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Transfer of blastocysts with deviant morphological and morphokinetic parameters at early stages of in-vitro development: a case series

Reproductive BioMedicine Online, Volume 28, Issue 4, Pages 424–435, April 2014

Abstract

Time-lapse imaging is increasingly applied as an adjunct to reproductive medicine. The gained information of the morphological and morphokinetic variables before the onset of transcription are supposed to be good predictors for the selection of the best embryo for transfer and are often seen in line with clinical outcomes. This retrospective case series investigated the outcome of transferred blastocysts that did not fulfil the proposed embryo scores at early cleavage or at later stages of development. The observations were made by time-lapse imaging. This study reports the birth of 16 healthy children after day-5 blastocyst transfer, of which at least one of the transferred embryos originated from deviant morphology and/or kinetic cleavage patterns. This case series suggests that some blastocysts derived from embryos with poor conventional morphological score and/or suboptimal morphokinetics can be successfully transferred and might result in live births. Such results might raise awareness that discarding embryos based only on early events is not a suitable approach to give patients the chance to conceive. In conclusion, to date only the transfer of viable embryos after culturing them until day 5 guarantees optimal embryo selection and helps to prevent embryo wastage.

In assisted reproduction treatment, the overall success rates are still low. Optimal embryo selection is essential for IVF therapy to provide the highest chances for implantation. Therefore, several models were postulated for prediction of embryo quality. Many strategies have been applied, focusing on oocyte, zygote and early cleavage-stage morphology in a static manner. With the implementation of time-lapse technology, the information that is available in regard to embryonic development has increased tremendously. Thus, new morphokinetic markers have been established as embryo selection criteria. However, this brings the danger of wasting low-scored embryos. We here report the birth of several healthy babies after transfer of day-5 embryos that originated from oocytes or embryos that did not fulfil the morphokinetic criteria. With regard to the high costs of an IVF cycle and the emotional stress for the patient, surplus embryos should not be discarded but cryopreserved for transfer in another IVF cycle. Although the likelihood of pregnancy is lower compared to normal-scored embryos, there is still a chance of reaching blastocyst stage and successful implantation. According to our experience, the best option regarding embryo selection is still embryo culture until day 5.

Keywords: blastocyst culture, case series, day-5 transfer, embryo scoring, morphokinetics, time-lapse imaging.

Introduction

In view of the still low success rates of assisted reproduction treatment, the high cost of an IVF cycle and the implementation of single-embryo transfer, it is an obligation to accurately select those embryos having the highest implantation potential. Thus, usually, only embryos with good morphology are used for transfer or cryopreservation respectively. A large number of embryos with poor quality or deviant kinetics are discarded because they are considered to be less viable ( Ren et al., 2012 ). However, it should be noted that the prediction of viability and implantation capacity has always been challenging in the field of reproductive medicine.

The current static and sequential evaluation of the morphology using light microscopy is the most common way to select embryos. Conventional morphological parameters including oocyte morphology, pronuclear scoring, early cleavage on day 1 and morphological appearance of embryos on days 2 and 3 (number, size, evenness of blastomeres, degree of fragmentation, presence of vacuoles, multinucleation, etc.) are the standard embryo selection criteria (Depa-Martynow et al, 2007, Liu et al, 2008, Meseguer et al, 2011, Scott, 2003, Sela et al, 2012, Sohrabvand et al, 2011, Van Montfoort et al, 2004, and Wharf et al, 2004).

With regard to oocyte morphology, it is well accepted that a high percentage of the oocytes retrieved from stimulated cycles exhibit one or more deviant morphological characteristics ( Figueira et al., 2010 ). Thus, several morphological peculiarities of the oocytes were used as poor prognostic markers, such as cytoplasmic granularity, vacuolization of the ooplasm, central clustering of the smooth endoplasmic reticulum (SER) and abnormalities of the zona pellucida or refractile bodies.

Granulated cytoplasm of the oocyte, besides inclusions, vacuolization and others parameters, is one of the prominent cytoplasmic dismorphisms (reviewed inBalaban and Urman, 2006 and Ebner et al, 2006). This was negatively related to day-2 embryo quality ( Rienzi et al., 2008 ) and is suggested to have a negative impact on the oocyte’s developmental potential ( Ubaldi and Rienzi, 2008 ). Nevertheless, Fancsovits et al. (2012) did not find a correlation between the occurrence of cytoplasmic granularity and fertilization rate or zygote morphology, although some type of granulation resulted in a lower cleavage rate and more fragmented embryos.

