Table of Contents    
Year : 2019  |  Volume : 10  |  Issue : 3  |  Page : 92-98  

Relationship between morphology of tripronuclear embryo and chromosomal abnormalities potential in intracytoplasmic sperm injection cycles

1 Division of Reproductive Endocrinology and Infertility, Department of Obstetrics and Gynecology, Faculty of Medicine, Universitas Indonesia, Jakarta, Indonesia
2 Department of Community Medicine, Faculty of Medicine, Universitas Indonesia, Jakarta, Indonesia
3 Division of Reproductive Endocrinology and Infertility, Department of Obstetrics and Gynecology, Faculty of Medicine; Human Reproductive, Infertility and Family Planning Research Center, Indonesian Medical Education and Research Institute, Universitas Indonesia, Jakarta, Indonesia

Date of Web Publication14-Jan-2020

Correspondence Address:
Budi Wiweko
Division of Reproductive Endocrinology and Infertility, Departement of Obstetrics and Gynecology, Faculty of Medicine, Universitas Indonesia, Jakarta; Indonesian Medical Education and Research Institute, Universitas Indonesia, Jakarta
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/jnsbm.JNSBM_19_19

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Introduction: Tripronuclear (3PN) zygote is a frequently observed abnormal outcome in vitro fertilization/intracytoplasmic sperm injection (ICSI) technology. They are usually discarded because of concerns about their abnormal chromosomal constitution. However, in rare cases when availability of the embryos is limited, evidence-based information on the suitability 3PN zygotes for implantation will be valuable. Materials and Methods: In this study, we analyzed the chromosomal constitution of 3PN to investigate the relationship between their morphology and chromosomal status. Thirty 3PN zygotes developed into embryos from 18 cycles were reviewed during 6 months. Biopsies were performed on day 5/6 and were subsequently screened for chromosomal status using a next-generation sequencing method. Results: Of the 30 3PN screened, 66.7% were chromosomally abnormal. At the cleavage stage, there was no association between any of the morphological features and chromosomal status. In contrast, at the blastocyst stage, a grade <3 blastocyst expansion had a significantly higher chromosomal abnormality (90%, P = 0.05) than the other grades of expansion. Embryos with grade non A (grade B and C) for both inner cell mass and tropectoderm, had significantly higher chromosomal abnormalities (100%, P = 0.001 and 93.3%, P = 0.001, respectively). Conclusion: We concluded that chromosomal status and 3PN morphology are linked at the blastocyst stage, and thus morphological assessment of 3PN blastocysts can be used in conjunction with preimplantation genetic screening to select which embryo should be transferred when no other embryos from two pronuclear ICSI zygotes are available.

Keywords: Chromosomal abnormality, embryo morphology, next-generation sequencing, preimplantation genetic screening, tripronuclear

How to cite this article:
Jasirwan SO, Iffanolida PA, Andika Santawi VP, Friska D, Wiweko B. Relationship between morphology of tripronuclear embryo and chromosomal abnormalities potential in intracytoplasmic sperm injection cycles. J Nat Sc Biol Med 2019;10, Suppl S1:92-8

How to cite this URL:
Jasirwan SO, Iffanolida PA, Andika Santawi VP, Friska D, Wiweko B. Relationship between morphology of tripronuclear embryo and chromosomal abnormalities potential in intracytoplasmic sperm injection cycles. J Nat Sc Biol Med [serial online] 2019 [cited 2020 Oct 28];10, Suppl S1:92-8. Available from:

