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Nuclear transfer of an oocyte into the cytoplasm of another enucleated oocyte has shown that embryogenesis and implantation are influenced by cytoplasmic factors. We report a case of a 30-year-old nulligravida woman who had two failed IVF cycles characterized by all her embryos arresting at the two-cell stage and ultimately had pronuclear transfer using donor oocytes. After her third IVF cycle, eight out of 12 patient oocytes and 12 out of 15 donor oocytes were fertilized. The patient's pronuclei were transferred subzonally into an enucleated donor cytoplasm resulting in seven reconstructed zygotes. Five viable reconstructed embryos were transferred into the patient's uterus resulting in a triplet pregnancy with fetal heartbeats, normal karyotypes and nuclear genetic fingerprinting matching the mother's genetic fingerprinting. Fetal mitochondrial DNA profiles were identical to those from donor cytoplasm with no detection of patient's mitochondrial DNA. This report suggests that a potentially viable pregnancy with normal karyotype can be achieved through pronuclear transfer. Ongoing work to establish the efficacy and safety of pronuclear transfer will result in its use as an aid for human reproduction.
). It was shown that, in couples that experienced repeated implantation failure as a result of poor embryo development, ooplasm transfer from donor oocytes at metaphase II (MII) stage into patient MII oocytes can be compatible with fertilization and pregnancy. These case series reports provided some interesting findings, but evidence of improved embryo development and implantation after ooplasmic augmentation remained elusive. Both nuclear and cytoplasmic deficiencies have been shown to be responsible for poor oocyte quality by contributing to meiotic defects and subsequent impaired embryo development (
Birth after the injection of sperm and the cytoplasm of tripronucleate zygotes into metaphase II oocytes in patients with repeated implantation failure after assisted fertilization procedures.
). In assisted reproduction, it has been shown that the ooplasm of mature oocytes from young women could be applied to restore normal growth and viability in developmentally compromised embryos, where the underlying cause was attributed to ooplasmic deficiency (
Birth after the injection of sperm and the cytoplasm of tripronucleate zygotes into metaphase II oocytes in patients with repeated implantation failure after assisted fertilization procedures.
) that normal meiosis can occur after the transfer of germinal vesicle into an enucleated host oocyte. We have shown in mice that oocytes reconstructed by germinal vesicle transfer into a cytoplasm of the same developmental stage mature normally in vitro through the MII stage (
). These data corroborate that a healthy cytoplasm is required for a normal nuclear function. We report herein a patient who had repetitive embryo arrest at the two-cell stage after two failed IVF cycles, and was subsequently able to conceive a normal pregnancy after pronuclear transfer into a donor cytoplasm.
Materials and methods
Clinical presentation
A 30-year-old nulligravida healthy woman had two failed IVF cycles characterized by all her embryos arresting at the two-cell stage. She was diagnosed with unexplained infertility after a complete work-up showing a normal ovarian reserve, normal semen analysis and patent fallopian tubes. The patient consented to the use of cytoplasm from a donated oocyte. The procedures used in this study were verbally approved by Sun Yat-Sen University Hospital Ethics Committee in China. Ovarian stimulation was then carried out and was synchronized for the patient and her oocyte donor so that their MII stage oocytes were retrieved within a 2-h period. After intracytoplasmic sperm injection, eight out of 12 patient oocytes and 12 out of 15 donor oocytes were fertilized with the sperm of the patient's partner. All pronuclei were then removed from the donor zygotes and discarded. The patient's (male and female) procnuclei were removed from each of her zygotes and transferred subzonally into the donor cytoplast (enucleated zygote).
Pronuclear karyoplast fusion
Electrofusion was then carried out as previously described (
Mechanisms by which a lack of germinal vesicle (GV) material causes oocyte meiotic defects: a study using oocytes manipulated to replace GV with primary spermatocyte nuclei.
