Introduction
The development of multicellular organisms is regulated not only by cell proliferation and differentiation but also by the elimination of unwanted cells with minimal disruption to the organism (Liu et al., Reference Liu, He, Mele, Veeck, Davis and Rosenwaks2000). Apoptosis is a well known mechanism by which such unwanted cells are removed and, therefore, is important in normal embryonic development as well as the elimination of abnormal cells to sustain tissue homeostasis. Aneuploidy in preimplantation embryos mostly results in implantation failure or miscarriage in the first trimester with the exception of some syndromic live births, such as Down’s syndrome or Turner’s syndrome (MacLennan et al., Reference MacLennan, Crichton, Playfoot and Adams2015). Therefore, any change in the number of chromosomes in the sperm or oocyte can affect the outcome of a pregnancy (MacLennan et al., Reference MacLennan, Crichton, Playfoot and Adams2015). Genome integrity is based on the homogenous distribution of replicated chromosomes to daughter cells during cell division. Apoptosis is a key process in ensuring this genomic integrity and escape from apoptotic pathways often results in neoplastic growths. The main mechanism of apoptosis is based on cytochrome c release from the mitochondria that is regulated tightly by Bcl2 and Bcl2 family proteins. Some members of this family, such as Bcl2, Bcl-XL and MCl-1, carry anti-apoptotic features whereas BAX and BAK are well known pro-apoptotic members of the family (Nguyen et al., Reference Nguyen, Marcellus, Roulston, Watson, Serfass, Murthy Madiraju, Goulet, Viallet, Bélec, Billot, Acoca, Purisima, Wiegmans, Cluse, Johnstone, Beauparlant and Shore2007). BAX and BAK are members of the Bcl-2 family and core regulators of the intrinsic pathway of apoptosis. Upon apoptotic stimuli, they are activated and oligomerized at the mitochondrial outer membrane to mediate its permeabilization, which is considered a key step in apoptosis (Clarke and Allan, Reference Clarke and Allan2009).
Apoptosis is triggered when pro-apoptotic family members with a BH3 domain, such as BAD, BID, and BIM, are activated during cellular stress via changes in expression or through post-translational modifications. Then pro-apoptotic proteins, BAX and BAK, induce changes in the mitochondrial outer membrane permeability leading to the release of cytochrome c from the organelle’s intermembrane space. Free cytochrome c then forms a complex with Apaf-1 called the apoptosome, which activates caspase-9 and triggers a cascade of apoptotic events (Pop et al., Reference Pop, Timmer, Sperandio and Salvesen2006; Riedl and Salvesen, Reference Riedl and Salvesen2007; Taylor et al., Reference Taylor, Cullen and Martin2008; Clarke and Allan, Reference Clarke and Allan2009).
Mitochondria play an active role in apoptosis. First, most Bcl-2 family protein/protein interactions take place at mitochondria. Second, BAX and BAK form toroidal pores composed of proteins and lipids at the mitochondrial outer membrane, suggesting that mitochondrial composition participates in pore formation. Third, a crucial step in apoptosis execution is the release of intermembrane space proteins to the cytosol. Fourth, BAX/BAK-induced mitochondrial outer membrane permeability in the absence of caspases triggers the release of mitochondrial DNA, leading to the production of type I interferons and causing a pro-inflammatory type of cell death. Therefore, it is likely that mitochondrial architecture, lipid composition and protein constitution are key elements in the complex regulation of apoptosis. It is well known that BAX and BAK colocalize in specific mitochondrial apoptotic foci upon activation (Clarke and Allan, Reference Clarke and Allan2009).
