Abstract
Graphical abstract
Abstract
Mitochondrial DNA (mtDNA) from embryonic cells is released into the spent culture medium (SCM) during cellular processes, providing a potential biomarker of embryo health. Analysing mtDNA levels in SCM enables a non-invasive evaluation of embryo quality and potential developmental abnormalities. In this retrospective study, we aimed to investigate the relationship between relative mtDNA copy number in embryo SCM and key factors, including embryo fragmentation, morphological quality and chromosomal abnormalities. Fertilised embryos produced through intracytoplasmic sperm injection were cultured to the blastocyst stage in an incubator. Embryo fragmentation was assessed on day 3 using the Istanbul criteria, while morphological grading was evaluated on day 5 using the Gardner criteria. On day 5, trophectoderm (TE) biopsies were performed for preimplantation genetic testing for aneuploidy, followed by embryo cryopreservation and collection of embryo SCM. The mtDNA was quantified using quantitative PCR. Statistical analyses using the Mann–Whitney U and Kruskal–Wallis tests (significance at P < 0.05) showed that relative mtDNA copy number did not significantly differ among embryos with fragmentation levels <10%, 10–25% and >25% (P > 0.05). For blastocyst grading, which evaluates the inner cell mass (ICM) and TE, no significant difference was observed in relative mtDNA copy number between grades B and C for ICM (P = 0.190) and TE (P = 0.289). Furthermore, a trend towards higher relative mtDNA levels was observed in aneuploid than in euploid embryos, although the difference was not statistically significant. Thus, relative mtDNA copy number in SCM may not accurately reflect embryo characteristics, such as fragmentation, morphological grading or chromosomal abnormalities.
Lay summary
This study examined whether the amount of mitochondrial DNA (mtDNA) in the fluid used to culture embryos in the laboratory could indicate embryo quality. We assessed various factors, including the appearance of the embryos, the presence of fragmented cells and the occurrence of chromosomal abnormalities. Fertilized eggs were cultured until they developed into blastocysts, and the amount of mtDNA in the culture fluid was measured using a machine that detects genetic material. The results revealed no clear association between mtDNA levels and embryo appearance or fragmentation. Although embryos with chromosomal abnormalities had slightly more mtDNA, the difference was not statistically significant. These findings suggest that mtDNA in the culture fluid may not be a reliable marker for assessing embryo quality or chromosomal status.
Introduction
Cell-free DNA (cfDNA) refers to DNA fragments released into the bloodstream or bodily fluids, such as cerebrospinal fluid, pleural fluid, urine and saliva, during cell death. It can serve as an indicator of various abnormalities in the body, including autoimmune diseases, inflammation and cancer, and is a valuable diagnostic test in obstetrics. cfDNA-based approach is non-invasive and does not disturb cells or cause cellular damage; it is therefore becoming popular as a promising biomarker. Moreover, it is a simple, repeatable and effective method for monitoring disease progression (Yan et al. 2021, Chen et al. 2022). In obstetrics, one of the most widely adopted applications of cfDNA is non-invasive prenatal testing (NIPT), which screens for foetal genetic abnormalities. This approach involves analysing foetal cfDNA, which is derived from placental cells and circulates in the maternal bloodstream. NIPT enables the detection of foetal chromosomal abnormalities without the need for invasive procedures that may pose a risk to the foetus (Suciu et al. 2019), and is highly beneficial in the field of assisted reproductive technology, particularly for assessing the cfDNA present in embryo spent culture medium (SCM). Preimplantation genetic testing for aneuploidy (PGT-A) is a technique used to detect chromosomal abnormalities in embryos before implantation and enables the selection of viable embryos by identifying embryos with normal chromosomal configurations, thereby increasing the likelihood of successful implantation and reducing the risk of miscarriage (Fodina et al. 2021, Kasaven et al. 2023). This procedure involves embryo biopsy, typically performed on trophectoderm (TE) cells, which are the outer layer of the blastocyst that later forms the placenta (Pan et al. 2020, Tocci 2020). The biopsied cells are then analysed for chromosomal abnormalities, providing critical information to support informed decision-making during assisted reproductive procedures. This approach facilitates the selection of genetically healthier embryos, thereby reducing the need for multiple embryo transfer (ET) attempts and improving the overall efficiency of assisted reproductive treatments (Basille et al. 2009, Fesahat et al. 2020). Biopsy is a widely used but invasive technique for PGT-A, involving the removal of a portion of the embryo, which can compromise its viability, and requires highly skilled embryologists. To address these limitations, non-invasive