   Introduction Top

One of the main purposes of in vitro fertilization (IVF) is to obtain an embryo with high implantation potential to achieve a healthy live singleton birth. The earliest embryonic identification and selection method employs morphological assessment based on pronuclear determination, which is routinely done both following insemination in conventional IVF as well as in intracytoplasmic sperm injection (ICSI). Formation of two pronuclear (2PN) zygotes 16–18 h after conventional insemination or ICSI signifies normal fertilization. A tripronuclear (3PN) zygote is a form of pronuclear aberration as a result of abnormal fertilization that frequently occurs in IVF technology and accounts for 5%–8.1% of fertilizations in conventional IVF and 4%–6.5% in ICSI.[1],[2],[3] The primary cause of 3PN is thought to be a result of dispermic fertilization that most commonly occurs in conventional IVF due to an immature or postmature oocyte, the breakdown of the zona pellucida, or high sperm concentrations.[4] This condition is not associated with age or ovarian stimulation type. In contrast, dispermy should not occur in ICSI because only a single sperm is injected into each oocyte. Therefore, the incidence of 3PN in ICSI is caused mostly by retention of a second polar body of oocyte following ICSI.[5],[6] A previous study reported that high response to ovarian stimulation and a high level of estradiol on the day of human chorionic gonadotropin (hCG) administration also are associated with the incidence of 3PN embryos in ICSI-IVF.[5]

In routine IVF/ICSI practice, 3PN zygotes are usually discarded because they are deemed to have abnormal chromosomal constituents and may develop into triploidy, aneuploidy, and even mosaic abnormality due to abnormal fertilization. Several previous studies have reported that the blastocyst rate from 3PN embryos was 10.75%–62.5%.[7],[8],[9],[10] Through preimplantation genetic screening (PGS) applied as an embryonic genetic assessment, it is reported that some embryos developing from 3PN zygotes had normal chromosomal composition.[2],[7],[8],[11],[12],[13] Two case reports even stated that a single euploid 3PN embryo transfer had resulted in a healthy birth.[8],[14] Therefore, PGS assessment of 3PN embryos may be beneficial in selecting embryos with normal chromosomal composition, especially when there are no normal 2PN embryos available. Thus, PGS in conjunction with morphology evaluation can be used to generate valuable information about whether to transfer or discard specific embryos. In this study, using next-generation sequencing (NGS), we analyzed the chromosomal constitution of embryos developed from 3PN zygotes to investigate the relationship between the morphology of 3PN embryos and their chromosome status.

   Materials and Methods Top

Ethics approval and source of embryos

This study was approved by ethics committee of Faculty of Medicine, University of Indonesia. All preimplantation 3PN embryos' samples were obtained from couples undergoing ICSI procedures only to exclude polyspermy by conventional IVF at the Yasmin IVF Clinic (Dr. Cipto Mangunkusumo Hospital, Jakarta) and the Daya Medika Clinic (Jakarta) between January and July 2018 after receiving written informed consent.

Ovum pickup and fertilization

Retrieval of cumulus-oocyte complexes was performed at 36-h post-hCG injection. The complexes were subsequently cultured for 4–5 h in medium supplemented with 40 IU/ml HSA solution (Vitrolife) to facilitate further denudation and maturation. ICSI was performed when the oocyte reached the Phase II metaphase.

Pronuclear checking and embryo culture

Assessment and recording of the pronuclei were performed using an inverted microscope at 18–20 h post-ICSI. The 3PN embryos that subsequently cleaved were cultured in ISM medium 1 until afternoon on day 2 of culture. Then, the 3PN embryos were transferred to Blast Assist Medium until day 5/6. Embryos that showed arrested development after 24 h were not cultured further and were discarded. The morphology of each embryo was recorded and assessed daily based on cell count, the percentage of fragmentation, and equal blastomere size. Embryos that reached the blastocyst stage were assessed using Gardner's score based on the degree of expansion, intracellular mass (ICM), and trophectoderm (TE). Embryos that reached the blastocyst stage on day 5 or 6 were selected for further analysis.

Embryo biopsy

Embryo biopsies were performed on day 5 or 6 for chromosome analysis using an NGS method. The selected day 5 or 6 blastocysts were biopsied on 3–6 TE cells using a laser (Octax).