During cell division, duplicated chromosomes must be segregated accurately into daughter cells to prevent aneuploidy. The organization of microtubules into a bipolar mitotic spindle structure, the proper attachment of chromosomes to spindle microtubules at kinetochores and an appropriate length of time in mitosis, to ensure that all chromosomes correctly attach to spindle microtubules, are required for normal chromosome segregation (Orr et al., Reference Orr, Godek and Compton2015). The spindle assembly checkpoint (SAC) is a highly controlled signalling network that delays mitotic progression until all kinetochores are connected to spindle microtubules, therefore promoting correct chromosomal segregation. The SAC is crucial for cell and organism viability in higher eukaryotes to prevent chromosomal missegregation. However, abnormal SAC functioning allows for premature cell-cycle advancement to anaphase and significantly raises the risk of whole-chromosome missegregation, which leads to aneuploidy (Orr et al., Reference Orr, Godek and Compton2015). The molecular mechanism of SAC activation involves the kinetochores of both mitotic and meiotic chromosomes being enriched with BUB and MAD proteins (Maciejewska et al., Reference Maciejewska, Polanski, Kisiel, Kubiak and Ciemerych2009). Mitotic arrest deficiency has been shown to be associated with MAD2, which is a key player in the SAC signal transduction cascade (Shi et al., Reference Shi, Hu, Luo, Liu, Jiang, Zhang, Wang, Yan and Weng2011). Furthermore, it has been shown that if the SAC pathway is not satisfied for an extended period of time, the members of this checkpoint are capable of triggering apoptotic signalling cascades to avoid aneuploidies and introducing genomic instability to the individual. Previous research predominantly suggests that cyclin B levels constantly drop when SAC is triggered. Once a certain threshold is obtained for cyclin B it can induce apoptosis by inducing caspase 9 and downstream apoptotic signals. Moreover, cyclin B is theorized to induce further upstream apoptotic signals that in turn activate mitochondrial pathways of apoptosis, namely BAX and BAK (Brito and Rieder, Reference Brito and Rieder2006; Clarke and Allan, Reference Clarke and Allan2009). Conversely, cyclin B is not the only SAC pathway member that can induce apoptosis, as MAD2 has also been suggested to show pro-apoptotic characteristics in addition to its role within the SAC pathway (Cheung et al., Reference Cheung, Jin, Ling, Wong, Wang, Tsao and Wang2005; Vogel et al., Reference Vogel, Hager and Bastians2007; Wang et al., Reference Wang, Yin, Du, Chen, Liang, Zhang, Du, Wu, Ding and Fan2010).
In recent years, optimization of culture medium and passage numbers has improved blastocyst formation and pluripotency while reducing rates of apoptosis in IVF clinics (Park et al., Reference Park, Oh, Ji, Choi, Kim, Kim and Kim2022). However, aneuploidies are still far from complete elimination in ART approaches and cause significant rates of miscarriages and/or failure of treatment due to implantation disruptions. Additionally, even in natural conception, aneuploidies can be observed in ∼4–5% of clinical pregnancies, causing miscarriages (Fragouli et al., Reference Fragouli, Alfarawati, Spath, Jaroudi, Sarasa, Enciso and Wells2013). While the causes of aneuploidy in IVF settings have been investigated previously, how are these aneuploidies countered by the growing embryo or how can they persist are questions remain to be firmly answered. Current theories of aneuploidy removal at the blastocyst-stage embryos involve the exclusion of aneuploid cells from compaction or ‘passive’ removal of aneuploid cells due to developmental arrest and stunted viability (Fragouli et al., Reference Fragouli, Alfarawati, Spath, Jaroudi, Sarasa, Enciso and Wells2013; Lagalla et al., Reference Lagalla, Tarozzi, Sciajno, Wells, Di Santo, Nadalini, Distratis and Borini2017, Reference Lagalla, Coticchio, Sciajno, Tarozzi, Zacà and Borini2020). Nevertheless, in somatic cells, DNA damage or aneuploidies are predominantly removed by apoptotic pathways. CHEK1-mediated cell-cycle arrest and consequent apoptosis activation are usually the first response against DNA damage. In contrast, the SAC pathway is generally the respondent during the mitotic phase and in the presence of aneuploidies (Clarke and Allan, Reference Clarke and Allan2009). Failure to remove abnormal cells via such pathways usually results in carcinogenesis. In embryos, apoptosis has been known to occur in response to DNA damage (Singla et al., Reference Singla, Iwamoto-Stohl, Zhu and Zernicka-Goetz2020; Zhang et al., Reference Zhang, Xie, Li, Song, Li, Li, Li, Fan, Zhou, Wang, Sun and Ou2023). However, an apoptotic reply in response to aneuploidies is yet to be confirmed. Similarities between carcinogenic pathways and embryogenic pathways suggest that they can be within the realm of possibilities (Ma et al., Reference Ma, Zhang, Wang, Yang, Yang and Qin2010; Smith and Sturmey, Reference Smith and Sturmey2013; Manzo, Reference Manzo2019). Therefore, this study aimed to determine the relationship between the expression of genes involved in apoptosis in human aneuploid and euploid blastocysts, thereby gaining an understanding of mechanisms that may influence aneuploidies in embryos.
