      • Martini E.
      Fluorescence in situ hybridization analysis of two blastomeres from day 3 frozen-thawed embryos followed by analysis of the remaining embryo on day 5.
      ) and another found 8.4% of the embryos diagnosed as abnormal on day 3 to be normal on day 5. In one study, an additional 17.5% of the embryos diagnosed as abnormal on day 3 were subsequently found to contain both normal and abnormal cells on day 5 (
      • Staessen C.
      • Platteau P.
      • Van Assche E.
      Comparison of blastocyst transfer with or without preimplantation genetic diagnosis for aneuploidy screening in couples with advanced maternal age: a prospective randomized trial.
      ).
      Mosaicism in the embryo has the potential to result in misdiagnosis after PGS (
      • Goosens V.
      • Rycke M.D.
      • Vos A.D.
      • et al.
      Diagnostic efficiency, embryonic development and clinical outcome after the biopsy of one or two blastomeres for preimplantation genetic diagnosis.
      ,
      • Michiels A.
      • Assche E.V.
      • Liebars I.
      • Steirteghem A.V.
      • Staessen C.
      The analysis of one or two blastomeres for PGD using fluorescence in-situ hybridization.
      ,
      • Munné S.
      • Sandalinas M.
      • Escudero T.
      • Marquez C.
      • Cohen J.
      Chromosome mosaicism in cleavage-stage human embryos: evidence of a maternal age effect.
      ). Meiotic errors in the oocyte and abnormalities occurring during the first cleavage division which result in 100% aneuploidy in all cells of the embryo, account for as little as 9% of aneuploidy in day-3 embryos (
      • Munné S.
      • Weier H.
      • Grifo J.
      • Cohen J.
      Chromosome mosaicism in human embryos.
      ,
      • Trussler J.
      • Pickering S.
      • Ogilvie C.
      Investigation of chromosomal imbalance in human embryos using comparative genomic hybridization.
      ,
      • Ziebe S.
      • Lundin K.
      • Loft A.
      • et al.
      FISH analysis for chromosomes 13, 16, 18, 21, 22, X and Y in all blastomeres of IVF pre-embryos from 144 randomly selected donated human oocytes and impact on pre-embryo morphology.
      ). Post-zygotic errors occurring in the second- and third-cleavage divisions and resulting in mosaicism, have been found in 15–57% of day-3 embryos (
      • Baart E.B.
      • Van Opstal D.
      • Los F.J.
      • Fauser B.C.J.M.
      • Martini E.
      Fluorescence in situ hybridization analysis of two blastomeres from day 3 frozen-thawed embryos followed by analysis of the remaining embryo on day 5.
      ,
      • Baart E.B.
      • Martini E.
      • Van Den Berg I.
      • et al.
      Preimplantation genetic screening reveals a high incidence of aneuploidy and mosaicism in embryos from young women undergoing IVF.
      ,
      • Coonen E.
      • Derhaag J.G.
      • Dumoulin J.C.M.
      • et al.
      Anaphase lagging mainly explains chromosomal mosaicism in human preimplantation embryos.
      ,
      • Harper J.C.
      • Coonen E.
      • Handyside A.
      • Rm W.
      • Ah H.
      • Delhanty J.D.A.
      Mosaicism of autosomes and sex chromosomes in morphologically normal monospermic preimplantation human embryos.
      ,
      • Munné S.
      Preimplantation genetic diagnosis and human implantation – a review.
      ,
      • Munné S.
      • Weier H.
      • Grifo J.
      • Cohen J.
      Chromosome mosaicism in human embryos.
      ,
      • Munné S.
      • Sandalinas M.
      • Escudero T.
      • Marquez C.
      • Cohen J.
      Chromosome mosaicism in cleavage-stage human embryos: evidence of a maternal age effect.
      ,
      • Trussler J.
      • Pickering S.
      • Ogilvie C.
      Investigation of chromosomal imbalance in human embryos using comparative genomic hybridization.
      ,
      • Wells D.
      • Delhanty J.D.A.
      Comprehensive chromosomal analysis of human preimplantation embryos using whole genome amplification and single cell comparative genomic hybridization.
      ). Yet this does not explain the higher delivery rates and no difference in SAB rates in patients with few embryos not having PGS since most mosaicism is eliminated prior to the first trimester (
      • Bielanska M.
      • Tan S.L.
      • Ao A.
      Chromosomal mosaicism throughout human preimplantation development in vitro: incidence, type, and relevance to embryo outcome.
