First pilot study of maternal spindle transfer for the treatment of repeated in vitro fertilization failures in couples with idiopathic infertility.

OBJECTIVES: To gain insights into the technical feasibility of maternal spindle transfer (MST) applied in the context of repeated in vitro fertilization (IVF) failures for the treatment of idiopathic infertility. DESIGN: A prospective pilot study. SETTING: IVF center. PATIENT(S): Twenty-five infertile couples with multiple previous unsuccessful IVF cycles (range, 3-11), no previous pregnancy, and no history of mitochondrial DNA (mtDNA) disease participated. The study focused on women <40 years, with previous IVF attempts characterized by a pattern of low fertilization rates and/or impaired embryo development. Couples with severe male-factor infertility were not eligible. Oocyte donors with previous successful IVF outcomes were matched with patients according to standard practice. INTERVENTION(S): We performed MST by transferring metaphase II spindles from the patients' oocytes into the previously enucleated donor oocytes, followed by intracytoplasmic sperm injection, in vitro embryo culture, blastocyst biopsy, and vitrification. Only euploid blastocysts were considered for embryo transfer. MAIN OUTCOME MEASURE(S): Outcome measures included oocyte fertilization, blastocyst development, clinical pregnancy and live birth, incidence of mitochondrial carryover and potential mtDNA reversal, as well as general health of the children born. RESULT(S): Twenty-eight MST cycles produced 6 children (19 embryo transfers, 7 clinical pregnancies). Pediatric follow-up of the children, performed at intervals from birth to 12-24 months of age, revealed their development to be unremarkable. DNA fingerprinting confirmed that the nuclear DNA of MST children was inherited from both parents, without any contribution from the oocyte donor. For 5 of the children, mtDNA was derived almost exclusively (>99%) from the donor. However, 1 child, who had similarly low mtDNA carryover (0.8%) at the blastocyst stage, showed an increase in the maternal mtDNA haplotype, accounting for 30% to 60% of the total at birth. CONCLUSION(S): This pilot study provides the first insights into the feasibility of applying MST for patients with idiopathic infertility and repeated IVF failures. Reconstructed oocytes produced embryos capable of implanting, developing to term and producing apparently healthy newborns/children. However, claims concerning the efficacy of MST with respect to infertility treatment would be premature considering the limitations of this study. Importantly, mtDNA reversal was detected in one child born after MST, a finding with possible implications for mitochondrial replacement therapies. CLINICAL TRIAL REGISTRATION NUMBER: Pilot trial registry number, ISRCTN11455145. The date of registration: 20/02/2018. The date of enrolment of the first patients: 18/03/2018.

Evaluation of the impact of laser-assisted hatching techniques on the hatching process of mouse blastocysts using time-lapse microscopy.

OBJECTIVE: To study the effect of zona opening (ZO) and 2 zona thinning (ZT) techniques on the hatching process of mouse embryos using a last-generation laser system and time-lapse microscopy (TLM). DESIGN: Prospective randomized study. SETTING: Private research center. ANIMALS: A total of 267 F1 hybrid (B6/CBA) mice embryos were included. INTERVENTION(S): Morulae were randomly selected and the zona pellucida (ZP) manipulated using a laser system according to 4 experimental groups: control (ZP intact, n = 59), ZO (25 µm hole, n = 70), ZT25 (25% perimeter thinned, n = 71), and ZT35 (35% perimeter thinned, n = 67). Embryo development was monitored by TLM until day 6. MAIN OUTCOME MEASURE(S): Time to first breach the ZP, hatching time, time to complete hatching, multiple breaching, multiple hatching, loss of cells, hole size, and embryo quality were analyzed. RESULT(S): No significant differences in the proportion of completely hatched embryos were found among groups. However, the time (average hours ± SD) to complete hatching was significantly delayed in the control group compared with all laser-treated groups: 118.3 ± 9.5 hours in the ZT25 group, 116.6 ± 8.7 hours in the ZT35 group, and 120.4 ± 9.9 hours in the ZO group. The applied laser techniques did not interfere with the quality of the blastocysts at day 5/6 of culture. CONCLUSION(S): ZO, ZT25, and ZT35 embryos hatched significantly earlier than the zona intact group without increasing the multiple hatching rates, suggesting an improvement of the hatching process. This study found that the pattern of the hatching process after ZT and ZO differs.

Maternal spindle transfer overcomes embryo developmental arrest caused by ooplasmic defects in mice.

