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Blastocyst quality affects the success of blastocyst-stage embryo transfer

      Abstract

      Objective:
      To determine the relationship between blastocyst quality and the results of embryo transfer at the blastocyst stage.
      Design:
      Retrospective case analysis.
      Setting:
      Tertiary care private hospital IVF center.
      Patient(s):
      A total of 350 blastocyst-stage embryo transfer cycles.
      Intervention(s):
      In vitro culture to the blastocyst stage was undertaken in 350 ICSI cycles where four or more cleavage-stage embryos were available on day 3.
      Main Outcome Measure(s):
      Relationship between blastocyst quality and implantation and clinical and multiple pregnancy rates.
      Result(s):
      Transfer of at least one grade 1 or grade 2 blastocyst or one hatching blastocyst was associated with very high implantation and pregnancy rates. However, transfer of grade 3 blastocysts yielded very low implantation and pregnancy rates.
      Conclusion(s):
      There appears to be a strong correlation between blastocyst quality and success of blastocyst transfer.

      Keywords

      Transfer of the human embryo at the blastocyst stage is becoming more common in the practice of assisted reproduction (
      • Meldrum D.R
      Blastocyst transfer—a natural evolution.
      ). The advantages of blastocyst-stage embryo transfer include better synchronization between the endometrium and the embryo and the possible selection of embryos with a higher implantation potential (
      • Olivennes F
      • Hazout A
      • Lelaidier C
      • Freitas S
      • Franchin R
      • de Ziegler D
      • et al.
      Four indications for transfer at the blastocyst stage.
      ). Transfer of the blastocyst has been associated with higher implantation rates as demonstrated in a recent prospective randomized study (
      • Gardner D.K
      • Schoolcraft W.B
      • Wagley L
      • Schlenker T
      • Stevens J
      • Hesla J
      A prospective randomized trial of blastocyst culture and transfer in in-vitro fertilization.
      ).
      The implantation potential of the individual blastocyst, however, is less well defined. Although there appears to be a clear relationship between morphologic grading of cleavage-stage embryos and their implantation potential, the same relationship has not been defined for blastocysts (
      • Ziebe S
      • Peterson K
      • Lindenberg S
      • Andersen A.G
      • Gabrielsen A
      • Andersen A.N
      Embryo morphology or cleavage stage how to select the best embryo for transfer after in vitro fertilization.
      ). Dokras et al. proposed a classification for blastocyst-stage embryos that takes into consideration parameters such as cavitation, expansion of the blastocele cavity, and characteristics of the inner-cell mass (
      • Dokras A
      • Sargent I.L
      • Barlow D.H
      Human blastocyst grading an indicator of developmental potential.
      ). The validity of this classification has been confirmed by measuring β-hCG secreted by the individual blastocyst grown on culture cells. The relationship between blastocyst grade and implantation will undoubtedly aid the clinician during embryo transfer through a selection process that will minimize the incidence of multiple pregnancies.
      In this study we analyzed 350 blastocyst-stage embryo transfers performed on day 5 or 6. Women who had four or more cleavage-stage embryos on day 2 were offered the opportunity to undergo embryo transfer at the blastocyst stage. The implantation potential of an individual blastocyst was assessed using the blastocyst grading system described by Dokras et al. (
      • Dokras A
      • Sargent I.L
      • Barlow D.H
      Human blastocyst grading an indicator of developmental potential.
      ).

      Material and methods

       Patients

      Between November 1998 and June 1999, 489 embryo transfers at the blastocyst stage were performed. Analysis of the data was confined to 350 transfers that involved blastocysts only (early, expanded, or hatching). Treatment cycles that resulted in the transfer of morulae only or morulae with blastocysts were excluded. Because approximately 85% of the treatment cycles performed in our center are ICSI, only these were included in the analysis of results. Institutional Review Board approval was not obtained because the study was not prospective and was confined to a retrospective review of case records.