Materials and methods
Ethical approval was granted by the Near East University Scientific Research Ethics Committee (YDU/2021/96–1432). All patients were informed about the details of the study and provided written consent when they graciously accepted to contribute. The samples were human surplus embryos from patients attending the British Cyprus IVF Centre, Nicosia Cyprus. In total, 32 samples obtained from 21 patients were involved in this study. Of these 32 samples, 13 blastocysts were euploid (control) and 19 blastocysts were aneuploidy, respectively. Next-generation sequencing (NGS) was performed for the detection of aneuploidy via preimplantation genetic testing for aneuploidy (PGT-A). Details of the aneuploidies of samples included in this study can be found in Table 1.
Table 1. List of aneuploidy status of the study sample population
Briefly, first the female partners underwent controlled ovarian stimulation and oocytes were obtained, followed by intracytoplasmic sperm injection (ICSI). The embryos that were developed to the blastocyst stage were biopsied and NGS was performed. Embryos included in this study showed no signs of structural defects such as translocations, deletions, duplications, inversions nor any other major anomalies aside from various aneuploidies.
Ovarian stimulation and embryo development
Controlled ovarian hyperstimulation (COH) was performed using a GnRH antagonist protocol. On the day of the menstrual cycle, recombinant FSH (150–300 IU, Gonal F, Serono) and/or hMG (75–150 IU, Merional, IBSA) were administered. From the sixth day of stimulation, the ovarian response was monitored by transvaginal ultrasound and by measuring the level of serum progesterone (P4) and estradiol (E2). The daily administration of 0.25 mg GnRH antagonist (Cetrotide, Serono) was applied until the day of ovulation trigger when the leading follicle size exceeded 13 mm. For the ovulation trigger, 250 mg hCG (Ovitrelle, Serono) or 0.2 mg triptorelin (Gonapeptyl, Ferrin) was administered and oocyte retrieval was planned 35 h after the trigger administration. The oocytes were denuded with 1/3 v/v hyaluronidase (hyaluronidase, 90101, Irvine Scientific, USA). Semen samples were prepared as described by Coban et al. (Reference Coban, Serdarogullari, Yarkiner and Serakinci2020) and the selected sperm was injected into the oocyte. A fertilization check was performed on the next day (16–18 h post-ICSI) and two pronuclei with two polar bodies were considered to be a normal fertilization case (Alpha Scientists in Reproductive Medicine and ESHRE Special Interest Group of Embryology, 2011). For the day 3 embryo morphology assessment, the grading system including the analysis of the number of blastomeres, blastomere evenness, degree of fragmentation, nucleus details and cytoplasmic characteristics, was used as described by Ciray et al. (Reference Ciray, Aksoy, Goktas, Ozturk and Bahceci2012). Morphological assessment of the embryos at the blastocyte stage (days 5 and 6) were performed as described by Gardner and Schoolcraft (Reference Gardner and Schoolcraft1999). Embryos at the blastocyst stage were biopsied and the blastocysts were vitrified (Vit Kit-Freeze; 90133-SO, Irvine Scientific, USA). The biopsied samples were processed for aneuploidy screening by NGS in Igenomix. The genetic diagnosis services were carried out by IGENOMIX FZ LLC. Aneuploidy screening was performed by Ion ReproSeqPGS Kit analyzing all 24 chromosomes using Ion S5 System instruments (Thermo Fisher Scientific, Inc., MA, USA) and the data were analyzed by Ion Reporter (IRv5.4) software.