methods for assessing embryo abnormalities have been developed. Current research suggests that cfDNA in embryo SCM originates from the breakdown of embryonic cells during cell division, particularly due to cellular fragmentation (Stigliani et al. 2013, Brouillet et al. 2020, Shitara et al. 2021). This fragmentation, often associated with genetic abnormalities, such as aneuploidy and mosaicism, may reflect defective embryonic processes and impair subsequent development. Severe fragmentation has also been linked to reduced mitochondrial DNA (mtDNA) levels and altered mitochondrial structures, which can diminish ATP production and impede embryonic growth. The utilization of mtDNA from embryo SCM to evaluate embryo quality and detect chromosomal abnormalities remains a topic of debate among embryologists and researchers. Although it offers a non-invasive alternative, its reliability and clinical significance are still under investigation (Huang et al. 2019, Zhang et al. 2019b , Kobayashi et al. 2020, Sheaba et al. 2022). Some studies suggest a correlation between mtDNA levels and embryo quality, highlighting its potential as a non-invasive alternative to PGT-A (Stigliani et al. 2013, Shamonki et al. 2016), whereas others argue that mtDNA levels may not reliably predict in vitro fertilization (IVF) outcomes and may reflect physiological stress rather than genetic health, potentially leading to misclassification of embryos (Winstanley et al. 2024). Furthermore, the techniques used to extract and analyse cfDNA from embryo SCM are technically challenging and prone to inter-laboratory variability, which could affect the reproducibility and accuracy of the results (Peng et al. 2024). The potential discarding of viable embryos based on genetic indicators that may not definitively determine their developmental potential raises ethical issues (Kaye 2023). The clinical application of mtDNA analysis requires further validation through rigorous, large-scale studies to establish its efficacy and address the existing challenges. Consequently, in the present study, we aimed to investigate the correlation between the relative mtDNA copy number in embryo SCM and embryo quality and chromosomal abnormalities to provide further insights into its potential as a non-invasive biomarker.
Methods
Ethical approval
The protocol for the present study was approved by the Institutional Review Board under approval number SWUEC-M-079/2565E. Informed consent was obtained from all participants before their inclusion in the study. Embryo SCM was collected and analysed.
Enrolment of participants and ovarian stimulation
Eleven infertile women aged 25–45 years underwent intracytoplasmic sperm injection (ICSI) at the Paragon Fertility Clinic, Thailand. The participants were carefully screened to exclude individuals with underlying medical conditions, including diabetes, endocrine disorders, immunological diseases or haematological abnormalities. Controlled ovarian stimulation was performed using human chorionic gonadotropin (hCG) administration to promote follicular development and facilitate oocyte retrieval for IVF using the ICSI technique. Once the follicles reached maturation, ovulation was triggered by injecting hCG. Mature metaphase II oocytes were retrieved and cumulus cells were enzymatically removed using hyaluronidase to ensure optimal preparation for fertilisation via ICSI.
Embryo culture and medium collection
Cumulus cell removal was performed to reduce maternal DNA contamination in SCM. After oocyte retrieval, cumulus cells and corona radiata were removed using ICSI Cumulase® (ORIGIO, Denmark), followed by mechanical pipetting and multiple washes in fresh medium. During ICSI, the embryologist re-examined oocytes to ensure complete removal of cumulus cells. A total of 40 MII oocytes were fertilised using ICSI within an IVF working chamber maintained at 37°C with 5% CO2 and 5% O2 (Pulse Science, Denmark). The resulting zygotes were cultured individually in 20 μL drops of continuous medium (Life Global, Denmark) until the blastocyst stage, which typically occurred over 5–6 days in an incubator. Embryonic development was monitored using an Olympus IX73 inverted microscope. Embryo grading was performed at two key time points. On day 3 post-ICSI, cleavage-stage embryos were evaluated for fragmentation based on the Istanbul criteria (Alpha Scientists in Reproductive Medicine and ESHRE Special Interest Group of Embryology 2011): <10% fragmentation, 10–25% fragmentation and >25% fragmentation. On day 5 post-ICSI, blastocyst stage embryos were assessed according to the Gardner’s criteria (Gardner et al. 2000), focussing on the quality of the inner cell mass (ICM) and TE, which were graded as A, B or C. At the blastocyst stage 3–5, TE cells were extracted via biopsy, collected in 1× phosphate buffered saline and sent to the laboratory for PGT-A using next-generation sequencing techniques. The remaining embryos were cryopreserved for future uterine transfer. In addition, 20 μL SCM from each embryo (n = 40) was carefully aliquoted into DNase- and RNase-free vials and immediately frozen at −80°C for subsequent DNA extraction.