       Stimulation protocol and oocyte retrieval

      Controlled ovarian hyperstimulation was undertaken using subcutaneous buserelin acetate (Suprefact proinjection, Hoechst AG, Frankfurt am Main, Germany) in a long protocol combined with pure FSH (Metrodin, 75, I.F. Serono, Rome, Italy). Treatment with buserelin acetate (0.3 mg/d) was commenced on day 20 or 21 of the preceding cycle and continued until the day of treatment with hCG. Treatment with 2–6 ampules of FSH, depending on the patients’ previous or anticipated response, was initiated on the third day of the menstrual cycle. The treatment was then individualized in a step-down fashion. When the leading follicle reached 20 mm in mean diameter with a serum estradiol level of 200–300 pg/mL per mature follicle 10,000 U of hCG (Profasi HP 5000; I.F. Serono, Rome, Italy) was administered. Oocyte retrieval was performed 36 hours after the injection of hCG. ICSI was performed according to conventional protocols and only on MII oocytes.

       In vitro culture of embryos to the blastocyst stage and embryo grading

      In vitro culture of embryos was undertaken as previously described (
      • Gardner D.K
      • Schoolcraft W.B
      • Wagley L
      • Schlenker T
      • Stevens J
      • Hesla J
      A prospective randomized trial of blastocyst culture and transfer in in-vitro fertilization.
      ,
      • Balaban B
      • Urman B
      • Sertac A
      • Alatas C
      • Aksoy S
      • Nuhoglu A
      Progression of excess embryos to the blastocyst stage predicts pregnancy and implantation rates after intracytoplasmic sperm injection.
      ). A sequential media system (grade 1 and grade 2 media from Scandinavian Science AB Products, Gotenborg, Sweden) designed for further embryonic development was used. Embryos were cultured in groups; in grade 1 medium until day 3 and in grade 2 medium until the blastocyst stage. Culture medium was refreshed every day.
      Cleavage-stage embryos were graded as follows: grade 1 embryo, no fragmentation with equal-size homogenous blastomeres; grade 2 embryo, <20% fragmentation with equal-size homogenous blastomeres; grade 3 embryo, 20%–50% fragmentation with unequal-size blastomeres; grade 4 embryo, >50% fragmentation with unequal-size blastomeres.
      Blastocyst grading was according to Dokras et al. (
      • Dokras A
      • Sargent I.L
      • Barlow D.H
      Human blastocyst grading an indicator of developmental potential.
      ). Grade 1 blastocysts were characterized by early cavitation, resulting in the formation of an eccentric and then expanded cavity lined by a distinct inner-cell mass region and trophoectoderm layer (Fig 1). Grade 2 blastocysts exhibited a transitional phase where single or multiple vacuoles were seen, which over subsequent days developed into the typical blastocyst appearance of the grade 1 blastocysts (Fig. 2). Grade 3 blastocysts were defined as blastocysts with several degenerative foci in the inner-cell mass with cells appearing dark and necrotic (Fig. 3).
      Figure thumbnail GR1
      Figure 1Expanded grade 1 blastocyst on day 5 (large arrow). This embryo showed early cavitation on day 4. Arrested and degenerating embryos (small arrows).
      Balaban. Blastocyst quality and embryo transfer. Fertil Steril 2000.
      Figure thumbnail GR2
      Figure 2Grade 2 blastocysts (asterisks); late developing blastocysts compared with grade 1 blastocysts. These exhibit a transitional phase before expansion. During this phase single or multiple vacuoles with sharp concave borders appear. These are usually seen starting from day 4 of development. There appears to be no morphologic difference from grade 1 blastocysts when the distinct region of the inner-cell mass and trophoectoderm have formed. Expanded grade 1 blastocysts (large arrows); early blastocysts (double arrows). Grading cannot be well distinguished between 1 and 2 until the expansion phase.
      Balaban. Blastocyst quality and embryo transfer. Fertil Steril 2000.
      Figure thumbnail GR3
      Figure 3Grade 3 blastocyst. These show several degenerative foci in the inner-cell mass.
      Balaban. Blastocyst quality and embryo transfer. Fertil Steril 2000.

