The press-assisted fusion scheme greatly reduces the amount of HVJ-E required in mitochondrial replacement techniques

doi:10.21203/rs.3.rs-2292711/v1

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The press-assisted fusion scheme greatly reduces the amount of HVJ-E required in mitochondrial replacement techniques

https://doi.org/10.21203/rs.3.rs-2292711/v1

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Background

Mitochondrial replacement techniques (MRTs) afford pathogenic mitochondria carried women an opportunity to have related disease-free offspring with a genetic link. Among the fusion methods, HVJ-E-induced fusion has been considered the most promising method for MRTs clinical translation. Although HVJ-E has been confirmed to have no RNA activity, a decrease in blastocyst quality was observed in several MRTs studies with HVJ-E-induced fusion scheme. Nevertheless, HVJ-E has not been proven to be a single factor affecting embryonic development in MRTs. Safety has been the biggest obstacle for its clinical application.

Methods

Pronuclear transfer (PNT) was performed on mouse zygotes and human abnormal zygotes (3PN,1PN) with the traditional HVJ-E-induced fusion (original HVJ-E) and press-assisted HVJ-E-induced fusion (1%HVJ-E). Fusion rates and residual amount of HVJ-E (the relative HVJ-E fluorescence intensity) in reconstructed mouse and human zygotes were assessed. Cleavage rate, blastocyst formation rate, intracellular ROS levels and double-stranded DNA breaks (γH2A.X) of reconstructed mouse zygotes in traditional fusion and press-assisted fusion groups were assessed.

Results

No significant differences were observed in the fusion rates of the press-assisted fusion and traditional fusion group in mouse zygotes and human 3PN/1PN zygote. The relative HVJ-E fluorescence intensity of the press-assisted fusion group was greatly lower than traditional fusion group in mouse and human. The relative ROS fluorescence intensity and the γH2A.X loci of the press-assisted fusion group were lower than that in the traditional group. The blastocyst formation rates in the press-assisted fusion were higher than hat in the traditional fusion group.

Conclusions

In this study, we proved high concentration of HVJ-E used in traditional HVJ-E fusion scheme is an independent factor affecting embryonic development in MRTs, which might be caused by enhanced DNA damage due to increased ROS levels in reconstructed embryos. In order to minimize the amount of HVJ-E attached to the reconstructed zygotes without reducing the fusion efficiency in MRTs, we designed a new scheme for HVJ-E-induced fusion: the press-assisted fusion, which is beneficial to decrease the adverse factors affecting embryo development in MRTs.

Mitochondrial replacement

Pronuclear transfer

HVJ-E

ROS

DNA damage

Preimplantation embryonic development

Mitochondrial diseases are lethal or disabling caused by mitochondrial DNA (mtDNA) mutations, and there is no cure at present[1–4]. Mitochondrial replacement techniques (MRTs) are considered the most promising choice for pathogenic mitochondria carried women to have related disease-free offspring [5–9]. In 2015, UK became the first country in the world to approve the clinical use of MRTs. Despite not having the appropriate legislation, strategies have been implemented in other countries. In 2016, it was reported that a woman carrying a deleterious mutation in mtDNA (m.8993T > G) responsible for Leigh syndrome gave birth to a healthy boy after oocyte spindle transfer in Mexico[10].

In the past decade, there has been considerable research on risks of mitochondrial carry-over, and the possible consequent ‘mitochondrial genetic drift’ [5, 7–9, 11–13]. Nevertheless, risks derived from micromanipulation, especially influences brought by the reagents used in oocyte/zygote-related manipulation, were rarely studied [14].

In MRTs, fusion between enucleated oocytes and small cell body containing main genetic material is a critical step. And inactivated Sendai virus (Hemagglutinating virus of Japan, HVJ) Envelope (HVJ-E) induced fusion and electrofusion are the two main fusion methods. Despite not importing foreign proteins, electrofusion can easily cause chromosome abnormality or premature activation of oocyte due to its obvious electric shock interference, which extremely limits the availability of reconstructed embryos[7, 15]. In contrast, HVJ-E-induced fusion has no such risk[7]. Meanwhile, many studies have confirmed that viral RNA has been inactivated, and there is no risk of genome replication and protein synthesis[6, 16]. HVJ-E-induced fusion has been widely employed to fuse the karyoplast with the cytoplast[5, 6, 8, 17–20]. Nonetheless, research about the effects of HVJ-E on reconstructed embryos is nearly empty at present and lack systematical studies. Disturbingly, it has been demonstrated that HVJ-E could increase reactive oxygen species (ROS) levels in various types of cells in a dose dependent manner, which could cause DNA damage in cells incubated with HVJ-E [21, 22]. A high concentration of HVJ-E used in traditional fusion methods in MRTs may adversely affect embryo development. More information on its safety is required prior to its clinical translation.