PGT-A
PGT-A was performed by scientists at Next-Generation Genomic Co., Ltd (NGG, Thailand) using 3–5 TE cell samples from each embryo. Library preparation was performed using the EmbryoMap Sample Prep Kit (Vitrolife, Sweden), following the manufacturer’s protocol, and sequencing was performed on a MiSeq platform. The results were reported as copy number variations, providing detailed insights into chromosomal integrity by identifying insertions or deletions across the 23 pairs of chromosomes. These data were used to assess the chromosomal status of the embryos for potential abnormalities.
DNA extraction, amplification and determination of relative mtDNA copy number from SCM
Statistical analysis
The relative mtDNA copy number in fresh medium and embryo SCM was expressed as the median and interquartile range (IQR) and compared using the Mann–Whitney U test performed with the GraphPad Prism (version 10.1). The median of mtDNA copy number of day 5 and 6 blastocysts was compared. The Kruskal–Wallis test was conducted to evaluate the relationship between relative mtDNA copy number and embryo fragmentation. In addition, the association between the relative mtDNA copy number in embryo SCM and blastocyst grade, and chromosomal abnormalities, was assessed using the Mann–Whitney U test. Statistical significance was set at P-value <0.05.
Results
Background of the patient and embryo development
The participants’ mean age was 32.8 ± 5.6 years. The body mass index was 18.9 ± 1.6 kg/m2. The baseline AMH was 4.07 ± 2.89. The average number of OPU cycles was 1.63 ± 0.9, and the cumulative ET was 0.91 ± 0.83. The average number of MII oocyte underwent ICSI was 18.63 ± 7.3 and fertilization rate was 80.7 ± 11.3%. The individual patient’s background is presented in Supplementary Table 1 (see section on Supplementary materials given at the end of the article). Embryo development was assessed on day 3 of culture using inverted microscopy, with fragmentation grading conducted in accordance with the Istanbul criteria. The findings indicated that all embryos had reached the cleavage stage, each exhibiting a varying number of cells, as presented in Supplementary Table 2.
Quantification of relative mtDNA copy number in embryo SCM
Fresh medium not exposed to the embryos (n = 3) that served as the control group. No mtDNA copy numbers were detected in the control samples, whereas variations in relative mtDNA copy numbers were observed in the 40 embryo SCM samples, as presented in the Supplementary Table 3. The relative mtDNA copy number in embryo SCM was significantly higher than that in the fresh medium (P < 0.05), as illustrated in Fig. 2A. Although the number of embryos in the present study was limited, the median of relative mtDNA copy number of day 5 and 6 blastocyst was compared. The results showed no statistically significant difference in mtDNA copy number between the medium collected on day 5 and 6 blastocysts (57.4 (IQR: 110.03) and 41.62 (IQR: 116.46), respectively, (P = 0.770)).
Relative mtDNA copy number and embryo fragmentation
Fragmentation analysis was performed using inverted microscopy (Fig. 1A), with the fragmentation grade of each embryo presented in Supplementary Table 2, and revealed that the median relative mtDNA copy numbers were 40.25 (IQR: 117.5) for embryos with <10% fragmentation (n = 11), 40.41 (IQR: 203.31) for embryos with 10–25% fragmentation (n = 19) and 80.33 (IQR: 128.8) for embryos with >25% fragmentation (n = 10). Statistical analysis showed no significant differences in the mtDNA copy numbers across these fragmentation categories (P = 0.704), as illustrated in Fig. 2B.
Embryo morphology on day 3 and day 5. (A) The degree of embryo fragmentation was assessed using inverted microscopy and categorised into three groups. Scale bar = 100 μm. (B) Blastocyst grading was performed using inverted microscopy. The ICM is indicated by a star, while the TE is marked by a black arrow. Scale bar = 100 μm.