Next-generation sequencing assessment

All single embryonic cell samples were purified to obtain a high concentration of DNA. A whole-genome amplification protocol was performed for all individual samples using PicoPlex technology (Rubicon Genomics, Inc., Ann Arbor, MI, USA). After whole-genome amplification, each sample was processed to prepare DNA libraries for sequencing on a MiSeq system according to the manufacturer's instructions (Illumina, San Diego, CA, USA) for PGS. Briefly, the diluted DNA (0.2 ng/μl) was tagmented using the Illumina Nextera XT transposase (amplicon tagment mixture and tagment DNA buffer) through a limited-cycle polymerase chain reaction (PCR) to amplify the inserted DNA. To enable dual-indexed sequencing, the index sequences were added to the samples then subsequently were amplified using the Nextera PCR Master Mix (NPM).

The PCR products were purified using AMPure XP beads (Beckam Coulter, USA). Then, the DNA libraries were normalized to equalize the quantity of each sample in the final pooling using the library normalization additive and beads. The normalized samples with equal volumes were pooled, denatured, and then sequenced. The Miseq Reagent Kit v.3 (Illumina, Inc., San Diego, CA, USA, RH-101-1001) was used on a MiSeq System (Illumina). The generated bioinformatics data were analyzed using BlueFuse Multi software (Illumina).

Statistical analysis

Statistical analysis was performed using SPSS Statistics version 22.0. Continuous data are presented as mean ± standard deviation and 95% confidence interval (CI) or median (minimum–maximum). Categorical variables are presented as n (%) and 95% CI. Data were analyzed by Chi-square or Fisher's exact test and odds ratio. P < 0.05 was considered as statistically significant.

   Results Top

From January to June 2018, TE biopsy with NGS was performed on 33 3PN blastocysts obtained from 18 couples who underwent IVF/ICSI at Yasmin IVF Clinic. Of the 33 blastocysts, two were degenerated and could not be biopsied, and one produced an inconclusive result due to DNA amplification failure. Hence, we analyzed 30 3PN embryos from 16 couples. The general characteristics of the 16 couples are provided in [Table 1]. The mean female age was 32.56 ± 2.73 years, and the most common causes of infertility were male factors (n = 6), namely oligozoospermia (n = 3), oligoasthenozoospermia (n = 2), and teratozoospermia (n = 1), while the female factors were polycystic ovary syndrome (n = 5), unexplained infertility (n = 3), and endometriosis (n = 2). The frequency of 3PN in each patient ranged from 4.55% to 46.15% with a median of 11.11%. Of the 30 embryos, 10 had normal/euploid chromosomes (33.3%) and 20 had abnormal chromosomes (66.7%), most of which were triploids (43.3%). The detailed distribution of chromosomal status is shown in [Table 2]. No correlation was observed between female age or infertility causes and chromosomal abnormality in the 3PN embryos [Table 3].
Table 1: General characteristics of the couples who donated tripronuclear blastocysts after undergoing in vitro fertilization/intracytoplasmic sperm injection (n=16)

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Table 2: Chromosomal status of the 30 tripronuclear embryos used in the study

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Table 3: Chromosomal abnormality according to female age and infertility causes

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Chromosomal status of tripronuclear embryos according to cleavage stage morphology

The morphological criteria of 3PN embryos on day three was evaluated based on blastomere numbers, symmetry, and degree of fragmentation. Embryos were graded according to the scoring system of Veeck.[15] We found that all Grade III 3PN embryos (100%, n = 3) had abnormal chromosomal status, while the proportions of Grade II and Grade I 3PN embryos with abnormal chromosomal status were 75% and 66.7%, respectively. However, we did not see any difference between the groups (P = 0.467) [Figure 1].
Figure 1: Chromosomal status of tripronuclear embryos on day 3 according to Veeck's classification criteria