       Embryo transfer

      Embryo transfer was performed 5 or 6 days after oocyte retrieval. Observation of hatching was not awaited for embryo transfer, however, whenever there was a hatching blastocyst (Fig. 4) this was included in the batch of embryos selected for transfer. Three to four (in women older than 35) blastocysts were transferred using the Wallace or Frydman embryo transfer catheters. All embryo transfers were performed under transabdominal ultrasound guidance. Tetracycline 200 mg bid (Monodoks; DEVA, Istanbul, Turkey) and methylprednisolone 16 mg/d (Prednol 16 mg; Mustafa Nevzat Ilaç Sanayi, Istanbul, Turkey) were administered for 5 days starting from the day of oocyte retrieval. The luteal phase was supplemented with intravaginal natural progesterone at a dose of 600 mg divided in three doses (Utrogestan; Laboratories Besins Iscovesco, Paris, France) starting on the day of oocyte retrieval.
      Figure thumbnail GR4
      Figure 4Early hatching grade 1 blastocyst (asterisks); late hatching grade 1 blastocyst (arrow).
      Balaban. Blastocyst quality and embryo transfer. Fertil Steril 2000.
      Pregnancy was defined as two β-hCG titers assessed 12 and 14 days after embryo transfer that showed appropriate doubling. Clinical pregnancy was defined as the presence of a gestational sac(s) with a viable embryo shown on vaginal ultrasonography performed approximately 24 days after embryo transfer.

       Statistics

      Results were analyzed using one-way analysis of variance and χ2 tests. A P value of <.05 was considered statistically significant.

      Results

      Of the 350 blastocyst-stage embryo transfer cycles, 319 were performed on day 5 and 31 were performed on day 6. Of the day 6 transfers only two were undertaken to further observe slow-cleaving embryos, whereas 25 were performed to avoid Sunday and holiday transfers. Day 5 and day 6 transfers were analyzed together. Cycles were stratified according to the homogeneity of transferred embryos. Heterogeneous cycles involved at least one grade 1 or grade 2 blastocyst. Homogenous cycles were comprised of all blastocysts of the same grade. Transfer cycles involving hatching blastocysts were also analyzed separately.

       Cleavage-stage embryo quality and blastocyst quality

      The relationship between embryo quality on day 3 and blastocyst quality on day 5 is shown in Table 1. Significantly more grade 1 and grade 2 cleavage-stage embryos progressed to the blastocyst stage compared with grade 3 and grade 4 embryos (P<.05). However, once blastocyst stage was reached, the quality of the individual blastocyst was not significantly affected by the quality of the cleavage-stage embryo that it originated from (P>.05).
      TABLE 1Rate of blastocyst formation from cleavage-stage embryos and the relationship between embryo quality on day 3 and blastocyst quality on day 5.
      G1=grade 1; G2=grade 2, etc. NA=not applicable; NS=not significant.
      ,
      Balaban. Blastocyst quality and embryo transfer. Fertil Steril 2000.
      G1+G2 day 3 embryosG3+G4 day 3 embryosP value
      No. of ETs22931220NA
      Blastocysts (%)1,356/2,293 (59.1)317/1,220 (25.9)<.05
      G1+G2 blastocysts (%)812/1,356 (59.8)178/317 (56.1)NS
      G3 blastocysts (%)544/1,356 (40.2)139/317 (43.9)NS
      legend G1=grade 1; G2=grade 2, etc. NA=not applicable; NS=not significant.
      legend Balaban. Blastocyst quality and embryo transfer. Fertil Steril 2000.