In this study, we took autologous pronuclear transfer (PNT) for minimizing the interference caused by other factors and evaluated the possible ROS and DNA damage caused by HVJ-E during fusion. Then we designed a new scheme for HVJ-E-induced fusion, which achieved high fusion efficiency and higher blastocyst formation rates with the reduced amount of HVJ-E attached to the reconstructed zygotes.

Ethical approval

The study was approved by the Institutional Ethical Review Committee of Shanghai Ninth People’s Hospital Affiliated with Jiao Tong University School of Medicine (Approval number: SH9H-2018-T57-1).

Zygotes collection and handling

Mouse zygotes were collected from wild-type C57BL/6 female mice (4-5 weeks old) mated with wild-type C57BL/6 males (8-10 weeks old). Ovulation was induced by the intraperitoneal injection of 10 IU of pregnant mare serum gonadotrophin (PMSG, Ningbo No.2 Hormone Factory, China) and 10 IU human chorionic gonadotrophin (hCG, Ningbo No.2 Hormone Factory, China) at a 48-hour interval. Zygotes were isolated 19-21 hours post hCG administration in modified human tubal fluid media (mHTF, 90126, IrvineScientific, USA).

Cumulus oocyte complex (COC) was added to pre-heated mHTF (containing 5 μg/ml hyaluronidase / H4272, Sigma, Germany) and incubated for 20-30 seconds. In order to get rid of cumulus mass, the COCs were quickly transferred to fresh mHTF and repeatedly blown and sucked with a slim pipette. Then the cumulus-free fertilized oocytes were rinsed three times with fresh mHTF. Subsequently, zygotes with two pronucleus were selected and placed under a humidified atmosphere of 37°C and 5% CO₂ in KSOM (MR-101, Millipore, Germany).

For human, tri-pronuclear (3PN) zygotes from in vitro fertilization (IVF) or intracytoplasmic sperm injection (ICSI) and mono-pronuclear (1PN) zygotes from ICSI at day 1 were collected and placed in G1 PLUS medium (10128, Vitrolife, Sweden) and maintained at 37 °C and 6.0% CO₂. Informed consents were obtained from the participants for the use of 3PN or 1PN zygotes for research.

Pronucleus karyoplast removal

Zygotes were transferred to operation solution (mHTF with 2.5 μM cytochalasin B/CB, 14930-96-2, Sigma, Germany) for 15 minutes before operation to enhance karyoplast removal. Manipulations were carried out within an inverted microscope (Nikon Eclipse Ti, Japan) equipped with a micromanipulation system (Nikon NARISHIGE NT-88-V3, Japan). Zygotes were fixed with a holding pipette and the zonal pellucida were punched through with a microsurgical laser (ZILOS-tk, Hamilton Thorne, USA). A 20μm -flat biopsy pipette (Sunlight Medical, USA) was inserted under the zona pellucida, then the second polar body (PB2) was removed with the biopsy pipette. The pronucleus and surrounding cytoplasm were carefully removed from zygotes as a membrane-bound karyoplast.

The traditional HVJ-E-induced fusion

Membrane-bound karyoplasts were transferred by a biopsy pipette into a 1μl drop of original HVJ-E (GenomONE-CF EX HVJ Envelope Cell Fusion Kit, ISHIHARA SANGYO KAISHA, Japan, Supplementary Fig. S1, Left) for 40 seconds, then pronuclear karyoplasts and a small volume of the suspension approximately equal to the volume of the karyoplast inhaled into the pipette. The biopsy pipette was inserted under the zona pellucida through the ablation in the zona pellucida, then membrane-bound karyoplasts and suspension of HVJ-E were blown into the perivitelline space to ensure good contact between the karyoplast and enucleated zygote membrane. Pronuclear karyoplast will fuse with the enucleated zygote within 10 minutes up to 1 hour after transfer.