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As shown in [Table 4], the chromosomal abnormalities in the 3PN embryos according to the the number of blastomeres were not significantly different between the three groups (P = 0.845). We also found no significant relationship between the symmetry of the blastomeres or degree of fragmentation with chromosomal abnormalities' potential (P = 0.440 and P = 0.682 respectively), although embryos with unequal size and >10% fragmentation were likely to have abnormal chromosomal compositions.
Table 4: Chromosomal status of tripronuclear embryos in relation to blastomere size, symmetry, and degree of fragmentation

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Chromosomal status of tripronuclear embryos according to their blastocyst stage morphology

Unlike the cleavage stage, all morphological variables of the 3PN blastocysts were associated with their chromosome status. Blastocysts displaying an expansion degree <3 had a higher probability of being chromosomally abnormal (90%, odds ratio [OR] = 25, 95% CI: 1.8–333.3, P = 0.013), compared with expansion degree 4, as shown in [Figure 2].
Figure 2: Chromosomal status of tripronuclear blastocysts according to the expansion degree. *P < 0.05

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We observed that all non-Grade A ICM blastocysts were chromosomally abnormal, and there was a significantly higher euploidy rate in embryos with a morphologically Grade A ICM (41.1%, P = 0.001) [Figure 3]. Similarly, blastocysts that had the high-quality TE scores had a significantly greater chance of being chromosomally normal than those with low TE scores. Thus, a non-Grade A TE was highly associated with an increase in the incidence of chromosome abnormality (93.3%, OR = 21.00, 95% CI: 2.15–204.61, P = 0.001) [Figure 4]. Our observations showed that the high-quality blastocysts of 3PN embryos had the highest probability of being chromosomally normal.
Figure 3: Chromosomal status according to the intracellular mass quality of tripronuclear blastocysts. **P < 0.01

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Figure 4: Chromosome status according to the trophectoderm quality of tripronuclear blastocysts. *P < 0.05

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   Discussion Top

In ICSI cycle, 3PN may occur due to digynic fertilization attributed to the second meiosis failure and nonextrusion of the second polar body. The present study was conducted in the ICSI cycle with the incidence rate of 3PN occurrences ranging from 4.55% to 46.15% with a median of 11.1% per cycle. This incidence rate is slightly higher than the rates reported by Macas et al.[11] and Staessen and Van Steirteghem,[2] respectively. However, comparable results were obtained in a recent study conducted by Matt et al. that showed 10% incidence of 3PN rate in IVF patients with sperm donors.[16]

Embryos derived from 3PN zygotes have a greater risk of chromosomal abnormalities, especially the risk of triploidy. However, 3PN embryos may have normal in vitro developmental potential. Moreover, previous studies have shown that the proportion of diploid 3PN embryos was relatively high, so they could be considered for reproductive purposes.[2],[7],[10],[13] Two recent publications have even reported live births from 3PN embryos after chromosomal screening using NGS and array comparative genomic hybridization.[14],[16] However, none of these studies analyzed the relationship between chromosome status of 3PN embryos and the morphological parameters of their development. The present study is a preliminary study that aimed to investigate the relationship between chromosome status of 3PN embryos and standard morphological parameters. We used NGS to assess the chromosome status of 3PN biopsied blastocysts. The overall numbers of normal chromosome were 33.3%, which is lower than that reported by Grau et al. in 2011 (52%).[10] The remaining chromosomes were abnormal with the largest composition of triploidy (43.3%). The differences in numbers of normal chromosomes may be because different techniques were used, i.e., NGS versus fluorescent in situ hybridization (FISH). One of the limitations of FISH is its inability to identify abnormalities in all chromosomal regions. We found a higher euploidy rate (25%) than the one reported by Chen et al.[13] This apparent discrepancy may be because of the different diagnostic tools used in the previous study (FISH) and also because of the different timings of the 3PN embryo biopsies (cleavage stage). As explained, diploid zygotes originating from ICSI may be due to the mechanism of ploidy natural correction that occurs in the early development of 3PN embryos before embryonic genomic activation.[10],[17] Other theories have suggested that an inadequate number of centrioles relative to the number of pronuclei prevents all three pronuclei apposition.[18] In addition, the excess numbers of pronuclei after ICSI are derived mostly from the second polar body, which does not contain genetic material, hence resulting in the diploid embryos.[13] Another explanation is the possible presence of haploid nucleus extrusion, which subsequently undergoes degeneration followed by normal division involving a bipolar spindle.[19]