       Heterogenous and grade 3 blastocyst transfer cycles

      Results were stratified according to the quality of transferred blastocysts (Table 2). Variables such as mean female age, two pronuclear fertilizations, grade of cleavage-stage embryos on day 3, and the rate of blastocyst formation did not differ in cycles where at least one grade 1 blastocyst was transferred (group 1), cycles where at least one grade 2 blastocyst was transferred (group 2), or cycles where only grade 3 blastocysts were transferred (group 3; Table 2). Implantation rate per embryo and clinical pregnancy and multiple pregnancy rates were significantly decreased in group 3 (Table 2). Very high multiple pregnancy rates were observed in groups 1 and 2. Although significantly more embryos per patient were transferred into group 3, significantly less multiple implantations were encountered.
      TABLE 2Patient characteristics and results of blastocyst transfer according to blastocyst grade: results from heterogenous transfers and grade 3 blastocyst transfers.
      G1=grade 1, G2=grade 2, etc.
      ,
      a There is a statistically significant difference between group 3 vs. groups 1 and 2.
      ,
      Balaban. Blastocyst quality and embryo transfer. Fertil Steril 2000.
      VariableTransfer cycles with 1 or more G1 blastocysts (group 1)Transfer cycles with 1 or more G2 blastocysts (group 2)Transfer cycles with only G3 blastocysts (group 3)
      No. of cycles (%)113 (32.2)139 (39.7)98 (28.1)
      Mean age (y)32.232.331.7
      No. of oocytes1,6142,0111,450
      No. of pronucleate embryos that fertilized (%)1,130 (70)1,428 (71)1,029 (71)
      No. of embryos that cleaved (%)1,099 (97.2)1,396 (97.7)1,018 (98.9)
      No. of G1+G2 embryos on day 3 (%)737 (67)894 (64)662 (65)
      No. of G3+G4 embryos on day 3 (%)362 (33)502 (36)356 (34)
      Blastocysts formed538/1,099 (48.9%)656/1,396 (46.9%)479/1,018 (47%)
      No. of blastocysts transferred (mean)a369 (3.2)473 (3.4)392 (4)
      No. of embryos that implanted/no. of ETs (%)a166/369 (44.9)173/473 (36.5)28/392 (7.1)
      No. of multiple pregnancies/total no. of pregnancies (%)a63/113 (55.7)67/139 (48.2)13/98 (13.3)
      No. of clinical pregnancies/no. of ETs (%)a45/63 (71.4)43/67 (64.1)2/13 (15.3)
      No. of higher order pregnancies/total no. of pregnancies (%)20/63 (31.7)25/67 (37.3)0
      legend G1=grade 1, G2=grade 2, etc.
      legend a There is a statistically significant difference between group 3 vs. groups 1 and 2.
      legend Balaban. Blastocyst quality and embryo transfer. Fertil Steril 2000.

       Blastocyst transfer cycles of homogenous quality

      When cycles where only grade 1 or grade 2 blastocysts were transferred were analyzed separately clinical pregnancy rates were very high (68.7% in group 1 and 61.7% in group 2). Likewise, 56.2% and 46.4% embryo implantation rates were achieved, respectively. When three grade 1 blastocysts were transferred, 20 (90.9%) of 22 treatment cycles resulted in a multiple pregnancy. Similarly, a mean number of 3.2 grade 2 blastocysts resulted in a 68.9% (20 of 29) multiple pregnancy rate (Table 3).
      TABLE 3Results from homogeneous blastocyst transfers.
      a There is a statistically significant difference between group 3 vs. groups 1 and 2.
      ,
      Balaban. Blastocyst quality and embryo transfer. Fertil Steril 2000.
      VariableCycles where only grade 1 blastocysts were transferredCycles where only grade 2 blastocysts were transferredCycles where only grade 3 blastocysts were transferred
      No. of cycles324798
      No. of blastocysts transferred (mean)96 (3)155 (3.2)392 (4)
      No. of clinical pregnancies/no. of ETs (%)22/23 (68.7)29/47 (61.7)13/98 (13.3)
      No. of embryos that implanted/no. of ETsa (%)54/96 (56.2)72/155 (46.4)28/392 (7.1)
      No. of multiple pregnancies/total no. of pregnancies (%)a20/22 (90.9)20/29 (68.9)2/13 (15.3)
      legend a There is a statistically significant difference between group 3 vs. groups 1 and 2.
      legend Balaban. Blastocyst quality and embryo transfer. Fertil Steril 2000.