Citation: Reproduction and Fertility 6, 2; 10.1530/RAF-25-0001
Box plots of relative mtDNA copy number and various embryo quality factors. (A) The relative mtDNA copy number in embryo SCM was significantly higher than that in the fresh medium (*P < 0.05). (B) The relative mtDNA copy number across the three fragmentation categories shows no significant differences. The relative mtDNA copy number with both the blastocyst ICM (C) and TE grades (D) shows no significant differences. (E) The relative mtDNA copy number with preimplantation genetic testing for aneuploidy revealed no significant differences.
Citation: Reproduction and Fertility 6, 2; 10.1530/RAF-25-0001
Relative mtDNA copy number and blastocyst grading
The present study utilised inverted microscopy to assess blastocyst quality, focussing on two key components: the ICM and TE, as illustrated in Fig. 1B. Due to the random selection of samples, there were insufficient embryos with grade A in both the ICM and TE grading categories (n = 1) for statistical analysis. Grade A embryos were excluded and statistical comparisons were conducted only between grade B and C embryos. For ICM, the median relative mtDNA copy number was 7.9 (IQR: 122.51) for grade B embryos (n = 22) and 58.62 (IQR: 100.94) for grade C embryos (n = 17). For TE, the median relative mtDNA copy number was 58.62 (IQR: 119.38) for grade B embryos (n = 25) and 17.32 (IQR: 115.98) for grade C embryos (n = 14). Statistical analysis indicated no significant differences in the relative mtDNA abundance in the embryo SCM between the morphological quality grades of blastocysts, specifically for ICM (P = 0.190) and TE (P = 0.289), as shown in Fig. 2C and D.
Relative mtDNA copy number and PGT-A
The PGT-A results for each embryo are summarised in Supplementary Table 3. The median relative mtDNA copy number was 40.33 (IQR: 65.49) for euploid embryos (n = 18) and 80.24 (IQR: 120.86) for aneuploid embryos (n = 22). Although statistical analysis indicated a trend towards higher relative mtDNA levels in aneuploid embryos than in euploid embryos, this difference was not statistically significant, as illustrated in Fig. 2E.
Discussion
The use of embryo SCM to evaluate embryo abnormalities offers a non-invasive alternative to traditional embryo biopsy techniques. This approach was developed to minimise the potential damage associated with biopsies, which can impair embryonic growth or result in other abnormalities. Embryo SCM contains several components, including cfDNA, RNA, microRNA and various metabolites, which may provide valuable insights into embryo quality, implantation potential and chromosomal abnormalities. This study focussed on cfDNA, which comprises both gDNA and mtDNA. Notably, the amount of mtDNA is higher than that of gDNA, highlighting its potential as a key marker for assessing embryo characteristics (Hammond et al. 2017). Scientists hypothesise that elevated mtDNA levels in embryo SCM may result from cellular breakage during division, which leads to the release of cellular organelles, including mitochondria. This hypothesis aligns with the findings of Stigliani et al. (2013) who reported a correlation between high mtDNA copy numbers in embryo SCM and severe fragmentation. Fragmentation occurs during the cleavage stage of embryonic development due to irregular cell division, resulting in portions of the cytoplasm pinching off. This process can lead to the loss of critical organelles, such as mitochondria, which are essential for cellular energy production, and may negatively affect embryonic development. However, in the present study, no significant association was found between mtDNA copy number and fragmentation. This discrepancy may be attributed to differences in the timing of embryo SCM collection; in our study, it was collected on day 5 after the embryos were cultured, further following fragmentation grading on day 3. This delay may have allowed additional developmental processes to occur, potentially altering the mtDNA levels through secretion or reabsorption by blastomeres. In contrast, Stigliani et al. (2013) collected embryo SCM immediately after fragmentation grading on day 3, likely capturing a more accurate relationship between mtDNA levels and fragmentation status. This discrepancy highlights the importance of standardising sample collection protocols for mtDNA analysis in future research (Stigliani et al. 2013). Therefore, the timing of medium collection modulates DNA concentration in SCM governed by embryonic developmental stage, cellular turnover and embryo quality. Advanced-stage embryos, such as day 6 blastocysts, may exhibit increased DNA release, while compromised embryos may shed more due to cellular fragmentation. However, this trend was not observed in our study, consistent with findings by Hammond et al. (2017) and Kobayashi et al. (2020).