Numerous previous cytogenetic studies, such as FISH and karyotype analysis, in 3PN embryos revealed that triploid, diploid, or severely abnormal karyotypes could occur. In the present study, we found that the majority of chromosomal abnormalities of 3PN zygotes in ICSI were triploidy (43.3%), followed by mosaicism (13.3%), and aneuploidy (10%). These results are consistent with several studies that compared abnormal chromosomal complement in 3PN embryos resulting from the ICSI cycle with the complement from conventional IVF.[2] Because only one sperm centriole plays a role in the mitotic division in ICSI, the high incidence of triploids indicates a regular pattern of bipolar division resulting in regular chromosomal segregation and two triploid symmetrical blastomeres. Whereas, complex mosaic chromosomes in 3PN embryos most likely originate from abnormal spindle formation, which leads to chaotic division in the first mitosis. This condition occurs frequently in conventional IVF due to dispermic fertilization or diploid sperm fertilization because there are two sperm centrosomes that can form tripolar spindles giving rise to irregular chromosome distribution.[2]

Unlike previous studies that mostly performed cytogenetic analyses in the zygote stage, day 3, or both day 3 and day 5,[10],[13],[14],[20] we performed the chromosome analysis in the blastocyst stage only, with the consideration that embryo genomic activation occurs when the embryo reaches the 4–8 cell stage.[21] By extending the embryo culture into the blastocyst stage, the cytogenetic results obtained are the actual chromosomal status of the embryo. We found no relationship between the 3PN embryo morphology at the cleavage stage and the chromosomal abnormality, whereas high-quality blastocysts were significantly related to a normal chromosome. Other studies have searched for an association between chromosome status and embryo morphology. For example, embryos at the cleavage stage were selected on day 3 according to morphological criteria, which consisted of blastomere numbers, fragmentation, and blastomere symmetry.[22],[23],[24] However, most of these studies were performed in advanced-age populations and used FISH, which might lead to an inaccurate classification of euploid embryos. Hence, none of the morphological parameters were found to be strong predictors of chromosomal status at the cleavage stage. Several recent studies have investigated links between morphological criteria at the cleavage stage and aneuploidy using 24 chromosome screening methods.[25],[26],[27],[28] Some of them concluded there was a relationship between the number of blastomeres and degree of fragmentation with aneuploidy rate.[25],[27] However, two recent studies revealed that embryo morphological criteria on day 3 was a weak predictor of embryo ploidy.[27],[28] Similarly, we observed no relationship between 3PN embryo morphology on day 3 according to Veeck's criteria and the chromosome status.

Although not significantly different, we found a tendency of increased chromosomal abnormalities 1.6 times higher in the slow-cleaving embryos (<7 cells) and 1.2 times in the fast-cleaving embryos (>9 cells) compared with the normal-cleaving embryos. This result is in accordance with a report by Phan et al. who observed significantly increased incidence (1.5–1.2 times) of chromosomal abnormalities in slow (<7 cells) and fast (>9 cells) development embryos on day 3 of the cleavage stage.[25] Kroener et al. also confirmed that chromosomal abnormalities increased in embryos <7 cells and >9 cells compared with embryos 7–9 cells, with the highest proportion chromosomal abnormalities in embryos >9 cells.[26]