The press-assisted fusion

1/4 volume of karyoplast was contacted suspension of HVJ-E for 40 seconds in a 1μl drop of 1% HVJ-E dilution (Supplementary Fig. S1, Right). Then only pronuclear karyoplasts were aspirated into the biopsy pipette. The pipette was inserted under the zona pellucida through the ablation in the zona pellucida. Pronuclear karyoplasts were pushed out to make it full contact with the enucleated zygote membrane for 2 minutes. Fusion of the pronuclear karyoplast with the enucleated zygote occurred within 10 minutes up to half an hour.

Statistical analysis of cleavage and blastocyst formation rates

Reconstructed zygotes were transferred to KSOM and maintained at 37°C and 5% CO₂ for subsequent observation and analysis. We calculated the cleavage rates approximately 24h after zygotes were isolated. And after approximately 120h, blastocyst formation rates were calculated. When the blastocyst cavity was ≥1.5 blastocyst volume, the blastocyst was considered to have formed.

Measurement of intracellular ROS levels

Reconstructed zygotes were incubated at 37°C for 30 min with ROS Orange Working Solution (ab186028, Abcam, USA). Stained zygotes were monitored at Ex/Em = 540/570 nm for the total fluorescence intensity. The relative fluorescence intensity after background subtraction was compared in our study. The total fluorescence intensity in the control group was defined as 100%.

Embryo fixation and immunostaining

Zygotes and embryos were fixed overnight with 2% (w/ v) PFA in PBS, at 4°C. Then, a permeabilizing solution of 0.25% (v/v) Triton X-100 was applied at room temperature to the zygotes and embryos, and then blocked in PBS containing 3% bovine serum albumin at 4℃ overnight. The primary antibody was incubated overnight at 4°C. Primary antibodies included polyclonal chicken Sendai virus (ab33988, Abcam, USA), monoclonal mouse anti-γH2A.X (05-636, EMD Millipore, USA) and polyclonal rabbit anti-H3K9me3 (ab8898, Abcam, USA). Then, samples were incubated with Goat Anti-Chicken IgY H&L (Alexa Fluor® 647, ab150171, Abcam, USA), Goat Anti-Mouse IgG H&L (FITC, ab7064, Abcam, USA), Goat Anti-Rabbit IgG H&L (TRITC, ab6718, Abcam, USA) at room temperature for one hour, and the nuclei were stained with DAPI (WH1149, Biotechwell, China). A Nikon A1 confocal microscope was used to image the samples after staining.

For anti-Sendai virus, measurements were obtained by averaging the signal collected in a region of interest, which we set as the center of the zygotes and embryos. Based on the difference between the fluorescence intensity of the background and the detected fluorescence intensity, the relative fluorescence intensity was determined. The average fluorescence intensity of the control group was defined as 100%.

Statistical methods

All statistical analyses were carried out using the Statistical Package for Social Sciences (SPSS) version 23.0 (IBM SPSS, Armonk, NY). Measuring data is represented as mean ±standard error of the mean (X ± SD). The t-test, one-way ANOVA with LSD tests or χ2 test were used to compare groups, with P<0.05 considered statistically significant.

The press-assisted fusion maintains high fusion rates in mouse and human

For the new scheme, the process of obtaining the karyoplast is the same with the traditional method (Fig. 1A, a; Fig. 1B, a-c). Our improvements are mainly focused on the steps to promote fusion. For the traditional method, the whole pronuclear karyoplast is incubated in original HVJ-E (Supplementary Fig. S1, Left) and implanted directly into the perivitelline space (Fig. 1A, b). Whereas, for the new scheme, 1/4 volume of karyoplast (Fig. 1B, d) is incubated in 1/100 concentration of HVJ-E (Supplementary Fig. S1, Right); then using biopsy pipette to compress karyoplast moderately (strength and time), so that it can fully contact with enucleated oocytes (Fig. 1A, c; Fig. 1B, e).