The occurrence of central clustering of the SER is also linked with suboptimal outcome like a higher incidence of obstetric problems ( Ebner et al., 2008 ) and is considered to be possibly associated with multiple fetal anomalies ( Akarsu et al., 2009 ). Otsuki et al. (2004) showed that the presence of SER is associated with lower chances of a successful pregnancy. However, a recently published study encompassing 394 SER-positive and 6845 SER-negative intracytoplasmic sperm injection cycles stated that embryos derived from SER+ metaphase-II (MII) oocytes have the capacity to develop normally and may lead to healthy newborns ( Mateizel et al., 2013 ). Before this important study, which encompasses the highest number of SER cycles so far, it was even strongly recommended that oocytes displaying SER should not be inseminated ( Alpha Scientists in Reproductive Medicine and ESHRE Special Interest Group of Embryology, 2011 ).

In general, oocyte morphology often fails to predict fertilizing ability and developmental competence ( Rienzi et al., 2011 ). Only a few morphologically detectable features of the MII oocyte indicate compromised developmental ability. In a study including 5903 MII oocytes, Balaban et al. (1998) found that abnormal oocyte morphology does not result in an impairment of embryo quality, clinical pregnancy and implantation rates. In conclusion, the noninvasive identification of predictive markers for oocyte potential remains a difficult task.

Pronuclear (PN) scoring is mostly done by evaluating the number, size and distribution of nucleoli, cytoplasmic heterogeneity and the occurrence of a cytoplasmic halo (Depa-Martynow et al, 2007, Scott et al, 2000, and Tesarik et al, 2000). PN scoring was postulated to provide predictive value of embryo development, even of the chromosomal constitution and implantation potential of the embryo (Gianaroli et al, 2007, Montag and van der Ven, 2001, Salumets et al, 2003, Scott and Smith, 1998, Scott et al, 2000, Tesarik and Greco, 1999, and Wittemer et al, 2000). Therefore, PN scoring is widely used to predict embryo fate, but is of course, the subject of a controversial debate (James et al, 2006, Ludwig et al, 2006, and Payne et al, 2005). Many investigators have found a relationship between PN scoring and improved embryo development/blastocyst formation (Balaban et al, 2001, Nagy et al, 2003, Rienzi et al, 2002, and Zollner et al, 2002) and/or increased pregnancy and implantation potential (Montag and van der Ven, 2001 and Wittemer et al, 2000; reviewed by Zollner et al., 2003 ). These studies have shown that the most viable zygotes have PN of similar size that are centrally located, with each containing nucleolar precursor bodies (NPB) of equal size and number aligned at the pronuclear interphase in preparation for syngamy ( Alpha Scientists in Reproductive Medicine and ESHRE Special Interest Group of Embryology, 2011 ). Nevertheless, numerous other studies have failed to show that the addition of PN scoring to a cumulative scoring system can effectively improve the selection of viable embryos (reviewed by Skiadas and Racowsky, 2007 ).

Uneven cleavage is supposed to negatively affect the developmental capacity of the embryo (Hesters et al, 2008 and Scott et al, 2007) and has been correlated with multinucleation and aneuploidy due to failures in chromosomal replication ( Hardarson et al., 2001 ). The occurrence of multinucleation, which is defined by the presence of more than one nucleus in at least one blastomere of the embryo in an early cleavage stage, is also associated with impaired developmental capacity (Van Royen et al, 2003 and Yakin et al, 2005). Nevertheless, multinucleation is a common phenomenon that can be observed in about 34% (day 2 and/or day 3) of all patients’ embryos ( Van Royen et al., 2003 ). While these embryos are usually excluded from transfer, it has also been documented that binucleated cells on day 1 can cleave into chromosomally normal cells ( Staessen and Van Steirteghem, 1998 ). In regard to morphological specifics and selection criteria of early cleavage embryos, fragmentation is widely considered to be detrimental for further development ( Alikani et al., 1999 ). The cause(s) of such events remain controversial, although abnormal cytokinesis is frequently discussed as a reason ( Wong et al., 2010 ). In fact, the degree of fragmentation can hardly be quantified and, moreover, fragments can be reabsorbed by the embryo within a few hours (Lemmen et al, 2008 and Pribenszky et al, 2010).