The spare aneuploid embryos and the non-transferred euploid embryos obtained from patients who had consented to research were collected for this project. Briefly, DNA and RNA were extracted from the single blastocysts and cDNA was synthesized using the RNA samples. The expression levels of genes involved in the apoptosis pathway were analyzed by real-time PCR.
Nucleic acid extraction, cDNA synthesis, and real-time PCR analysis
Nucleic acid extraction was performed using the Hibrigen total nucleic acid isolation kit (Hibrigen, Turkey, cat. No. MG-TNA-01-10) following the manufacturer’s instructions. The purity and concentration of the extracted RNA were estimated using a NanoDrop spectrophotometer following the manufacturer’s protocol (Thermo-Scientific, Pittsburgh, USA). The Hibrigen cDNA synthesis kit (Hibrigen, Turkey, cat. No. MD-CDNA-01–100) was used for reverse transcription following the manufacturer’s instructions. The LightCycler® 480 SYBR Green I Master kit (Roche, Germany, ref. no. 04707516001) was utilized for real-time PCR following the manufacturer’s protocol. Melting curve analysis was performed following each real-time PCR to differentiate between the primer dimer and the product, respectively. Primer sequences are listed in Table 2 and PCR conditions are shown in Table 3. The ACTB gene was used as a housekeeping gene for normalization. In cases in which an aneuploidy would affect the expression level of a gene directly, those samples were excluded from the analysis. For example, samples 6 and 10 were excluded from the analysis of BAX expression for having an extra chromosome 19 and missing one respectively, as BAX is situated on chromosome 19. Therefore, BAX expression analysis was conducted with 17 aneuploid embryos and 13 euploid samples in total.
Table 2. List of primer sequences
Table 3. Real-time PCR conditions
Statistical analysis
GraphPad Prism v8 was used for all the statistical analysis.
Results
In total, 32 surplus blastocyst-stage embryos were used for this study. Trophectoderm biopsies were performed for all the blastocysts and NGS analyses were carried out. Of the 32 blastocysts, 19 were shown to have aneuploidies and the remaining 13 were classified as euploid. In this study, the possible regulatory effect of genes involved in apoptosis and the aneuploidy status of the embryos were investigated.
Briefly, the gene expression levels of BAX, BAK, and MAD2L1, which are involved in apoptosis, were analyzed by real-time PCR in euploid and aneuploid human blastocysts, respectively. The Ct values of all the analyses remained between 17–25 for MAD2L1, 24–28 for BAK and 21–29 for BAX as shown in Table 4. The expression level of the BAX gene was shown to be significantly higher in euploid compared with the aneuploid blastocysts as shown in Figure 1 (P < 0.05). Downregulation of BAX expression in aneuploid cells was found to be ∼1.5-fold. The expression levels of the BAK gene were similar in samples obtained from aneuploid and euploid blastocysts samples, respectively (P > 0.05). Last, the expression level of MAD2L1 was slightly higher in euploid blastocysts compared with the aneuploid blastocysts, respectively. However, this difference in the expression level was not statistically significant (P > 0.05).
Table 4. Ct Value that shows the expression level of the genes
Figure 1. Relative expression of BAX in aneuploid embryos compared with euploid embryos. ΔΔCt values were plotted on a logarithmic scale. When compared with euploid embryo cells, BAX expression shows an ∼1.5-fold decrease in aneuploid cells, suggesting a repression of apoptotic responses in these cells.
Discussion
Aneuploidy, mutations, or abnormal levels of gene expression during early preimplantation embryo development can disrupt crucial embryonic functions at specific stages and ultimately cause cleavage arrest or widespread apoptosis. Investigation of such genes and their function will surely be useful for gaining a greater understanding of the genetic circuitry of early human development. It will also provide an opportunity for the development of novel screening methods for evaluating genetic risk in reproduction. Therefore, this study aimed to investigate the relationship between the expression levels of genes involved in apoptosis in aneuploid and euploid human embryos.