To evaluate the efficiency of the new fusion scheme, PNTs were performed on mouse 2PN zygotes and human 3PN/1PN zygotes, and the fusion rates were calculated (Fig. 1C; Fig. 6A). In mouse, the fusion rate was 98.2% (n = 170) in the traditional fusion group and 96.5% (n = 200) in the press-assisted fusion group (Fig. 1C); In human, the fusion rate was 94.4% (n = 18) in the traditional fusion group and 90.0% (n = 20) in the press-assisted fusion group (Fig. 6A). There was no significant difference in the fusion rates between the press-assisted fusion group and traditional fusion group in mouse 2PN zygotes (P = 0.353) and human 3PN/1PN zygotes (P = 1.000), respectively.

We also tried to use the traditional fusion method with 1% HVJ-E in mouse 2PN zygotes, but the fusion rate of the 1% HVJ-E group (n = 25, fusion rate 8.0%) was extremely lower than the traditional fusion with original HVJ-E group (n = 170, fusion rate 98.2%; 1% HVJ-E vs original HVJ-E, P < 0.0001) (Fig. 1C).

The press-assisted fusion can greatly reduce the residual amount of HVJ-E on the membrane of reconstructed zygotes

In order to assess the residual rates of HVJ-E in reconstructed zygotes generated by the press-assisted fusion scheme, immunofluorescence staining of HVJ-E in reconstructed mouse 2PN zygotes and human 3PN/1PN zygotes were performed and its relative intensity were analyzed statistically (Fig. 2; Fig. 6B and C). In mouse, the relative HVJ-E fluorescence intensity of the control group was 1.00 ± 0.3×100% (n = 35)(Fig. 2B). In the traditional group, the relative HVJ-E fluorescence intensity was 21.24 ± 2.9×100% (n = 46; Control vs traditional group, P < 0.0001) (Fig. 2B). While, in the press-assisted fusion group, the relative HVJ-E fluorescence intensity was 3.33 ± 1.9×100% (n = 52; Control vs press-assisted fusion group, P < 0.0001; Press-assisted fusion group vs traditional group, P < 0.0001 ) (Fig. 2B). In human, the relative HVJ-E fluorescence intensity of the control group was 1.00 ± 0.2×100% (n = 10)(Fig. 6C). Meanwhile, the relative HVJ-E fluorescence intensity of the traditional group was 24.78 ± 2.6×100% (n = 15;Control vs traditional group, P < 0.0001) (Fig. 6C). However, in the press-assisted fusion group, the relative HVJ-E fluorescence intensity was 3.73 ± 1.1×100% (n = 15; Control vs press-assisted fusion group, P < 0.0001; Press-assisted fusion group vs traditional group, P < 0.0001 ) (Fig. 6C).

The press-assisted fusion can greatly reduce intracellular ROS caused by HVJ-E in reconstructed zygotes

In order to assess the adverse effects of HVJ-E on reconstructed zygotes, intracellular ROS levels were measured (Fig. 3A and B). The relative ROS fluorescence intensity of the control group was 100 ± 22.7% (n = 37) (Fig. 3B). In the traditional fusion group, the relative ROS fluorescence intensity was 175.9 ± 28.4% (n = 27; Control vs traditional fusion, P < 0.0001), while in the press-assisted fusion group, the relative ROS fluorescence intensity was 136.6 ± 23.4% (n = 32; Control vs press-assisted fusion, P < 0.0001; Traditional fusion vs press-assisted fusion, P < 0.0001) (Fig. 3B).

The press-assisted fusion can greatly reduce double-stranded DNA breaks caused by HVJ-E in reconstructed zygotes

Further investigation of the adverse effects of HVJ-E on embryo development was conducted by examining the γH2A.X loci (DNA damage marker) in the male and female pronucleus of reconstructed zygotes. H3K9me3 was used as the marker of the female pronucleus because it is absent in the male pronucleus before zygotic genome activation.

A comparison of the γH2A.X loci was carried out first in the male pronucleus (Fig. 4A and B).