In the further course of early development, the appearance of blastomeres with micronuclei are used as exclusion criteria with regard to transfer or cryopreservation and, thus, these embryos might be discarded because they do not fulfil the criteria to be considered as a ‘normal’ embryo.

The cell number at a certain stage of development is assumed to be an important marker of viability and embryo potential. Numerous studies have shown a direct correlation between the number of cells in day-3 embryos and implantation rates following day-3 transfer (Carrillo et al, 1998, Puissant et al, 1987, Racowsky et al, 2003, and Steer et al, 1992). Some authors have also associated cell number on day 3 with blastocyst formation rate ( Jones et al., 1998 ). Of all morphological characteristics that are typically assessed in cleavage-stage embryos, cell number is still believed to be the single most important indicator for embryo viability. Thus, top-quality embryos on day 2 or day 3 were mostly defined as embryos with 4 or 6–8 cells, even cell size and with less than 10 (mild) or 20% (moderate) fragmentation, respectively.

Actually, with the recent introduction of time-lapse recording in the clinical practice, the static evaluation is more and more replaced by dynamic assessment of the embryo development and it is now possible to continuously track this period until the day of transfer. Thus time-lapse imaging allows embryo observation in a more accurate and objective manner as well as the precise detection of irregular morphological or cell kinetic events occurring during the in-vitro development that cannot be detected by conventional static observation. For this reason, much more information is now available on embryo development during the period of in-vitro culture, acknowledging the fact that the static visual observations describe only a brief time span of the development ( Kirkegaard et al., 2012 ). In fact, this noninvasive system of continual monitoring now allows a broad and detailed evaluation of the developmental kinetics and morphological alterations over a long time. Thus, new embryo selection parameters were postulated in numerous studies by taking account of the kinetic observations (e.g. time point of PN formation or the time needed by the embryo to reach the 2-, 3- or 5-cell stage respectively (t2,t3,t5; Meseguer et al., 2011 ). Additionally a direct cleavage from 2- to a 3-blastomere embryo was assumed to have a poor outcome (Meseguer et al, 2011 and Rubio et al, 2012). With the implementation of time-lapse imaging systems in assisted reproduction treatment, more and more embryos are selected for transfer on the basis of these observed morphokinetic parameters. Even more morphokinetic variables have been postulated as selection criteria to distinguish aneuploid embryos from euploid ones ( Campbell et al., 2013 ). But until now, there is obviously no unique algorithm for the prediction of embryonic developmental competence to be applied as a standard tool in every laboratory ( Chamayou et al., 2013 ).

Nevertheless, several algorithms were introduced for use on day-2 and/or day-3 embryos to discriminate between the ‘good-quality’ embryos and those having low implantation potential ( Meseguer et al., 2011 ). The embryos that do not fulfil the criteria are not selected for embryo transfer on day 2 or 3. The question is whether an embryo selection procedure that is based only on early events during in-vitro culture constitutes a suitable approach as it completely neglects the late paternal effect (Tesarik, 2005 and Vanderzwalmen et al, 1991) and does not take into account that most of the paternal genome is silenced until its activation at day 2/3. In the case of abnormal early development, one option is prolonged in-vitro culture until day 5 along with the observation of the outcome of embryonic development. This manuscript reports several cases of time-lapse imaging, demonstrating that embryos with poor scores between days 1 and 3, according to the mentioned criteria, can reach the blastocyst stage and their subsequent transfer can result in the birth of healthy babies.

Materials and methods

The gonadotrophin-releasing hormone (GnRH) long protocol was applied for all patients with daily injections of triptorelin (0.1 mg/day, Decapeptyl; Ferring Arzneimittel, Vienna, Austria), beginning in the midluteal phase of the preceding cycle for down-regulation of the pituitary gland. Human menopausal gonadotrophin (HMG; Merional; IBSA, Lugano, Switzerland) 2–4 × 75 IU/day was used for follicle stimulation. Oocyte retrieval and intracytoplasmic morphologically selected sperm injection (IMSI) were performed as previously described by Vanderzwalmen et al. (2008) . Embryo culture was performed in a time-lapse incubation system (EmbryoScope; Unisense Fertilitech, Aarhus, Denmark) until transfer on day 5. Individual embryos were cultured in 25 μl of a single-step medium (LifeGlobal) under 5.8% CO2covered with oil. The medium was renewed on day 3. Images of the cultured embryos were taken every 20 min. Embryos were chosen for transfer on day 5 according to Gardner’s morphological criteria ( Gardner et al., 2000 ). Analysis of morphological and kinetic events of the embryos was performed retrospectively by two independent operators. Uneveness of blastomeres was estimated according to Hardarson et al. (2001) . The determination of time ranges for normal or deviant morphokinetics was performed according to Meseguer et al. (2011) and Dal Canto et al. (2012) . Pregnancy was determined biochemically by testing urinary β-human chorionic gonadotrophin 14 days after embryo transfer and fetal heartbeat by ultrasound 8–12 weeks after embryo transfer. Monozygotic twins were evaluated by ultrasound at gestation weeks 6–8 by the attending gynaecologist. All patients signed consent for publishing medical data. All works performed were in concordance with the principles for medical research according to the WMA declaration of Helsinki. Ethical committee approval was not necessary as all the techniques used are standard techniques in reproductive medicine.