). The donor's cytoplast with the male and female patient procnuclei were exposed to modified human tubal fluid medium with 10% serum supplemented with 7.5 µg/ml cytochalasin B (Sigma, St. Louis, MO, USA) for 15 min at room temperature to disrupt the microfilaments and increase plasma membrane flexibility before manipulation. The dish was then placed onto the stage of on Olympus IX71 inverted microscope equipped with micromanipulators. A slot was made in the zona pellucida of each oocyte by applying a sharp-tipped pipette that penetrates the zona while holding the oocyte against the wall of the holding pipette. This allowed the enucleation pipette to pass through the zona slot and approach the pronuclei before gently applying negative pressure to aspirate the procnuclei. Once a pronucleus was separated from the cytoplasm, it was transferred into the perivitelline space of the donor's enucleated cytoplast. The membrane fusion between both pronuclei and the donor's cytoplast was initiated by placing it into a fusion medium (0.3 M mannitol, 0.1 mM CaCl2, and 0.05 mM MgSO4) between platinum electrodes aligned in response to AC (6–8 V) current for 5–10 s before an electrical pulse (1.8–2.5 kV/cm DC for 50 µs) was delivered by a Model 2001 Electro Cell Manipulator BTX (Holliston, MA). The formed complexes were rinsed three times in modified human tubal fluid and then incubated in human tubal fluid medium at 5% CO2 and 37°C. Membrane fusion usually occurred 30 min after the start of the electric pulse.
DNA extraction and mitochondrial genotype analysis
Nuclear and cytoplasmic DNA profiles were analysed in blood samples from the patient, the oocyte donor and the fetuses (after pregnancy loss). Nuclear DNA fingerprinting was carried out at 5 microsatellite loci with subtraction of the husband's genotype, as previously described (
). Mitochondrial (mt) DNA was analysed by amplification and sequencing of a 524-bp segment in the D-loop region (16024–577). In brief, DNA was extracted from the embryos using the DNeasy Blood and Tissue kit (Qiagen, Valencia, CA) according to the manufacturer's recommendation. The mtDNA genotyping analysis was carried out using restriction fragment length polymorphism method, as previously described (
Maternally inherited cardiomyopathy: clinical and molecular characterization of a large kindred harboring the A4300G point mutation in mitochondrial deoxyribonucleic acid.
Maternally inherited cardiomyopathy: clinical and molecular characterization of a large kindred harboring the A4300G point mutation in mitochondrial deoxyribonucleic acid.
). The PCR products were then purified using QIAquick PCR Purification Kit (Qiagen, Valencia, CA) according to the manufacturer's instructions. A 15 µl aliquot of the final product was digested overnight with the endonuclease RsaI (New England Biolabs, Ipswich, MA) and then electrophoresed on a 2.5% agarose gel and visualized by ethidium bromide staining under ultraviolet light.
Results
Electrofusion of the patient's karyoplast with the donor's cytoplast resulted in seven successfully reconstructed zygotes and one that degenerated (Figure 1). Upon the patient's request, given her poor history of embryo development, and given the poor embryo cryopreservation techniques at that time, the five reconstructed zygotes that cleaved to the four-cell stage at 48 h (after retrieval) were transferred to the patient's uterus. A triplet pregnancy with fetal heartbeats was achieved.
Figure 1Transfer of pronuclei into the cytoplast of a donor's oocyte. (a,b) Enucleation of a zygote at the two pronuclei stage; (c) two pronuclei transfer into the perivitelline space of the donor's cytoplast; (d) membrane fusion between the two pronuclei and the cytoplast; (e) embryo at the four-cell stage. Arrow indicates pronucleus. Scale bar, 25 µm. 2PN, two pronuclei.
Fetal reduction to a twin pregnancy was performed transvaginally 33 days after transfer. At 24 weeks of gestation, Fetus B delivered as a result of premature rupture of membranes, and did not survive owing to respiratory distress. At 29 weeks of gestation, Fetus C was delivered after intrauterine fetal demise due to cord prolapse. Normal karyotypes were found in the embryonic tissue (46, XY), the 24-week (46, XX) and 29-week (46, XY) old fetuses. Nuclear genetic fingerprinting confirmed that the nuclear DNA from the 24-week and the 29-week old fetuses matched that of the patient's nuclear genetic fingerprinting. The mtDNA profiles of fetal red blood cells were identical to those of the donor's mtDNA with no detection of patient's mtDNA.