Chromosome segregation failures in female meiosis result in aneuploidy in the developing oocyte and embryo, making them one of the primary genetic causes of spontaneous abortions and developmental disorders in humans. It is well acknowledged that aneuploidy of meiotic origin increases considerably as women age, and current research suggests that the majority of abnormalities originate in meiosis I. It is becoming increasingly clear that abnormalities are mostly tolerated until embryonic genome activation at the blastocyst stage. A study by Fragouli et al. (Reference Fragouli, Alfarawati, Spath, Jaroudi, Sarasa, Enciso and Wells2013) showed that in an IVF setting aneuploidy rates can be observed in up to 83% of embryos at the morula stage that rapidly descends to approximately 60% of embryos when blastocyst-stage samples are analyzed. Several studies about the mechanism of maternal age-related aneuploidy have been presented, including a failed SAC in meiosis I, failures in early meiosis, and low sister chromatid cohesion with age (Chiang et al., Reference Chiang, Schultz and Lampson2012). Therefore, it is very important to investigate the expression levels of genes involved in the SAC not only in oocytes but also in the resulting embryos as potential defects may be persistent in the embryos originating from such oocytes.
Important systems for regulating genomic stability, such as cell arrest, cell-cycle checkpoints, and apoptosis, are lacking or insufficient throughout early human embryo development, resulting in an increased rate of aneuploidies (Mantikou et al., Reference Mantikou, Wong, Repping and Mastenbroek2012). Apoptosis, is required for the human body to operate properly, including cell turnover, immune system development and regulation, embryonic development, and gametogenesis (Vartak et al., Reference Vartak, Iyer, Santhoshkumar, Sharma, Mishra, Goldsmith, Srivastava, Srivastava, Karki, Surolia, Choudhary and Raghavan2017). DNA damage has the potential to affect a wide range of biological activities, including cell-cycle control, DNA repair, and apoptosis (Bazrgar et al., Reference Bazrgar, Gourabi, Yazdi, Vazirinasab, Fakhri, Hassani and Valojerdi2014). Numerous numbers of studies have focused on investigating the expression of genes involved in DNA repair. They discovered that it was increased in low-quality preimplantation human embryos with complicated aneuploidies (Wells et al., Reference Wells, Bermúdez, Steuerwald, Malter, Thornhill and Cohen2005; Jaroudi and SenGupta, Reference Jaroudi and SenGupta2007; Jaroudi et al., Reference Jaroudi, Kakourou, Cawood, Doshi, Ranieri, Serhal, Harper and SenGupta2009; Kakourou et al., Reference Kakourou, Jaroudi, Tulay, Heath, Serhal, Harper and SenGupta2013). As a result, DNA repair pathways are more activated in these embryos compared with cell-cycle control and apoptotic pathways. This suggests that DNA repair, rather than cell proliferation or apoptosis, and may be the main mechanism of DNA damage in poor-quality embryos with complicated aneuploidy (Bazrgar et al., Reference Bazrgar, Gourabi, Yazdi, Vazirinasab, Fakhri, Hassani and Valojerdi2014). More specifically, it has been reported that the base excision repair (BER) mechanism is active in the zygote stage with sperm and oocyte BER enzymes (Lord and Aitken, Reference Lord and Aitken2015). Therefore, oxidative damage is predicted to be repaired. Paternal double-stranded breaks in the embryo are suggested to be repaired by a homologous repair mechanism using the female’s genomic complement (Ma et al., Reference Ma, Marti-Gutierrez, Park, Wu, Lee, Suzuki, Koski, Ji, Hayama, Ahmed, Darby, Van Dyken, Li, Kang, Park, Kim, Kim and Gong2017). Non-homologous end joining does not seem to be active at this stage. After embryonic genome activation, the embryo may be more prone to damage and checkpoint kinase 1 (Chk1) may be involved in the control regulating the cell-cycle arrest (Ma et al., Reference