Results

Case 1

A 40-year-old woman with endometriosis underwent programmed ovarian stimulation in August 2011. Her husband was previously diagnosed with oligoasthenoteratozoospermia (OAT) III.

After 10 days of follicle stimulation with HMG, nine oocytes were retrieved, of which six were fertilized. After IMSI, all oocytes were cultured in the EmbryoScope and four reached the blastocyst stage. The two blastocysts with the highest classification were denoted as 3AB and 5BB according to the criteria of Gardner et al., 2000 and were thus selected for embryo transfer. Fetal heartbeat was detected 4 weeks later. The patient gave birth to two healthy girls (52 cm, 2960 g and 51 cm, 2650 g, respectively).

In the retrospective analysis, one embryo revealed multinucleation at the 2-cell stage in one of the two blastomeres ( Figure 1 A and Table 1 ); however, blastocyst formation occurred ( Figure 1 B). The other embryo ( Figure 1 C) revealed a high degree of fragmentation and a prolonged time period to the 5-cell stage (t5 = 58.4 h; reference 50.7 ± 6.1 h for embryos developing to blastocyst ( Dal Canto et al., 2012 ) ( Figure 1 D). Additionally, the trophectoderm revealed a low cell number ( Figure 1 D) and embryo was classified as 3BC blastocyst.

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Figure 1 Case 1. (A, B) First embryo: multinucleation in one blastomere at the 2-cell stage after 58.4 h (A) and development at 115.3 h (B). (C, D) Second embryo:t5 = 58.4 h (C); after 115.3 h trophectoderm consisting of a few cells (D). Bar = 50 μm.

Table 1 Summary of morphological and morphokinetic observations, quality of transferred blastocysts and data of babies born.

Case Abnormal morphology Abnormal kinetics Quality of transferred blastocysts Babies born
SER Granularity PN score Uneven blastomeres BN/MN/MCN Fragmentation Blastocyst formation t2 t5 cc2 Others
I                         Dizygotic
          Yes, MN in 1 blastomere at 2-cell stage             5BB F (52 cm, 2960 g), healthy
            High Trophectoderm with low cell number   Prolonged (58.4)     3BC F (51 cm, 2650 g), healthy
 
II       Yes, at 2-cell stage         Shortened (38.5)     ebl M (48 cm, 2800 g), healthy
        Yes, at 2-cell stage           Long (12.7)   4BC F (48 cm, 2810 g), healthy
 
III                         Dizygotic
  Yes, before PN formation                     4AA M (45 cm, 2170 g), healthy
              2 blastomeres were not incorporated in blastocyst         4BA M (47 cm, 2430 g), healthy
 
IV     Poor   Yes, MN in 1 blastomere at 2-cell stage             4AB M (36 cm, 1420 g), healthy
      Poor         Shortened (21.0)       4BB F (37 cm, 1420 g), healthy
 
V   High, large areas                   ebl F (49 cm, 3360 g), healthy
 
VI                         Dizygotic
      Poor       ICM with bridge formation         4AB M (48 cm, 2410 g), healthy
      Poor   Yes, BN in 1 blastomere at 2-cell stage       Late (65.1) Long (13.9)   3BB M (50 cm, 2940 g), healthy
 
VII                         Dizygotic
      Poor   Yes, MCN in 1 blastomere at 2-cell stage             3CB M (43 cm, 1990 g), healthy
      Poor       1 blastomere was not incorporated in blastocyst     Short (7.9)   4CB M (46 cm, 2330 g), healthy
 
VIII                         Dizygotic
        Yes, at 8-cell stage   High         1st direct cleavage into 3 blasto-meres 4CC F (51 cm, 3000 g), healthy
                  Late (61.4)     3BC F (50 cm, 2780 g), healthy
 
IX             2 blastomeres were not in-corporated in the blastocyst       Divisions back and forward to 2-cell stage 4BC M (53 cm, 3605 g), healthy

Values in parentheses are time in h. Blastocyst quality was assessed according to Gardner et al. (2000) .BN = binucleation, MCN = micronucleation, MN = multinucleation; PN = pronuclear.