      ). It has been reported that mosaicism is only present in 5% of aneuploid spontaneous abortions at 6–20 weeks (
      • Hassold T.
      Mosaic trisomies in human spontaneous abortions.
      ) and in 1–2% of viable pregnancies screened by chorionic villi sampling (
      • Ledbetter D.
      • Zachary J.
      • Simpson J.
      • et al.
      Cytogenetics results from the US Collaborative Study on CVS.
      ,
      • Wang B.
      • Rubin C.
      • Williams J.
      Mosaicism in chorionic villus sampling: an analysis of incidence and chromosomes involved in 2612 consecutive cases.
      ). Some research suggests that, depending on the type and proportion of aneuploid cells that exist, small amounts of aneuploid cells in early embryos may not be detrimental to the overall potential of the embryo (
      • Bielanska M.
      • Tan S.L.
      • Ao A.
      Chromosomal mosaicism throughout human preimplantation development in vitro: incidence, type, and relevance to embryo outcome.
      ,
      • Ziebe S.
      • Lundin K.
      • Loft A.
      • et al.
      FISH analysis for chromosomes 13, 16, 18, 21, 22, X and Y in all blastomeres of IVF pre-embryos from 144 randomly selected donated human oocytes and impact on pre-embryo morphology.
      ) and that abnormal cells within a mosaic embryo may self correct (
      • James R.
      • West J.
      A chimaeric animal model for confined placental mosaicism.
      ,
      • Kola I.
      • Trounson A.
      • Dawson G.
      • Rogers P.
      Tripronuclear human oocytes: altered cleavage patterns and subsequent karyotypic analysis of embryos.
      ,
      • Munné S.
      • Velilla E.
      • Colls P.
      • et al.
      Self-correction of chromosomally abnormal embryos in culture and implications for stem cell production.
      ).
      Patients having PGS with less than six embryos are more likely to have no euploid embryos as a result of a misdiagnosis. These patients are at significantly higher risk of not having an embryo transfer simply because they have fewer embryos to diagnose and less room for error. Therefore, for poor-prognosis patients with a low number of embryos, the risk of having no embryo transferred after PGS appears to be greater than that of having an aneuploid conception without the use of PGS.
      This study was a retrospective analysis. As a result, it is not without limitations. Frozen–thawed embryos were combined with fresh embryos in 24 (8%) of the PGS cycles. This may interfere with the proper interpretation of these results since no frozen–thawed embryos were used in the patients not having PGS. Different numbers of embryos were also transferred on different days within all groups, creating a potential bias in results. Patients opting out of PGS had an average of 2.4 embryos transferred on either day 2 or day 3 and their results were compared with PGS patients having an average of 1.8 embryos transferred on either day 4 (2005 only) or day 5. However, this did not result in a significant difference in implantation rates between the two groups (Table 2). Also, this bias in embryo transfer data does not affect the delivery rates per oocyte retrieval between group 1 and group 2 (15% versus 23%; P < 0.005; Table 3). Lastly, patients in group 2 (no PGS) had significantly less use of agonist stimulations and increased use of antagonist protocols compared with patients undergoing PGS. Agonist stimulation protocols were more likely to be used in patients with a history of better ovarian response to stimulation medication while antagonist protocols were more likely to be used for poorer prognosis patients. This difference may reflect yet another bias in the two groups although patients proceeding with PGS had higher use of agonist suppression protocols and less use of antagonist protocols suggesting the bias would err towards higher delivery rates in this group. Instead, there was a reduced delivery rate per oocyte retrieval in this group.
      This study stresses the importance of careful, timed SES in the early embryo. Multiple studies looking at embryo morphology and aneuploidy have found correlations between single, independent morphology parameters and PGS results (
      • Balaban B.
      • Yakin K.
      • Urman B.
      • Isiklar A.
      • Tesarik J.
      Pronuclear morphology predicts embryo development and chromosome constitution.
      ,
      • Baltaci V.
      • Satiroglu H.
      • Kabukcu C.
      • et al.
      Relationship between embryo quality and aneuploidies.
      ,
      • Bielanska M.
      • Tan S.L.
      • Ao A.
      Chromosomal mosaicism throughout human preimplantation development in vitro: incidence, type, and relevance to embryo outcome.
      ,
      • Coskun S.
      • Hellani A.
      • Jaroudi K.
      • Al-Mayman H.
      • Al-Kabra M.