Ma, Marti-Gutierrez, Park, Wu, Lee, Suzuki, Koski, Ji, Hayama, Ahmed, Darby, Van Dyken, Li, Kang, Park, Kim, Kim and Gong2017). DNA damage in the embryos overall may result in repair, implantation failure or passing on to the offspring (Khokhlova et al., Reference Khokhlova, Fesenko, Sopova and Leonova2020). The outcome is influenced by the damage type and severity; in such severe DNA damage may result in abnormal blastocysts with embryonic loss (Shoukir et al., Reference Shoukir, Chardonnens, Campana and Sakkas1998; Dumoulin et al., Reference Dumoulin, Coonen, Bras, Van Wissen, Ignoul-Vanvuchelen, Bergers-Jansen, Derhaag, Geraedts and Evers2000; Seli et al., Reference Seli, Gardner, Schoolcraft, Moffatt and Sakkas2004). BCL-2 family proteins, involved in an anti-apoptotic pathway, are expressed in zygotes as well as blastocysts and therefore in cases in which their expression is impaired, implantation failure may be observed (Opferman and Kothari, Reference Opferman and Kothari2018). Mismatch repair genes, MSH2, MS3 and PMS1, were also shown to be expressed in human oocytes and blastocysts, suggesting that this pathway may also be active (Jaroudi et al., Reference Jaroudi, Kakourou, Cawood, Doshi, Ranieri, Serhal, Harper and SenGupta2009).
In contrast, Singla et al. (Reference Singla, Iwamoto-Stohl, Zhu and Zernicka-Goetz2020) discovered that aneuploid mouse cells generated at the 4–8-cell stage are progressively reduced via apoptosis from the early blastocyst stage to early post-implantation from the mosaic embryo’s epiblast. In diploid cells, cellular protein quality control systems, such as the proteasome machinery and autophagy, remove the misfolded or unfolded proteins to reduce cytotoxicity and enhance healthy cell survival. After several mitotic divisions, chronic protein misfolding upregulates autophagy to the point in which it mediates cell death rather than protects the cell. This prevents the aneuploid cell from advancing further in the formation of the epiblast (Singla et al., Reference Singla, Iwamoto-Stohl, Zhu and Zernicka-Goetz2020). The findings of this study revealed that the BAX gene was expressed significantly differently between aneuploid and euploid embryos, respectively. Therefore, this may suggest that the BAX gene is functioning to activate the apoptotic pathway for the elimination of cells and eventually the whole embryo in euploid cases. Conversely, this mechanism is possibly hindered in aneuploidies. Conversely, there was no statistical difference in the expression levels of BAK and MAD2L1 genes. Therefore, it seems the activation of apoptotic pathways is more complicated in preimplantation embryos.
One of the limitations of this study was that the fragmentation of the embryos was not investigated. Fragmentation can be associated with apoptosis. Apoptotic fragments of the embryos are expected to remain detectable if aneuploid cells are eliminated by apoptotic pathways. Therefore, in future studies, we are aiming to also include this assessment in our study. Another drawback of our study was the limited number of genes investigated. However, further studies are being performed to cover more genes in the apoptosis pathway as well as the spindle checkpoint. Conclusions of this preliminary study suggest that there may be a suppression of BAX-mediated apoptosis in aneuploid embryos however this intercepting mechanism is yet to be identified. This possible prevention of apoptosis might be the factor contributing to the persistence of aneuploidies in embryos that in turn may hinder success rates of pregnancies either by natural conception or via assisted reproductive technologies. Therefore, this study forms the basis of future studies. Further steps might also include the inclusion of translational studies to confirm that decreased BAX transcription is indeed able to exert influence on protein levels, as well as investigating possible suppressors of BAX/BAK-mediated apoptosis such as the Akt pathway and/or Bcl2 and other anti-apoptotic Bcl2 family members.