Case 2

A 33-year-old patient underwent ovarian stimulation programme in her first IVF attempt in March 2011. Her partner was previously diagnosed with asthenoteratozoospermia.

After 15 days of follicle stimulation with HMG, 20 oocytes were retrieved. After IMSI on 14 MII oocytes, eight of them were fertilized. Due to a robust presenting zona pellucida, assisted zona hatching was performed on day 4 with Fertilase laser (MTG, Bruckberg, Germany) ( Figure 2 B, E). Two embryos (4BC and an early blastocyst) were chosen for transfer on day 5 according to the quality criteria described ( Figure 2 C, F). The patient gave birth to a healthy boy (48 cm, 2800 g) and a healthy girl (48 cm, 2810 g) after an uneventful pregnancy in pregnancy week 37.

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Figure 2 Case 2. Both embryos reveal uneven blastomeres on day 2 (A, D); assisted zona hatching was performed on day 4 (B, E); outcome on day 5 (C, F). Bar = 50 μm.

In the retrospective analysis, both embryos showed uneven blastomeres on day 2 ( Figure 2 A, D and Table 1 ). Moreover, one embryo revealed a strongly shortenedt5period (38.5 h; reference 50.7 ± 6.1 h for embryos developing to blastocyst, Dal Canto et al., 2012 ) and the other one revealed a rather long second cell cycle (cc2, defined ast3 − t2; 12.7 h; reference 10.5 ± 1.9 h, Dal Canto et al., 2012 ).

Case 3

A 36-year-old patient with endometriosis started with the ovarian stimulation programme in May 2011. After 12 days of follicle stimulation with HMG, 16 oocytes were retrieved and 12 injected MII oocytes were fertilized. On day 5, two blastocysts, quality 4AA ( Figure 3 D) and 4BA ( Figure 3 J), were selected for transfer. The patient gave birth spontaneously to two dizygotic healthy boys (45 cm, 2070 g; 47 cm, 2430 g) in pregnancy week 38.

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Figure 3 Case 3. (A–D) First embryo: SER cluster before 2 pronuclei Formation (A–C); quality of transferred blastocyst (D). (E–J) Second embryo: two blastomeres were not incorporated in the blastocyst. Bar = 50 μm.

In the retrospective analysis of the transferred embryos, one oocyte showed a cluster of SER that disappeared before appearance of the 2 pronuclei ( Figure 3 A, B and Table 1 ). For the other embryo ( Figure 3 E–J), there was no evidence of any obvious abnormal events during early development. However, two blastomeres were not incorporated during blastocyst formation ( Figure 3 E–H and Table 1 ). This observation was only possible by time-lapse imaging. Especially at the expanded stage of the blastocyst, the excluded blastomeres appeared to be part of the trophectoderm since they were pushed to the zona pellucida ( Figure 3 I, J). At 92 h, this early blastocyst would usually not have been considered to be suitable for embryo transfer or cryopreservation due to unincorporated blastomeres.

Case 4

A 26-year-old patient whose husband had been diagnosed with asthenozoospermia (motility grades: 4% a, 15% b, 15% c, 66% d, according to WHO, 2010 ) underwent ovarian stimulation in July 2011. After 11 days of follicle stimulation with HMG and 1 day of coasting, eight oocytes were retrieved. Out of seven MII oocytes, six showed 2 pronuclei 1 day after IMSI. After 5 days of culture, six blastocysts were obtained and, on the patient’s request, two blastocysts, 4AB ( Figure 4 D) and 4BB ( Figure 4 H), were selected for embryo transfer. She delivered a healthy boy (36 cm, 1160 g) and a healthy girl (37 cm, 1420 g) in pregnancy week 30.

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Figure 4 Case 4. (A–D) First embryo: z-score of z3–4 (A, B), multinucleation at the 2-cell stage in one blastomere (C) and development to the blastocyst stage (D). (E–H) Second embryo: z-score of z3–4 (E–F), 2-cell-stage embryo (G) and development to the blastocyst stage (H). Bar = 50 μm.