In this study, we found that the relative mtDNA copy number was not associated with fragmentation grade, morphology grade or chromosomal abnormalities in embryos. This may be attributed to the mechanism of mtDNA replication during embryonic development. Before fertilisation, oocytes accumulate large numbers of mitochondria and mtDNA copies to provide sufficient energy for early development. After fertilisation, mtDNA levels in the embryo decrease until the blastocyst stage (Piko & Taylor 1987). At the blastocyst stage, mtDNA replication factors, which reactivate mtDNA replication, are upregulated. However, replication occurs primarily in TE cells, which form the placenta and mediate implantation for further embryonic development (St John et al. 2010). This finding aligns with that of Wang et al. (2021) who observed an association between mtDNA content and TE quality but not with ICM. Similarly, Klimczak et al. (2018) noted a relationship between mtDNA content and embryo morphological grade using TE biopsy. However, in our study, no association was found between mtDNA content and embryo morphology grades, which was consistent with other studies (Zhang et al. 2019a , Kobayashi et al. 2020, Ritu et al. 2022), but contradicted the findings of Wang et al. (2021) and Klimczak et al. (2018). This discrepancy may have occurred as we included only moderate- and poor-quality embryos. Consequently, the differences in mtDNA quantities across the groups were not statistically significant.
Regarding chromosomal abnormalities, previous studies have reported a correlation between mtDNA copy number and aneuploidy using blastomere biopsies (Tao et al. 2017, Ritu et al. 2022). This supports the ‘quiet embryo hypothesis’, which posits that embryos under stress or with limited energy reserves upregulate mitochondrial replication early to meet increased energy demands at the blastocyst stage (Cecchino & Garcia-Velasco 2019). Although this study found a trend towards higher mtDNA copy numbers in the aneuploid group, this difference was not statistically significant. These findings were consistent with those reported by Victor et al. (2017) and Zhang et al. (2019a). However, Fragouli et al. (2015) reported a significant association between mtDNA copy number and chromosomal abnormalities. This inconsistency may be due to the small sample size of this study (n = 40), which limited the statistical power to detect significant differences. Expanding the sample size in future studies may yield more conclusive results. Another potential factor contributing to the lack of correlation between mtDNA copy number in the embryo SCM and the embryo itself is contamination by maternal DNA (Hammond et al. 2017). Maternal DNA from incompletely removed cumulus cells may confound mtDNA measurements. Enhanced denuding techniques to ensure the complete removal of cumulus cells could improve the accuracy of the results. In addition, the sensitivity of the real-time PCR technique used for mtDNA detection in this study may have been insufficient. Employing digital PCR, which is a more precise and sensitive technique, could improve the detection of mtDNA in embryo SCM.
Conclusion
Based on the findings of this study, the relative mtDNA copy number in embryo SCM may not serve as a reliable biomarker for assessing blastocyst morphology or chromosomal abnormalities. Although a trend towards higher mtDNA levels was observed in the aneuploid group, its clinical utility remains uncertain. Factors, such as maternal DNA contamination, small sample sizes and limitations in mtDNA quantification techniques, may have contributed to these results. Future studies should aim to increase the sample size, minimise maternal DNA contamination through improved cumulus cell removal techniques and adopt more sensitive methods, such as digital PCR, to enhance the reliability and accuracy of mtDNA analysis in embryo SCM.
Supplementary materials
This is linked to the online version of the paper at https://doi.org/10.1530/RAF-25-0001.
Declaration of interest
The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported.
Funding
This work was supported by the Faculty of Medicine, Srinakharinwirot University (grant numbers, MED-RES-200; 265/2565, MED-STUDENT-50; 266/2565, MED-GRAD-150; 279/2567). RR is supported by Innovation and the Strategic Wisdom and Research Institute of Srinakharinwirot University (grant number 106/2568).
Author contribution statement
The study was conceptualized and designed by ST, AN and RR, who also contributed to data analysis and interpretation. Data collection and experiments were conducted by ST, KW, NN, KW, PB and PK, with PA handling sample preparation. ST, KW, NN, KW, PB, PK, AN, SP and RR contributed to data analysis, interpretation and manuscript preparation.
Acknowledgement
We would also like to thank the embryologists from Paragon Fertility Clinic in Bangkok, Thailand, for their kind assistance in collecting the spent embryo culture medium.