The developmental potential of early embryos is mainly dictated by the quality of the oocyte. Here, we explore the utility of the maternal spindle transfer (MST) technique as a reproductive approach to enhance oocyte developmental competence. Our proof-of-concept experiments show that replacement of the entire cytoplasm of oocytes from a sensitive mouse strain overcomes massive embryo developmental arrest characteristic of non-manipulated oocytes. Genetic analysis confirmed minimal carryover of mtDNA following MST. Resulting mice showed low heteroplasmy levels in multiple organs at adult age, normal histology and fertility. Mice were followed for five generations (F5), revealing that heteroplasmy was reduced in F2 mice and was undetectable in the subsequent generations. This pre-clinical model demonstrates the high efficiency and potential of the MST technique, not only to prevent the transmission of mtDNA mutations, but also as a new potential treatment for patients with certain forms of infertility refractory to current clinical strategies.

Infertility is a growing problem that affects millions of people worldwide. Medical procedures known as in vitro fertilization (IVF) help many individuals experiencing infertility to have children. Typically in IVF, a woman's egg cells are collected, fertilized with sperm from a chosen male and grown for a few days in a laboratory, before returning them to the woman's body to continue to develop. However, there are some women whose egg cells cannot develop into a healthy baby after they have been fertilized. Many of these patients use egg cells from donors, instead. This greatly improves the chances of the IVF treatment being successful, but the resultant children are not genetically related to the intended mothers. Previous studies suggested that a cell compartment known as the cytoplasm plays a crucial role in allowing fertilized egg cells to develop normally. A new technique known as maternal spindle transfer, often shortened to MST, makes it possible to replace the entire cytoplasm of a compromised egg cell. This is achieved by transplanting the genetic material of the compromised egg cell into a donor egg cell with healthier cytoplasm that has previously had its own genetic material removed. Using this technique, it is possible to generate human egg cells for IVF that have the genetic material from the intended mother without the defects in the cytoplasm that may be responsible for infertility. However, it is not clear whether this approach would be a safe and effective way to treat infertility in humans. Costa-Borges et al. applied MST to infertile female mice and found that the technique could permanently correct deficiencies in the cytoplasms of poor quality egg cells, allowing the mice to give birth to healthy offspring. Further experiments studied the offspring and their descendants over several generations and found that they also had higher quality egg cells and normal levels of fertility. These findings open up the possibility of developing new treatments for infertility caused by problems with egg cells, so experiments involving human egg cells are now being performed to evaluate the safety and effectiveness of the technique.

Effects of different laser-drilled openings in the zona pellucida on hatching of in vitro-produced cattle blastocysts.

OBJECTIVE: To examine whether the size of artificial openings in the zona pellucida effects the hatching process in in vitro-produced cattle embryos. DESIGN: We evaluated the course of hatching of cattle blastocysts with and without laser-drilled zona pellucida openings of different sizes. SETTING: University-based cattle reproductive biotechnology laboratory. INTERVENTION(S): Cattle blastocysts were produced in vitro. Laser treatment was performed 7 days after fertilization. In group I, a small opening of 7-15 microm in width and 40 microm in length was created. In group II, an opening of at least 40 by 40 microm was created. Group III blastocysts received no laser treatment. MAIN OUTCOME MEASURE(S): Hatching course, zona pellucida thickness, diameter. RESULT(S): By day 9, the rate of hatched blastocysts was 27.1%, 63,0%, and 42,9% in groups I, II, and III, respectively. The remainder in group I were entrapped during hatching in a typical figure eight shape. The hatching rate in group II was significantly different from the other groups. All laser-treated blastocysts (groups I and II) showed no change of zona pellucida diameter until day 9. CONCLUSION(S): These results suggest that the size of the artificial openings in the zona pellucida has an influence on the hatching process.

Evaluation of laser-assisted lentiviral transgenesis in bovine.

Lentiviral transduction of oocytes or early embryos is an efficient strategy to generate transgenic rodents and livestock. We evaluated laser-based microdrilling (MD) of the zona pellucida, which is a physical barrier for viral infection, and subsequent incubation in virus suspension as a new route for lentiviral transgenesis in bovine. Lentiviral vectors carrying an eGFP expression cassette were used to transduce oocytes or zygotes after MD as compared to the established subzonal virus injection technique (MI). The type of manipulation (MD vs. MI) did not affect cleavage rates, but had a significant effect on blastocyst rates (P < 0.001). MI of virus or sham-MI (buffer) resulted in higher blastocyst rates as compared to MD, both in the oocyte and zygote treatment groups. The latter exhibited higher rates of early cleavage (P < 0.05) and blastocyst rates (P < 0.01). The proportion of eGFP expressing blastocysts was higher after infection of oocytes (MD: 44 +/- 9%; MI: 67 +/- 8%) than after infection of zygotes (MD: 26 +/- 8%; MI: 26 +/- 9%). Overall efficacy (eGFP-positive blastocysts per treated oocytes or zygotes) was highest after MI of oocytes (18 +/- 2%). Our study demonstrates the feasibility of laser-assisted lentiviral gene transfer into bovine oocytes and zygotes. However, further optimization of the procedure is required, mainly to reduce the incidence of polyspermy after MD of oocytes and to eliminate negative effects of MD on early embryonic development.