Retrospectively analysed, one embryo indicated multinucleation in 1 blastomere on day 2 of culture ( Figure 4 C and Table 1 ). The other embryo exhibited a very shortt2(21 h; Figure 4 G and Table 1 ). Moreover, both embryos showed an unequal number of NPB at the pronuclear stage ( Figure 4 A, B, E, F). One embryo showed the same size for NPB almost aligning at the PN junction (z4–2; Figure 4 A, B). In the other embryo, the NPB differed in size and were not aligned ( Figure 4 E, F) (z3–4 according to Scott (2003) Figure 7 and Figure 8).

Case 5

A 41-year-old patient underwent ovarian stimulation in February 2011. After 9 days of follicle stimulation, 19 oocytes were retrieved. Sixteen of them showed a polar body as indication for MII. Eleven oocytes were fertilized normally, which was confirmed by the appearance of 2 pronuclei and 2 polar bodies. Seven fertilized oocytes were cultured in the EmbryoScope, and six were cultured in a standard incubator.

All oocytes showed a very deep granular area in the centre of the oocyte ( Figure 5 A–C and Table 1 ). On day 5, two early blastocysts were chosen for transfer, one from the time-lapse incubator and one from the conventional culture system. The patient gave birth spontaneously to a healthy girl (49 cm, 3360 g) in pregnancy week 39.

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Figure 5 Case 5. Large area of granularity (A–C). Bar = 50 μm.

In the retrospective analysis of the transferred embryo, out of the time-lapse incubator the oocyte presented, like all oocytes, a large area of granularity ( Figure 5 A–C).

Case 6

A 30-year-old patient underwent ovarian stimulation in April 2011. After 11 days of stimulation, eight MII oocytes could be retrieved. Six oocytes were fertilized and resulted in three blastocysts. Out of these, two blastocysts, 4AB and 3BB, were selected and on the patient’s request both were transferred ( Figure 6 C, H). The patient delivered healthy dizygotic male twins (48 cm, 2410 g; 50 cm, 2940 g) in pregnancy week 38.

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Figure 6 Case 6. (A–C) First embryo: different size and number of nucleolar precursor bodies at 17 h (A) and 22.9 h (B) and blastocyst showing an inner cell mass forming a bridge (C). (D–H) Second embryo: unequal size and number of nucleolar precursor bodies (z-score of z3–3) (D–F), multinucleation in one blastomere at 2-cell stage (G) and development to the blastocyst stage (H). Bar = 50 μm.

Retrospectively, one embryo showed NPB with different size and number ( Figure 6 A–B). This embryo had an otherwise uneventful development but revealed a cellular bridging of the ICM ( Figure 6 C and Table 1 ). The other embryo had an unequal size and number of NPB ( Figure 6 D–F) and one cell in the 2-cell stage had two nuclei ( Figure 6 G). cc2 was rather long (13.9 h) and the occurrence oft5was unusually late (65.1 h, Table 1 ; reference 50.7 ± 6.1 h for embryos developing to blastocyst, Dal Canto et al., 2012 ).

Case 7

In April 2011, a 36-year-old woman underwent oocyte retrieval after a stimulation period of 10 days. Her partner was diagnosed with asthenozoospermia (motility parameters: 0% grade a, 21% b, 40% c and 39% d, according to WHO, 2010 ). She had a history of four previous failed IVF attempts and one abortion. Out of five retrieved oocytes, four MII oocytes were fertilized. Two of the fertilized oocytes developed into blastocysts of quality 3CB and 4CB and were chosen for transfer ( Figure 7 D, H). In pregnancy week 39, the woman delivered two healthy dizygotic boys (43 cm, 1990 g; 46 cm, 2330 g).

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Figure 7 Case 7. (A–D) First embryo: 2 pronuclei stage with separated pronuclei at 17.0 h (A), embryo at 21.4 h (B), one blastomere showing a micronucleus at 34.5 h (C) and outcome after 115 h (D). (E–H) Second embryo: 2 pronuclei stage embryo at 17.1 h (E), 4-cell-stage embryo at 35.5 h (F), embryo at 102.7 h, one blastomere not incorporated within the blastocyst (G) and 115.0 h (H). Bar = 50 μm.