Compared with the control group (2.06 ± 1.51, n = 18), γH2A.X loci was increased in the traditional fusion group (14.33 ± 5.97, n = 15; Control vs, P < 0.0001), while no significant difference was detected in the press-assisted fusion group (4.18 ± 2.27, n = 17; Control vs, P = 0.091). As compared with traditional fusion, the number of γH2A.X loci decreased significantly in the press-assisted fusion group (Traditional fusion vs press-assisted fusion, P < 0.0001). Furthermore, the female pronucleus showed a similar trend (Fig. 4A and C). Compared with the control group (1.78 ± 1.11, n = 18), γH2A.X loci was increased in the traditional fusion group (10.47 ± 2.10, n = 15; Control vs, P < 0.0001), but no significant difference was detected in the press-assisted fusion group (2.82 ± 1.63, n = 17; Control vs, P = 0.064). While, the number of γH2A.X loci was significantly reduced in the press-assisted fusion group when compared with the traditional fusion group (Press-assisted fusion vs traditional fusion, P < 0.001).

The press-assisted fusion can improve blastocyst formation rate of mouse reconstructed zygotes

To evaluate the impact of the new method on embryonic development, the cleavage and blastocyst formation rates were calculated (Fig. 5A-C). The cleavage rate was 91.3% in the press-assisted fusion group (n = 92), 91.1% in the traditional fusion group (n = 79), and 93.8% in the control group (n = 112). No significant difference was observed in cleavage rates among the different groups (Fig. 5B). While, compared with the traditional group (61.1%, n = 72), the press-assisted fusion group (77.4%, n = 84) had higher blastocyst formation rate (Press-assisted fusion vs traditional fusion, P = 0.036). Meanwhile, the blastocyst formation rates in the new scheme group were similar to that in the control group (79.1%, n = 105; Press-assisted fusion vs Control, P = 0.860), while the blastocyst formation rates in traditional group was lower than that in the control group (Traditional fusion vs control, P = 0.011) (Fig. 5C).

HVJ-E persists on embryos developed from reconstructed zygotes

We wondered whether HVJ-E disappears from embryos developed from reconstructed zygotes during embryonic development. As shown in Supplementary Fig. S2, HVJ-E was detected on reconstructed zygotes, 2-cell embryos, 4-cell embryos, 8-cell embryos, morula and blastocysts after PNTs with traditional fusion scheme. The relative HVJ-E fluorescence intensity of reconstructed zygotes and embryos developed from reconstructed zygotes were all significantly higher than their control groups (Supplementary Fig. S2C). The residues of HVJ-E were decreased gradually from zygotes to 8-cell embryos (zygotes: 20.6 ± 3.3×100%, n = 23; 2-cell: 7.85 ± 2.8×100%, n = 15; 4-cell: 4.20 ± 0.8×100%, n = 15; 8-cell: 2.2 ± 0.5×100%, n = 15; Zygotes vs 2-cell, P < 0.0001; 2-cell vs 4-cell, P < 0.0001;4-cell vs 8-cell, P = 0.008) (Supplementary Fig.S2B). However, the relative HVJ-E fluorescence intensity of morula has no significant differences when compared with 8-cell embryos (Morula: 1.95 ± 0.7×100%, n = 15; 8-cell vs morula, P = 0.724) (Supplementary Fig. S2B). Meanwhile, the relative HVJ-E fluorescence intensity of blastocysts was also not significant differences when compared with morula (Blastocysts: 1.45 ± 0.3×100%, n = 15; Morula vs Blastocysts, P = 0.502) (Supplementary Fig. S2B).

MRTs afford pathogenic mitochondria carried women an opportunity to have related disease-free offspring with a genetic link. Since widespread adoption in MRTs research, HVJ-E-induced fusion has been considered the most promising fusion method for clinical translation[5, 8, 10, 17, 19, 20, 23–25]. However, its safety has been the biggest obstacle due to the lack of relevant research[14]. In this study, we have confirmed the adverse effects of high concentration HVJ-E on embryonic development, and optimized a new fusion scheme that can greatly reduce the amount of HVJ-E attached to the reconstructed zygotes in MRTs, which saved the low blastocyst formation rates caused by excess HVJ-E.