References
Alpha Scientists in Reproductive Medicine and ESHRE Special Interest Group of Embryology 2011 The Istanbul consensus workshop on embryo assessment: proceedings of an expert meeting. Hum Reprod 26 1270–1283. (https://doi.org/10.1093/humrep/der037)
Basille C , Frydman R , El Aly A , et al. 2009 Preimplantation genetic diagnosis: state of the art. Eur J Obstet Gynecol Reprod Biol 145 9–13. (https://doi.org/10.1016/j.ejogrb.2009.04.004)
Brouillet S , Martinez G , Coutton C , et al. 2020 Is cell-free DNA in spent embryo culture medium an alternative to embryo biopsy for preimplantation genetic testing? A systematic review. Reprod Biomed Online 40 779–796. (https://doi.org/10.1016/j.rbmo.2020.02.002)
Cecchino GN & Garcia-Velasco JA 2019 Mitochondrial DNA copy number as a predictor of embryo viability. Fertil Steril 111 205–211. (https://doi.org/10.1016/j.fertnstert.2018.11.021)
Chen Y , Gong Y , Dou L , et al. 2022 Bioinformatics analysis methods for cell-free DNA. Comput Biol Med 143 105283. (https://doi.org/10.1016/j.compbiomed.2022.105283)
Fesahat F , Montazeri F & Hoseini SM 2020 Preimplantation genetic testing in assisted reproduction technology. J Gynecol Obstet Hum Reprod 49 101723. (https://doi.org/10.1016/j.jogoh.2020.101723)
Fodina V , Dudorova A & Erenpreiss J 2021 Evaluation of embryo aneuploidy (PGT-A) and endometrial receptivity (ERA) testing in patients with recurrent implantation failure in ICSI cycles. Gynecol Endocrinol 37 17–20. (https://doi.org/10.1080/09513590.2020.1821231)
Fragouli E , Spath K , Alfarawati S , et al. 2015 Altered levels of mitochondrial DNA are associated with female age, aneuploidy, and provide an independent measure of embryonic implantation potential. PLoS Genet 11 e1005241. (https://doi.org/10.1371/journal.pgen.1005241)
Gardner DK , Lane M , Stevens J , et al. 2000 Blastocyst score affects implantation and pregnancy outcome: towards a single blastocyst transfer. Fertil Steril 73 1155–1158. (https://doi.org/10.1016/s0015-0282(00)00518-5)
Hammond ER , McGillivray BC , Wicker SM , et al. 2017 Characterizing nuclear and mitochondrial DNA in spent embryo culture media: genetic contamination identified. Fertil Steril 107 220–228.e5. (https://doi.org/10.1016/j.fertnstert.2016.10.015)
Huang L , Bogale B , Tang Y , et al. 2019 Noninvasive preimplantation genetic testing for aneuploidy in spent medium may be more reliable than trophectoderm biopsy. Proc Natl Acad Sci U S A 116 14105–14112. (https://doi.org/10.1073/pnas.1907472116)
Kasaven LS , Marcus D , Theodorou E , et al. 2023 Systematic review and meta-analysis: does pre-implantation genetic testing for aneuploidy at the blastocyst stage improve live birth rate? J Assist Reprod Genet 40 2297–2316. (https://doi.org/10.1002/rf2.13306)
Kaye DK 2023 Addressing ethical issues related to prenatal diagnostic procedures. Matern Health Neonatol Perinatol 9 1. (https://doi.org/10.1186/s40748-023-00146-4)
Klimczak AM , Pacheco LE , Lewis KE , et al. 2018 Embryonal mitochondrial DNA: relationship to embryo quality and transfer outcomes. J Assist Reprod Genet 35 871–877. (https://doi.org/10.1007/s10815-018-1147-z)
Kobayashi M , Kobayashi J , Shirasuna K , et al. 2020 Abundance of cell-free mitochondrial DNA in spent culture medium associated with morphokinetics and blastocyst collapse of expanded blastocysts. Reprod Med Biol 19 404–414. (https://doi.org/10.1002/rmb2.12344)
Pan Y , Hao G , Wang Q , et al. 2020 Major factors affecting the live birth rate after frozen embryo transfer among young women. Front Med 7 94. (https://doi.org/10.3389/fmed.2020.00094)
Peng H , Pan M , Zhou Z , et al. 2024 The impact of preanalytical variables on the analysis of cell-free DNA from blood and urine samples. Front Cell Dev Biol 12 1385041. (https://doi.org/10.3389/fcell.2024.1385041)