      • Qeba M.
      Nucleolar precursor body distribution in pronuclei is correlated to chromosomal abnormalities in embryos.
      ,
      • Edirisinghe W.
      • Jemmott R.
      • Smith C.
      • Allan J.
      Association of pronuclear Z score with rates of aneuploidy in in vitro-fertilised embryos.
      ,
      • Gamiz P.
      • Rubio C.
      • Santos M.J.D.L.
      • et al.
      The effect of pronuclear morphology on early development and chromosomal abnormalities in cleavage-stage embryos.
      ,
      • Gianaroli L.
      • Magli M.
      • Ferraretti A.
      • Fortini D.
      • Grieco N.
      Pronuclear morphology and chromosomal abnormalities as scoring criteria for embryo selection.
      ,
      • Gianaroli L.
      • Magli M.
      • Fiorentino F.
      • Baldi M.
      • Ferraretti A.
      Clinical value of preimplantation genetic diagnosis.
      ,
      • Gianaroli L.
      • Magli M.
      • Ferraretti A.
      • Lappi M.
      • Borghi E.
      • Ermini B.
      Oocyte euploidy, pronuclear zygote morphology and embryo chromosomal complement.
      ,
      • Hardarson T.
      • Hanson C.
      • Sjogren A.
      • Lundin K.
      Human embryos with unevenly sized blastomeres have lower pregnancy and implantation rates: indications for aneuploidy and multinucleation.
      ,
      • Kahraman S.
      • Kumtepe Y.
      • Sertyel S.
      • et al.
      Pronuclear morphology scoring and chromosomal status of embryos in severe male infertility.
      ,
      • Kligman I.
      • Benadiva C.
      • Alikani M.
      • Munné S.
      Fertilization and early embryology: the presence of multinucleated blastomeres in human embryos is correlated with chromosomal abnormalities.
      ,
      • Magli M.
      • Gianaroli L.
      • Munné S.
      • Ferraretti A.
      Incidence of chromosomal abnormalities from a morphologically normal cohort of embryos in poor-prognosis patients.
      ,
      • Moayeri S.
      • Allen R.
      • Brewster W.
      • Kim M.
      • Porto M.
      • Werlin L.
      Day-3 embryo morphology predicts euploidy among older subjects.
      ,
      • Rubio C.
      • Rodrigo L.
      • Mercader A.
      • et al.
      Impact of chromosomal abnormalities on preimplantation embryo development.
      ,
      • Sandalinas M.
      • Sadowy S.
      • Alikani M.
      • Calderon G.
      • Cohen J.
      • Munné S.
      Developmental ability of chromosomally abnormal human embryos to develop to the blastocyst stage.
      ,
      • Ziebe S.
      • Lundin K.
      • Loft A.
      • et al.
      FISH analysis for chromosomes 13, 16, 18, 21, 22, X and Y in all blastomeres of IVF pre-embryos from 144 randomly selected donated human oocytes and impact on pre-embryo morphology.
      ). The current study revealed an even greater correlation between SES from day 1 to day 3 and aneuploidy result, rather than individual morphological parameters. From this is concluded that SES may be more accurate than PGS as a means of selection for low-responding poor-prognosis patients with few embryos. Based on the results of this study, embryos selected through SES are more likely to be euploid than aneuploid and the risk of misdiagnosis after PGS in this patient population is eliminated.
      The application of SES combines the morphology of each embryo at each stage of development to provide a cumulative, rather than a static score of early embryo development. This develops an understanding of the individual embryo’s potential, which ultimately includes the chromosomal complement. Multiple studies have examined mechanisms such as karyokinesis without cytokinesis, abnormal alignment or incorrect packaging of chromosomes during and after mitosis (
      • Hardy K.
      • Winston R.
      • Handyside A.
      Binucleate blastomeres in preimplantation human embryos in vitro: failure of cytokinesis during early cleavage.
      ,
      • Pickering S.
      • Taylor A.
      • Johnson M.
      • Braude P.
      An analysis of multinucleated blastomere formation in human embryos.
      ,
      • Tesarik J.
      • Kopecny V.
      • Plachot M.
      Ultrastructural and autoradiographic observations on multinucleated blastomeres of human cleaving embryos.
      ,
      • Winston N.
      • Braude P.
      • Pickering S.
      The incidence of abnormal morphology and nucleocytoplasmic ratios in 2-,3- and 5-day human pre-embryos.
      ,
      • Winston N.