       Transfer of hatching blastocysts

      In cycles where all transferred blastocysts were hatching a 57.7% clinical pregnancy rate, a 43.9% implantation per embryo rate, and a 82.1% multiple pregnancy rate were achieved. In patients who had no hatching blastocyst available for transfer, respective rates were 27.8%, 19.1%, and 40.6% (P<.05 for all; Table 4).
      TABLE 4Pregnancy and implantation rates according to blastocyst hatching.
      a Group 2 significantly different from groups 1 and 3.
      ,
      b Group 2 significantly different from groups 1 and 3; group 1 significantly different from group 2.
      ,
      c Group 2 significantly different from groups 1 and 3.
      ,
      Balaban. Blastocyst quality and embryo transfer. Fertil Steril 2000.
      VariablePatients who had at least one hatching blastocyst available for transfer (group 1)Patients who had all blastocysts hatching available for transfer (group 2)Patients who had no hatching blastocyst available for transfer (group 3)
      No. of patients13897115
      Mean age (y)31.430.633.1
      No. of oocytes1,9561,4171,700
      2PN fertilization (%)1,360 (69.5)1,006 (70.9)1,221 (71.8)
      Cleavage (%)1,333 (98)983 (97.7)1,197 (98)
      G1+G2 embryos on day 3 (%)867 (65)659 (67)767 (64)
      G3+G4 embryos on day 3 (%)466 (35)324 (33)430 (36)
      Blastocysts formed on day 5 (%)627 (47)482 (49)564 (47)
      Blastocysts478 (3.4)328 (3.3)428 (3.7)
      No. of clinical pregnancies/no. of ETs (%)55/138 (39.8)56/97 (57.7)32/115 (27.8)
      Implantation rate per embryob (B)141/478 (29.4)144/328 (43.9)82/428 (19.1)
      No. of multiple pregnancies/total no. of pregnancies (%)c31/55 (56.3)46/56 (82.1)13/32 (40.6)
      legend a Group 2 significantly different from groups 1 and 3.
      legend b Group 2 significantly different from groups 1 and 3; group 1 significantly different from group 2.
      legend c Group 2 significantly different from groups 1 and 3.
      legend Balaban. Blastocyst quality and embryo transfer. Fertil Steril 2000.