Discussion
Some of the reasons for early embryonic developmental arrest remain unclear. Early embryo arrest has been proposed as a protective mechanism for preventing further development of abnormal embryos because almost one-half of arrested human embryos shows chromosomal abnormalities (
). Microtubules and microfilaments are indicators of embryonic normality because they represent the major cytoskeletal components and are associated with chromosomal condensation and formation of the mitotic spindle. Additionally, early developmental arrest in mouse embryos has been associated with elevated levels of free oxygen radicals, indicating that oxidative stress might be a reason for embryo arrest (
Radical solutions and cultural problems: could free oxygen radicals be responsible for the impaired development of preimplantation mammalian embryos in vitro?.
). Successful development of embryos reconstructed by nuclear transfer is dependent on a wide range of factors such as cell cycle coordination between the donor's and the recipient's oocytes, as in our case, in order to maintain normal ploidy (
). Our case report further indicated that the structural and functional integrity of the reconstructed zygote was not compromised by the micromanipulation and electrofusion procedures themselves making them technically successful.
Another potential application of pronuclear transfer is the treatment of genetic diseases caused by mtDNA mutations (
). Therefore, the replacement of mutated mtDNA with wild-type haplotypes would potentially represent a cure for these genetic mutations. Some of the mutated mitochondria adjacent to the nucleus (or pronucleus), however, are likely to be carried over into the reconstructed oocytes during its transfer, thus creating mtDNA heteroplasmy (
). To completely eliminate the risk of transmission of mutated mtDNA into the reconstructed oocytes, it is necessary to completely remove mtDNA from the patient carrying the genetic mutation when nuclear and pronuclear transfer is carried out. Interestingly in our case, the results of mtDNA analysis from the patient's blood, the donor's oocyte and the fetuses did not show any heteroplasmy using the pronuclear transfer technique.
In conclusion, pronuclei transfer in humans can produce a viable pregnancy with normal karyotype and minimal mtDNA heteroplasmy. This technique could potentially be used as a unique approach to prevent the transmission of mutated mtDNA from mothers to their children, and as an aid for women who produce embryos that arrest at the two-cell stage after IVF.
References
Almeida P.A.
Bolton V.N.
Cytogenetic analysis of human preimplantation embryos following developmental arrest in vitro.
Maternally inherited cardiomyopathy: clinical and molecular characterization of a large kindred harboring the A4300G point mutation in mitochondrial deoxyribonucleic acid.
Birth after the injection of sperm and the cytoplasm of tripronucleate zygotes into metaphase II oocytes in patients with repeated implantation failure after assisted fertilization procedures.
Radical solutions and cultural problems: could free oxygen radicals be responsible for the impaired development of preimplantation mammalian embryos in vitro?.
Mechanisms by which a lack of germinal vesicle (GV) material causes oocyte meiotic defects: a study using oocytes manipulated to replace GV with primary spermatocyte nuclei.
Dr Zhang completed his medical degree in at the Zhejiang University School of Medicine, and subsequently received his Master's Degree at Birmingham University in the UK. In 1991, Dr Zhang earned his PhD in IVF, and, after studying and researching the biology of mammalian reproduction and human embryology for nearly 10 years, became the first Fellow in the Division of Reproductive Endocrinology and Infertility of New York University's School of Medicine in 2001. Dr. Zhang continues his research in minimal stimulation IVF, non-embryonic stem cell research, long-term cryopreservation of oocytes, and oocyte reconstruction by nuclear transfer.
Article info
Publication history
Published online: August 01, 2016
Accepted:
July 25,
2016
Received in revised form:
July 20,
2016
Received:
July 16,
2015
Declaration: The authors report no financial or commercial conflicts of interest.
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