      • Johnson M.
      • Braude P.
      Assessment of the cellular DNA content of whole mounted mouse and human oocytes and of blastomeres containing single or multiple nuclei.
      ) and cytoskeletal and spindle malformation that may explain the correlations between abnormal embryo morphology and aneuploidy (
      • Balakier H.
      • Cadesky K.
      The frequency and developmental capability of human embryos containing multinucleated blastomeres.
      ,
      • Hardarson T.
      • Hanson C.
      • Sjogren A.
      • Lundin K.
      Human embryos with unevenly sized blastomeres have lower pregnancy and implantation rates: indications for aneuploidy and multinucleation.
      ,
      • Hardy K.
      • Winston R.
      • Handyside A.
      Binucleate blastomeres in preimplantation human embryos in vitro: failure of cytokinesis during early cleavage.
      ,
      • Hnida C.
      • Engenheiro E.
      • Ziebe S.
      Computer-controlled, multilevel, morphometric analysis of blastomere size as biomarker of fragmentation and multinuclearity in human embryos.
      ,
      • Kligman I.
      • Benadiva C.
      • Alikani M.
      • Munné S.
      Fertilization and early embryology: the presence of multinucleated blastomeres in human embryos is correlated with chromosomal abnormalities.
      ,
      • Magli M.
      • Gianaroli L.
      • Ferraretti A.
      Chromosomal abnormalities in embryos.
      ,
      • Pickering S.
      • Taylor A.
      • Johnson M.
      • Braude P.
      An analysis of multinucleated blastomere formation in human embryos.
      ,
      • Scott L.
      • Finn A.
      • O’Leary T.
      • McClellan S.
      • Hill J.
      Morphologic parameters of early cleavage-stage embryos that correlate with fetal development and delivery: prospective and applied data for increased pregnancy rates.
      ,
      • Staessen C.
      • Steirteghem A.V.
      The genetic constitution of multinuclear blastomeres and their derivative daughter blastomeres.
      ,
      • Van Royen E.
      • Mangelschots K.
      • De Neubourg D.
      • Laureys I.
      • Ryckaert G.
      • Gerris J.
      Calculating the implantation potential of day 3 embryos in women younger than 38 years of age: a new model.
      ,
      • Ziebe S.
      • Lundin K.
      • Loft A.
      • et al.
      FISH analysis for chromosomes 13, 16, 18, 21, 22, X and Y in all blastomeres of IVF pre-embryos from 144 randomly selected donated human oocytes and impact on pre-embryo morphology.
      ).
      Although some studies have reported that morphometrics is not a reliable method of selecting euploid embryos (
      • Eaton J.
      • Hacker M.
      • Harris D.
      • Thornton K.
      • Penzias A.
      Assessment of day-3 morphology and euploidy for individual chromosomes in embryos that develop to the blastocyst stage.
      ,
      • Magli M.
      • Gianaroli L.
      • Munné S.
      • Ferraretti A.
      Incidence of chromosomal abnormalities from a morphologically normal cohort of embryos in poor-prognosis patients.
      ,
      • Magli M.
      • Gianaroli L.
      • Ferraretti A.
      Chromosomal abnormalities in embryos.
      ,
      • Munné S.
      Chromosome abnormalities and their relationship to morphology and development of human embryos.
      ,
      • Munné S.
      • Chen S.
      • Colls P.
      • et al.
      Maternal age, morphology, development and chromosome abnormalities in over 6000 cleavage stage embryos.
      ), this is the first study to show significant relationships between the combined cumulative early morphology scores and subsequent euploidy. These data suggest that for poor-prognosis IVF patients, SES followed by embryo transfer on day-2 or day-3 results in the same delivery rates as de-selection through PGS. Conversely, PGS has proven to have a significantly negative effect on poor-prognosis patients with few embryos, with a reduced delivery rate.
      In the clinical IVF setting, it is important to maintain a standard of care that is comprised of individual treatment plans for each patient based on their reproductive history, concerns and goals. PGS may yet be a useful tool but only in a select population of patients that produce a high number of embryos and with a history of high order recurrent pregnancy loss (more than five), previous aneuploid conceptions or advanced maternal age (over 41 years). PGS may be useful in these patients as an additional tool to complement SES and deselect from a selected cohort of embryos with good morphology on days 1–3 to identify the most viable embryo for transfer. For the majority of IVF patients, PGS does not afford the benefit of increased delivery per patient.

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