The chromosomal composition of 3PN embryos is related to the regularity of chromosomal segregation. After ICSI, the majority of 3PN embryos that develop into uniform triploid blastomeres are a result of regular chromosomal segregation.[29],[30],[31] This is also evident from our results that found some equal blastomeres (58.8%) had abnormal chromosomes instead of normal chromosomes, with triploidy accounting for most of the chromosomal abnormalities. Unlike regular segregation, mosaicism and aneuploidy are most likely manifested by irregular mitotic division patterns. This is confirmed by our result, which demonstrated that most unequal 3PN embryos (76.9%) have chromosomal abnormalities. Although not significant, the unequal blastomere group had a 2.3 times likelihood of chromosomal abnormality compared with the equal blastomere group. This result is comparable to that reported by Phan et al. who revealed that the rate of aneuploid embryos (81.6%) was higher in the unequal blastomere group.[25] Similarly, a significant increase in mosaic, polyploidy, and haploidy numbers along with an increase in the percentage of fragmentation was reported.[22],[32] Contrary to these results, we found no statistically significant differences in chromosomal abnormalities between groups with fragmentation <10% and 10%–25% despite abnormal chromosomes being more prevalent in embryo with 10%–25% fragmentation. However, we found a lower fragmentation threshold (0%–25%) than previous studies. Interestingly, slight fragmentation (<20%) does not seem to affect human embryonic development. Using a time-lapse method, it was shown that fragmentation was a dynamic process in which fragments can be reabsorbed into newly cleaved blastomeres, indicating that fragmentation may be a part of normal embryo development.[33],[34],[35] However, moderate to severe degrees of fragmentation are often associated with multinucleation of blastomeres and chromosomal abnormalities, most often a mosaic chromosome that may affect the implantation process.[22],[32],[36],[37],[38]

Unlike the cleavage stage, we observed a strong association when blastocyst stage morphology was correlated with chromosome abnormality. All morphological parameters of a blastocyst stage 3PN embryo were predictive of chromosome abnormality, especially ICM and TE. We observed that all embryos with a non-Grade A ICM were chromosomally abnormal. Likewise, high abnormal chromosome numbers were found in 3PN embryos with a non-Grade A TE score (~90%, P < 0.001). Our results are consistent with Honnma et al. who investigate direct association between TE quality and clinical outcomes with higher rate of miscarriages observed in Grades B and C TE compared with Grade A, influenced by chromosomal abnormalities.[39]

   Conclusion Top

Our analysis revealed that only blastocyst morphology had a strong association with the potential for chromosomal abnormalities in 3PN embryos. Thus, if no 2PN euploid embryo is obtained after ICSI, a carefully selected 3PN embryo could be considered for reproductive purposes following chromosomal analysis and euploid confirmation, and after proper counseling to the recipients. Because the chromosomal abnormalities rate was still high despite the embryo having good morphological characteristics in the cleavage stage, the traditional morphological assessment of the cleavage stage based on standard assessment cannot be relied upon in selecting embryos. Conversely, the morphological parameters of the blastocyst could be used when selecting 3PN embryos because most good-quality blastocysts tended to have normal chromosomes.

A limitation of our study is the relatively small number of samples. As a result, we did not find a relationship between the morphological parameters of 3PN embryos at the cleavage stage and chromosomal abnormalities. However, our results did demonstrate a statistically significant relationship between the chromosomal abnormalities' potential and the quality of blastocysts.


The authors wish to thank the clinical, the paramedical, and the laboratory team of the Yasmin Fertility Clinic, Cipto Mangunkusumo Hospital and the 3rd ICE on IMERI Committee who had supported the peer review and manuscript preparation before submitting to the journal.

Financial support and sponsorship

The 3rd ICE on IMERI committee supported the peer review and manuscript preparation of this article.

Conflicts of interest

There are no conflicts of interest.

   References Top

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  [Figure 1], [Figure 2], [Figure 3], [Figure 4]

  [Table 1], [Table 2], [Table 3], [Table 4]


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