Retrospectively analysed, one embryo had a poor PN scoring with separated PN at 17.0 h, an unequal number and distribution of NPB ( Figure 7 A, B and Table 1 ), one blastomere showed a micronucleus at the 2-cell stage ( Figure 7 C). The other embryo had a very short cc2 of 7.9 h, ( Table 1 ; reference 10.28 to 10.51 h, Dal Canto et al., 2012 ). The scoring of the 2 pronuclei showed NPs different in number (2 vs. >7) and, size and NPs are aligned in one of the two PNs and scattered in the other with respect to the PN junction ( Figure 7 E). The 4-cell stage was quality 4B2 ( Figure 7 F) and at the blastocyst stage one blastomere was not incorporated in the blastocyst formation ( Figure 7 G and Table 1 ), which could be detected only in the time-lapse sequence.

Case 8

A 41-year-old patient underwent ovarian stimulation for a period of 10 days. Out of 26 oocytes retrieved on the day of oocyte retrieval 25 oocytes were in MII. Nineteen oocytes revealed 2 pronuclei 1 day later and 10 blastocysts of moderate quality could be cultured. Two blastocysts of quality 4CC ( Figure 8 E) and 3BC (data not shown) were transferred without any problems. The woman delivered spontaneously in pregnancy week 38 two healthy dizygotic girls (51 cm, 3000 g; 50 cm, 2780 g).

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Figure 8 Case 8. Direct cleavage from 1 to 3 cells (A–C), high degree of fragmentation on day 3 (D) and outcome on day 5 (E). Bar = 50 μm.

In the retrospective analysis, one embryo showed a direct cleavage from 1 to 3 cells ( Figure 8 a–c), revealing a high degree of fragmentation and, moreover, uneven blastomeres at the 8-cell stage ( Figure 8 D and Table 1 ). The other embryo did not show any noticeable aberrations but a rather latet5(61.4 h; Table 1 ).

Case 9

From a 32-year-old patient whose husband had been diagnosed with OAT, 13 MII oocytes could be retrieved after 12 days of stimulation using the long protocol. Eleven oocytes were fertilized regularly determined by the appearance of 2 pronuclei. Five blastocysts were obtained on day 5 after culture to the blastocyst stage. One blastocyst of quality 4BC ( Figure 9 F) was transferred on day 5, the others were vitrified. After an uneventful pregnancy, the women gave birth to a healthy boy in the 39th week of pregnancy (53 cm, 3605 g).

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Figure 9 Case 9. Day-2 embryo cleaving forward and backward between 26.6 and 36.5 h (A–D), on early day 5, two blastomeres are not included in the blastocyst formation (E) and outcome at 115.9 h (F). Bar = 50 μm.

Retrospectively analysed, the time-lapse record showed an embryo with divisions back and forward to a 2-cell embryo (26.8–36.5 h; Figure 9 A–D and Table 1 ). On day 5, two blastomeres were not included in the blastocyst formation ( Figure 9 E, F and Table 1 ).

Discussion

In the described cases, abnormal morphological and/or deviant morphokinetic patterns were observed at different stages of development. This case series demonstrates that, even when some embryos do not fulfil the conventional criteria, there can be a positive outcome that includes the formation of blastocysts, achievement of pregnancies and even the birth of healthy babies.

Although IVF techniques and protocols have dramatically improved, the overall success rates are still relatively low, and assisted reproduction units are still facing the challenge of improving pregnancy rates ( Souza Setti et al. 2010 ). Many approaches were described that were based on morphology or kinetics to select the best oocyte(s) and embryo(s) at early cleavage stages or at the blastocyst stage to increase pregnancy rates. Scoring and selection of gametes and embryos have a long tradition and are as old as the assisted reproduction treatment itself. However, the yielded results are conflicting and the outcome is a matter of never-ending and controversial debates.

Taking account of these findings, there is the fundamental question whether all these quality parameters or observed deviant events obtained by time-lapse imaging can be considered as ‘abnormal’ and justifies discarding of early cleavage embryos with ‘poor’ morphology or irregular/deviant cleavage events. Considering that those embryos that did not fulfil the morphokinetic criteria have the chance to implant, this raises the question of how to define a ‘good-quality’ embryo. Of course, such embryos might have lower capacity of implantation, but in the authors’ opinion, they should still not be discarded as long as the chance of birth of a healthy baby is given.