      Discussion

      The results of this study show that [1] the rate of progression to the blastocyst stage is correlated with cleavage-stage embryo quality; [2] the quality of the cleavage-stage embryo is not a determinant of blastocyst quality; [3] blastocyst quality is clearly associated with implantation and pregnancy; [4] good quality blastocysts (grades 1 and 2) yield very high implantation rates, hatching blastocysts having the highest implantation rate; and [5] bad quality blastocysts (grade 3) implant at a much lower rate.
      Overall, 47% of cleavage-stage embryos reached the blastocyst stage after 5–6 days of in vitro culture. The rate of blastocyst formation from ICSI embryos appears to be higher than that reported by Shoukir et al. and Dumoulin et al. (
      • Shoukir Y
      • Chardonnens D
      • Campana A
      • Sakkas D
      Blastocyst development from supernumerary embryos after intracytoplasmic sperm injection a paternal influence.
      ,
      • Dumoulin J.C.M
      • Coonen E
      • Bras M
      • van Wissen L.C.P
      • Ignoul-Vanvuchelen R
      • Bergers-Jansen J.M
      • et al.
      Comparison of in-vitro development of embryos originating from either conventional in vitro fertilization or intracytoplasmic sperm injection.
      ). The reasons for this may be the use of different culture systems or the inclusion of mild and moderate male factor cases in our study.
      Transfer of human embryos at the blastocyst stage results in higher implantation rates (
      • Gardner D.K
      • Schoolcraft W.B
      • Wagley L
      • Schlenker T
      • Stevens J
      • Hesla J
      A prospective randomized trial of blastocyst culture and transfer in in-vitro fertilization.
      ). More cleavage-stage embryos progress to the blastocyst stage in complex, sequential culture systems, and the development of such media obviated the need for coculture with feeder cells. Whether the quality of the cleavage-stage embryo is a determinant of blastocyst quality is not clear. Rijnders and Jansen found a limited predictive value of embryo morphology on day 3 for subsequent blastocyst formation (
      • Rijnders P.M
      • Jansen C.A.M
      The predictive value of day 3 embryo morphology regarding blastocyst formation, pregnancy and implantation rate after day 5 transfer following in vitro fertilization or intracytoplasmic sperm injection.
      ). In our study, there appeared to be a clear relationship between cleavage-stage embryo quality and the rate of progression to the blastocyst stage. Grade 1 and 2 cleavage-stage embryos progressed to the blastocyst stage significantly more often than their grade 3 and 4 counterparts. However, blastocyst quality appeared to be independent of cleavage-stage embryo quality.
      Despite enthusiastic reports regarding blastocyst culture and transfer, several aspects of the procedure still need to be studied in detail. Until recently, there was no study in the literature that reported the effectiveness of blastocyst transfer in an unselected patient population. Most, if not all, studies dealt with a very favorable patient population, i.e., good responders who yielded many cleavage-stage embryos.
      In a retrospective study, Del Marek et al. compared the results of day 3 transfer versus unselective blastocyst transfer in 790 cycles of IVF/ICSI (
      • Del Marek M.A
      • Langley B.S
      • Gardner D.K
      • Confer N
      • Doody K.M
      • Doody K.J
      Introduction of blastocyst culture and transfer for all patients in an in vitro fertilization program.
      ). Blastocyst transfer yielded higher implantation rates in women less than 39 years of age. Only 6.7% of the patients failed to reach the stage of embryo transfer when embryos were cultured for 5 days. However, this was still significantly more than the 2.9% cancellation rate when the women were scheduled to undergo embryo transfer on day 3.
      Milki et al. compared blastocyst transfer with day 3 transfer in similar patient populations and concluded that blastocyst transfer is associated with higher implantation and clinical pregnancy rates (
      • Milki A.A
      • Hinckley M.D
      • Fisch J.D
      • Dasig D
      • Behr B
      Comparison of blastocyst transfer with day 3 embryo transfer in similar patient populations.
      ). The investigators attributed better results with blastocyst transfer to better embryo selection, improved embryo-uterine synchrony, and decreased cervical mucus. Huisman et al. compared implantation and pregnancy rates after in vitro fertilization and transfer of a maximum of two embryos following 3 or 5 days of embryo culture (
      • Huisman G.J
      • Fauser B.C
      • Eijkemans M.J
      • Pieters M.H
      Implantation rates after in vitro fertilization of a maximum of two embryos that have undergone three to five days of culture.
      ). They demonstrated higher implantation rates when embryos were transferred at the blastocyst stage.
      The major shortcoming of the above and other studies in the literature is their nonrandomized nature. It is yet to be determined whether the transfer at the blastocyst stage itself or better embryo selection afforded after prolonging the culture period is responsible for the reported high implantation rates with blastocyst transfer. Balaban et al. observed excess embryos up to the blastocyst stage following transfer at the cleavage stage. It was clearly demonstrated in that study that embryos transferred into the uterus implanted at a very high rate when their in vitro observed counterparts reached the blastocyst stage (
      • Balaban B
      • Urman B
      • Sertac A
      • Alatas C
      • Aksoy S
      • Nuhoglu A
      Progression of excess embryos to the blastocyst stage predicts pregnancy and implantation rates after intracytoplasmic sperm injection.
      ). The results of that study indicated that the zygote is endowed with an inherent capacity to progress to further stages of embryonic development and this capacity is reflected in the sibling oocytes observed under in vitro conditions. Therefore, the embryo that has the potential to develop into the blastocyst is also the embryo that will implant more successfully.
      As shown in this study, quality appears to be correlated with the implantation potential of the individual blastocyst. Limiting the number of grade 1 and grade 2 blastocysts that are transferred is absolutely necessary to avoid a high multiple pregnancy rate. The transfer of three or more such blastocysts resulted in a very high multiple pregnancy rate, and we now adopt the policy of limiting the number of grade 1 or grade 2 blastocysts to two. However, when only grade 3 blastocysts are available for transfer, implantation, clinical pregnancy, and multiple pregnancy rates were very low. More blastocysts can safely be transferred in these patients. When all transferred embryos are hatching blastocysts an implantation rate of 44% was obtained. In this group the transfer of three blastocysts resulted in an 82% multiple pregnancy rate. A case can be made for transferring only one hatching blastocyst in the young and no more than two hatching blastocysts in the older patient. This assumption, however, has to be tested in clinical trials.
      In conclusion, there appears to be a very close relation between the success of blastocyst transfer and blastocyst quality. The grading system used herein can be used for determining the number of blastocysts that should be transferred to prevent multiple pregnancies without compromising the pregnancy rate.

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        Progression of excess embryos to the blastocyst stage predicts pregnancy and implantation rates after intracytoplasmic sperm injection.
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        Blastocyst development from supernumerary embryos after intracytoplasmic sperm injection.
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