Piko L & Taylor KD 1987 Amounts of mitochondrial DNA and abundance of some mitochondrial gene transcripts in early mouse embryos. Dev Biol 123 364–374. (https://doi.org/10.1016/0012-1606(87)90395-2)
Ritu G , Veerasigamani G , Ashraf MC , et al. 2022 Mitochondrial DNA levels in trophectodermal cells show No association with blastocyst development and pregnancy outcomes. J Hum Reprod Sci 15 82–89. (https://doi.org/10.4103/jhrs.jhrs_103_21)
Rooney JP , Ryde IT , Sanders LH , et al. 2015 PCR based determination of mitochondrial DNA copy number in multiple species. Methods Mol Biol 1241 23–38. (https://doi.org/10.1007/978-1-4939-1875-1_3)
Shamonki MI , Jin H , Haimowitz Z , et al. 2016 Proof of concept: preimplantation genetic screening without embryo biopsy through analysis of cell-free DNA in spent embryo culture media. Fertil Steril 106 1312–1318. (https://doi.org/10.1016/j.fertnstert.2016.07.1112)
Sheaba MH , Gadel-Rab AG , El-Bitar AMH , et al. 2022 Total and mitochondrial cell free DNA quantification in day 5 embryos culture media reflect embryos quality. Egypt J Hosp Med 89 4346–4352. (https://doi.org/10.21608/ejhm.2022.258440)
Shitara A , Takahashi K , Goto M , et al. 2021 Cell-free DNA in spent culture medium effectively reflects the chromosomal status of embryos following culturing beyond implantation compared to trophectoderm biopsy. PLoS One 16 e0246438. (https://doi.org/10.1371/journal.pone.0246438)
St John JC , Facucho-Oliveira J , Jiang Y , et al. 2010 Mitochondrial DNA transmission, replication and inheritance: a journey from the gamete through the embryo and into offspring and embryonic stem cells. Hum Reprod Update 16 488–509. (https://doi.org/10.1093/humupd/dmq002)
Stigliani S , Anserini P , Venturini PL , et al. 2013 Mitochondrial DNA content in embryo culture medium is significantly associated with human embryo fragmentation. Hum Reprod 28 2652–2660. (https://doi.org/10.1093/humrep/det314)
Suciu ID , Toader OD , Galeva S , et al. 2019 Non-invasive prenatal testing beyond trisomies. J Med Life 12 221–224. (https://doi.org/10.25122/jml-2019-0053)
Tao X , Landis JN , Krisher RL , et al. 2017 Mitochondrial DNA content is associated with ploidy status, maternal age, and oocyte maturation methods in mouse blastocysts. J Assist Reprod Genet 34 1587–1594. (https://doi.org/10.1007/s10815-017-1070-8)
Tocci A 2020 The unknown human trophectoderm: implication for biopsy at the blastocyst stage. J Assist Reprod Genet 37 2699–2711. (https://doi.org/10.1007/s10815-020-01925-0)
Victor AR , Brake AJ , Tyndall JC , et al. 2017 Accurate quantitation of mitochondrial DNA reveals uniform levels in human blastocysts irrespective of ploidy, age, or implantation potential. Fertil Steril 107 34–42.e3. (https://doi.org/10.1016/j.fertnstert.2016.09.028)
Wang J , Diao Z , Zhu L , et al. 2021 Trophectoderm mitochondrial DNA content associated with embryo quality and day-5 euploid blastocyst transfer outcomes. DNA Cell Biol 40 643–651. (https://doi.org/10.1089/dna.2020.6271)
Winstanley YE , Liu J , Adhikari D , et al. 2024 Dynamics of mitochondrial DNA copy number and membrane potential in mouse Pre-implantation embryos: responses to diverse types of oxidative stress. Genes 15 367. (https://doi.org/10.3390/genes15030367)
Yan YY , Guo QR , Wang FH , et al. 2021 Cell-free DNA: hope and potential application in cancer. Front Cell Dev Biol 9 639233. (https://doi.org/10.3389/fcell.2021.639233)
Zhang J , Xia H , Chen H , et al. 2019a Less-invasive chromosome screening of embryos and embryo assessment by genetic studies of DNA in embryo culture medium. J Assist Reprod Genet 36 2505–2513. (https://doi.org/10.1007/s10815-019-01603-w)
Zhang X , Sun Y , Dong X , et al. 2019b Mitochondrial DNA and genomic DNA ratio in embryo culture medium is not a reliable predictor for in vitro fertilization outcome. Sci Rep 9 5378. (https://doi.org/10.1038/s41598-019-41801-1)