Although HVJ-E has been demonstrated to have no RNA activity[6, 16], recent studies have detected a dose-dependent increase of ROS levels in some cells incubated with HVJ-E [21, 22, 26, 27]. In 2016, a preclinical study on human PNT achieved a high blastocyst formation rate through a series of changes in manipulation medium and process, including reduced the HVJ-E concentration tenfold [5]. However, as one of many manipulations and culture conditions altered by the authors, HVJ-E has not been proven to be a single factor affecting embryonic development. In the present study, the concentration of HVJ-E was evaluated without changing any other conditions, and the results showed that high concentration of HVJ-E certainly contributed to a significant increase of ROS levels in reconstructed zygotes. Additionally, DNA double-strand break assays also revealed that reconstructed fertilized oocytes which developed to the prokaryotic stage exhibited higher levels of DNA damage. In line with previous reports, DNA is more likely to be destroyed when experiencing excessive ROS[28], particularly in nucleus of oocytes and preimplantation embryos, which are more susceptible to oxidative stress[29, 30]. These conclusions suggest that it is necessary to reduce the concentration of HVJ-E in MRTs. Nevertheless, a reduction in HVJ-E concentration will significantly reduce the fusion rate, resulting in a decrease of MRTs efficiency.

In order to solve these problems, we optimized the conventional method and designed a new scheme of HVJ-E-induced fusion, which greatly reduced the concentration of HVJ-E used in manipulation with high fusion efficiency. For the traditional method, the membrane-bound karyoplast was directly implanted into the perivitelline space[31, 32]. Consequently, more HVJ-E is required to enhance the fusion probability of small karyoplasts, which benefit for reducing mitochondrial carry-over. For the new scheme, the most important step is to use biopsy pipette to compress karyoplast moderately (strength and time), so that it can fully contact with enucleated oocytes (Fig. 1A, c; Fig. 1B, e). In this way, effective contact of pronuclear karyoplasts and enucleated zygote membrane was sufficient. On this basis, under the control of biopsy pipette, full incubation of 1/4 volume of karyoplast (Fig. 1B, d) with 1/100 concentration of HVJ-E (Supplementary Fig. S1, Right) is enough to complete the subsequent high efficiency fusion. Collectively, the actual amount of inactivated virus carried by reconstructed zygote was greatly reduced (Fig. 2A and B). Further examination revealed that ROS levels in the reconstructed zygotes and DNA damage levels of the prokaryotes were significantly decreased in the press-assisted fusion group. Subsequent in vitro observations of embryo development also indicated that the blastocyst formation rates in traditional fusion group were lower than control group. While, the blastocyst formation rates of the new method group were improved compared to the traditional group. This suggests that reducing the amount of HVJ-E attached to the karyoplasts during fusion is beneficial to decrease the adverse factors affecting embryo development in MRTs.

Although the underlying mechanisms for the impact of HVJ-E on embryonic development remain unknown, it is quite likely that high ROS levels caused by high concentration of HVJ-E increased DNA breaks. As has been found in other cells[21, 22, 26, 27], in the present study, a higher level of ROS was observed in the traditional group, suggesting that high concentration of HVJ-E was associated with excessive ROS production in MRTs. Meanwhile, the same trend was found for DNA damage: DNA breaks of male and female pronuclei in the press-assisted fusion group were significantly less than that of the traditional group, and no significant difference with the control group.

In this study, residues of HVJ-E were detected persisted on embryos developed from reconstructed zygotes. The residues of HVJ-E were decreased gradually from zygotes to 8-cell embryos. However, no significant differences have been found between 8-cell embryos, morula and blastocysts. The results indicated that the effect of HVJ-E on embryos might be mainly in the period shortly after fusion.

Notably, due to the difficulty of obtaining normal human zygotes for experiments, we only verified residue removal effect of this method with 3PN/1PN human zygotes. The result proved that it could also significantly reduce residues of HVJ-E in human samples, which should be beneficial to the clinical application of HVJ-E, although the import of HVJ-E was not completely avoided. In addition, it achieved the same embryonic development outcomes as the control group in mouse models. Our study demonstrated that fusion with low concentrations of HVJ-E had little impact on the pronucleus of reconstructed zygotes and did not adversely affect the quality of blastocysts. Furthermore, based on our findings, HVJ-E gradually decreases in each blastomere as embryonic development progresses, signifying the effect of HVJ-E on embryonic development gradually decreases. A future investigation with more focus on inquiring whether HVJ-E has any influence on individual development is therefore suggested.