Which criteria should the embryo selection be performed – according to morphology or morphokinetics? What are the reliable parameters to predict embryo implantation? Recent studies demonstrated that the morphology score of cleavage stage cannot predict accurately the developmental potential of blastocyst formation (Graham et al, 2000, Guerif et al, 2007, and Rijnders and Jansen, 1998). Quality of early cleavage was found in some studies to be correlated with developmental capacity (Lee et al, 2012 and Salumets et al, 2003); however, not with pregnancy rates ( Lemmen et al., 2008 ).

The superiority of blastocyst culture and day-5 embryo transfer with regard to better embryo selection and improved uterine and embryonic synchronicity is reflected by increased implantation and live birth rates compared with cleavage-stage (day 2/3) embryo transfer. This has been well documented by numerous publications over the past years (Blake et al, 2007, Glujovsky et al, 2012, Papanikolaou et al, 2006, and Reh et al, 2010). Nevertheless, the majority of laboratories still perform static evaluation of their embryos on day 1, 2 or 3 before performing embryo transfer on day 3 or freezing them.

In this setting, blastocyst culture was superior to day-3 embryo transfer, and blastocyst culture is still providing the best information for selecting an embryo for transfer ( Zech et al., 2007 ). These findings are supported by a number of researchers (Graham et al, 2000, Papanikolaou et al, 2005, and Papanikolaou et al, 2008). Therefore, if the patient has at least four good day-3 embryos, extended culture until day 5 is recommended ( Papanikolaou et al., 2005 ). In fact, most of the applied morphological and kinetic selection parameters are still limited predictors of embryo quality on day 5. Embryo selection on day 2 or 3 neglects the fact that the embryonic genome activation does not occur before day 3. Putative negative or compensating effects are completely blanked by an embryo transfer at day 2 or 3.

In an oocyte donation programme or in young and good-prognosis patients, these limited selection criteria can be applied for selecting embryos to achieve a pregnancy, but in the authors’ opinion, blastocyst culture is the best way to attain high implantation rates and offer all patients the best possible and most effective chance of giving birth to a healthy child. It is, therefore, concluded that embryos which are routinely deselected due to abnormal findings obtained through conventional observation techniques such as low PN score, a high degree of fragmentation, unevenness of blastomeres, multinucleation or alterations in the kinetics of cleavage(s) should not be discarded but considered either for transfer or for cryopreservation after subsequent culture to day 5. Although embryos with irregular morphokinetics have a reduced implantation potential, it is nevertheless possible to achieve the birth of a healthy child. In a retrospective study, Shaw-Jackson et al. (2013) analysed the developmental capacity of poorly scored day-3 embryos and the further developmental potential after vitrification at blastocyst stage. They draw two important conclusions. Firstly, the cleavage-stage morphology has its limits for embryo selection, and second, embryo wastage can be reduced by extended culture to day 5 and subsequent vitrification. In another recently published study, Ren et al. (2012) examined the in-vitro blastocyst development of poor-morphological-scored cleavage stages and clinical outcomes after transfer of these blastocysts in warming cycles and stated that extended culture allows the reduction of embryo wastage. In fact, more blastocysts provide higher chances for the patient to achieve a pregnancy in successive vitrified embryo transfer cycles. More vitrified blastocysts could impact the cumulative pregnancy rate. With the introduction of several commercial aseptic vitrification devices, such as VitriSafe, there is increasing interest in cryostorage of oocytes and embryos at different stages of development ( Alpha Scientists in Reproductive Medicine, 2012 ). Even when the blastocyst quality is limited, according to this centre’s experience, not only top-quality embryos but also lower-quality blastocysts survive the aseptic vitrification process and have the capability to implant ( Wirleitner et al., 2013 ).

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Footnotes

a IVF Centers Prof. Zech-Bregenz, Austria

b Centre Hospitalier Inter Régional Edith Cavell (CHIREC), Braine-l’Alleud, Brussels, Belgium

c IVF Centers Prof. Zech-Salzburg, Austria

lowast Corresponding author.

fx1 Astrid Stecher obtained her degree in biology with special focus on molecular genetics and microbiology from the Karl-Franzens University of Graz, Austria in 1993. She joined the field of embryology in the team of H Zech in 1995 and she is currently the head of all laboratories of the IVF Centers Prof H Zech. Her major areas of interest are in-vitro culture conditions, early events of oocyte activation, assessment of embryo morphology and development, noninvasive selection methods in IVF and quality management in the IVF laboratory.