In summary, we highlight that high concentration of HVJ-E had adverse effects on blastocyst quality by increasing the DNA breaks of reconstructed zygotes due to high ROS levels. We optimized the conventional method and designed a new scheme of HVJ-E-induced fusion, which greatly reduced the amount of HVJ-E attached to the reconstructed zygotes, the ROS levels and DNA breaks, and thus improved embryo quality in MRTs. Therefore, this study can offer new insights into a better understanding of the effect of HVJ-E on embryonic development and might provide a basic ground for clinical translation of HVJ-E fusion in MRTs.

MRTs

mitochondrial replacement techniques

HVJ-E

Hemagglutinating virus of Japan Envelope

mtDNA

mitochondrial DNA

ROS

reactive oxygen species

PNT

pronuclear transfer

COC

cumulus oocyte complex

mHTF

modified human tubal fluid

hCG

human chorionic gonadotrophin

PMSG

pregnant mare serum gonadotrophin

2PN

bi-pronuclear

3PN

tri-pronuclear

1PN

mono-pronuclear

IVF

in vitro fertilization

ICSI

intracytoplasmic sperm injection

PB2

second polar body.

Ethics approval and consent to participate

All the animal experiments were conducted according to human-animal care standards and the claim of the Institutional Ethical Review Committee of Shanghai Ninth People’s Hospital Affiliated with Jiao Tong University School of Medicine. All the patients were informed of sample collection and usage. The abnormal zygotes were collected and used with the agreement with the Institutional Ethical Review Committee of Shanghai Ninth People’s Hospital Affiliated with Jiao Tong University School of Medicine.

Consent for publication

Written informed consent for publication was obtained from all participants.

Data Availability

The datasets supporting our findings are presented in the article.

Competing interests

The authors declare that they have no competing interests.

Funding

This research was funded by National Natural Science Foundation of China (NSFC) (grant no. 81871163, 81901478 and 82201886), and Research and Development Centre Project of Serve China (grant no. 2019001YZGT).

Authors’ contributions

Q.L., Y.K. and W.L. conceived and designed the experiments. M.M., S.J., W.J. and W.L. performed the experiments. C.C., K.L. and X.L. established the research methods. W.J. and D.L. collected the samples. M.M. and W.L. analyzed the data. M.M. and S.J. wrote the paper. All authors approved the final manuscript. All authors have read and agreed to the published version of the manuscript.

Acknowledgements

We gratefully acknowledge all the staff of the department of assisted reproduction at Shanghai Ninth People’s Hospital for their support and cooperation.

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No competing interests reported.

FigS1.tif
Fig. S1.HVJ-E original solution and 1% diluent were observed under a microscope. Left: Red star, original HVJ-E; Right: Green star, 1% HVJ-E.
FigS2.tif
Fig. S2 HVJ-E persists on embryos developed from reconstructed zygotes. (A) Representative images of the HVJ-E (anti-Sendai virus) fluorescence intensity in reconstructed zygotes, 2-cell embryos, 4-cell embryos, 8-cell embryos, morula and blastocysts. Anti-Sendai virus(purple). Scale bars, 20μm. (B) Quantification of the relative HVJ-E (anti-Sendai virus) fluorescence intensity (100%, Y-axis) in reconstructed zygotes (n = 23), 2-cell embryos (n = 15), 4-cell embryos (n = 15), 8-cell embryos (n = 15), morula (n = 15) and blastocysts (n = 15) (X-axis). Each dot represents one reconstructed zygote or one embryo in the group, and the bar and whiskers represent the mean and SD. One-way ANOVA, **P < 0.01, **P < 0.0001, ns: no significance. (C) Quantification of the relative HVJ-E (anti-Sendai virus) fluorescence intensity (100%, Y-axis) in reconstructed zygotes group (n = 23) and its control zygotes group (n = 15), 2-cell embryos group (n = 15) and its control 2-cell embryos group (n = 10), 4-cell embryos group (n = 15) and its control 4-cell embryos group (n = 11), 8-cell embryos group (n = 15) and its control 8-cell embryos group (n = 10), morula(n = 15) group and its control morula group (n=10), blastocysts group (n = 15) and its control blastocysts group (n = 10) (X-axis). Each dot represents one reconstructed zygote or one embryo in the group, and the bar and whiskers represent the mean and SD. t-test, *P < 0.001, ****P < 0.0001.

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The press-assisted fusion scheme greatly reduces the amount of HVJ-E required in mitochondrial replacement techniques

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