First babies conceived with Automated Intracytoplasmic Sperm Injection.

RESEARCH QUESTION: Can an automated sperm injection robot perform Automated Intracytoplasmic Sperm Injection (ICSIA) for use in human IVF? DESIGN: The ICSIA robot automated the sperm injection procedure, including injection pipette advancement, zona pellucida and oolemma penetration with piezo pulses, and pipette removal after sperm release. The robot was first tested in mouse, hamster and rabbit oocytes, and subsequently using discarded human oocytes injected with microbeads. A small clinical pilot trial was conducted with donor oocytes to study the feasibility of the robot in a clinical setting. The ICSIA robot was controlled by engineers with no micromanipulation experience. Results were compared with those obtained with manual ICSI conducted by experienced embryologists. RESULTS: The ICSIA robot demonstrated similar results to the manual procedure in the different animal models tested as well as in the pre-clinical validations conducted in discarded human oocytes. In the clinical validation, 13 out of 14 oocytes injected with ICSIA fertilized correctly versus 16 out of 18 in the manual control; eight developed into good-quality blastocysts versus 12 in the manual control; and four were diagnosed as chromosomally normal versus 10 euploid in the manual control. Three euploid blastocysts from the ICSIA robot group have been transferred into two recipients, which resulted in two singleton pregnancies and two babies born. CONCLUSIONS: The ICSIA robot showed high proficiency in injecting animal and human oocytes when operated by inexperienced personnel. The preliminary results obtained in this first clinical pilot trial are within key performance indicators.

Parameters of the Mouse Embryo Assay that affect detection of peroxides in mineral oil.

RESEARCH QUESTION: Can culture conditions influence the sensitivity of a Mouse Embryo Assay and its potential to detect peroxide-related toxicity in mineral oil samples? DESIGN: Protein type and concentration, embryo density and culture dish design were selected as the variables in the culture system with the potential to influence the assay's sensitivity. Fresh 1-cell mouse embryos were cultured under mineral oil samples with known peroxide concentrations. Protein type (human serum albumin [HSA] + α/ß-Globulins versus HSA versus bovine serum albumin [BSA]), concentration (5 mg/ml versus 0.5 mg/ml), embryo density (25 versus 3 µl/embryo) and culture dish (Petri versus micro-well dish) were adjusted to define the culture conditions with the highest sensitivity. RESULTS: High concentrations of peroxides can be easily detected by current quality control standards. However, for oil samples with a lower concentration of peroxides, supplementing the culture medium with 5 mg/ml of HSA + alpha/beta-globulins or with HSA resulted in an increased detection of embryo toxicity compared with when BSA was used as the protein supplement. The sensitivity of the assay was greatly reduced when embryos were cultured in groups and when certain micro-well dishes were used. CONCLUSIONS: Current quality control protocols may not be sensitive enough to identify low concentrations of peroxides, which, if undetected, can increase over time and become potentially harmful during gamete and embryo culture. The different parameters established in this study allow the sensitivity of the Mouse Embryo Assays to be optimized to specifically detect peroxides in mineral oil samples prior to their release into the market and their broad use in human IVF.

Pre-clinical validation of a closed surface system (Cryotop SC) for the vitrification of oocytes and embryos in the mouse model.

Vitrification is currently a well-established technique for the cryopreservation of oocytes and embryos. It can be achieved either by direct (open systems) or indirect (closed systems) contact with liquid nitrogen. While there is not a direct evidence of disease transmission by transferred cryopreserved embryos, it was experimentally demonstrated that cross-contamination between liquid nitrogen and embryos may occur, and thus, the use of closed devices has been recommended to avoid the risk of contamination. Unfortunately, closed systems may result in lower cooling rates compared to open systems, due to the thermal insulation of the samples, which may cause ice crystal formation resulting in impaired results. In our study, we aimed to validate a newly developed vitrification device (Cryotop SC) that has been specifically designed for being used as a closed system. The cooling and warming rates calculated for the closed system were 5.254 °C/min and 43.522 °C/min, respectively. Results obtained with the closed system were equivalent to those with the classic Cryotop (open system), with survival rates in oocytes close to 100%. Similarly, the potential of the survived oocytes to develop up to good quality blastocysts after parthenogenetic activation between both groups was statistically equivalent. Assessment of the meiotic spindle and chromosome distribution by fluorescence microscopy in vitrified oocytes showed alike morphologies between the open and closed system. No differences were found either between the both systems in terms of survival rates of one-cell stage embryos or blastocysts, as well as, in the potential of the vitrified/warmed blastocysts to develop to full-term after transferred to surrogate females.

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.

Effect of the enucleation procedure on the reprogramming potential and developmental capacity of mouse cloned embryos treated with valproic acid.

Mouse recipient cytoplasts for somatic cell nuclear transfer (SCNT) are routinely prepared by mechanical enucleation (ME), an invasive procedure that requires expensive equipment and considerable micromanipulation skills. Alternatively, oocytes can be enucleated using chemically assisted (AE) or chemically induced (IE) enucleation methods that are technically simple. In this study, we compared the reprogramming potential and developmental capacity of cloned embryos generated by ME, AE, and IE procedures and treated with the histone deacetylase inhibitor valproic acid. A rapid and almost complete deacetylation of histone H3 lysine 14 in the somatic nucleus followed by an equally rapid and complete re-acetylation after activation was observed after the injection of a cumulus cell nucleus into ME and AE cytoplasts. In contrast, histone deacetylation occurred at a much lower level in IE cytoplasts. Despite these differences, the cloned embryos generated from the three types of cytoplasts developed into blastocysts of equivalent total and inner cell mass mean cell numbers, and the rates of blastocyst formation and embryonic stem cell derivation were similar among the three groups. The cloned embryos produced from ME and AE cytoplasts showed an equivalent rate of full-term development, but no offspring could be obtained from the IE group, suggesting a lower reprogramming capacity of IE cytoplasts. Our results demonstrate the usefulness of AE in mouse SCNT procedures, as an alternative to ME. AE can facilitate oocyte enucleation and avoid the need for expensive microscope optics, or for potentially damaging Hoechst staining and u.v. irradiation, normally required in ME procedures.

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.

Blastocyst development in single medium with or without renewal on day 3: a prospective cohort study on sibling donor oocytes in a time-lapse incubator.

OBJECTIVE: To evaluate the efficiency of using a continuous (one-step) protocol with a single medium for the culture of human embryos in a time-lapse incubator (TLI). DESIGN: Prospective cohort study on sibling donor oocytes. SETTING: University-affiliated in vitro fertilization (IVF) center. PATIENT(S): Embryos from 59 patients. INTERVENTION(S): Culture in a TLI in a single medium with or without renewal of the medium on day-3. MAIN OUTCOME MEASURE(S): Embryo morphology and morphokinetic parameters, clinical pregnancy, take-home baby rate, and perinatal outcomes. RESULT(S): The blastocyst rates (68.3 vs. 66.8%) and the proportion of good-quality blastocysts (transferred plus frozen) obtained with the two-step (80.0%) protocol were statistically significantly similar to those obtained in the one-step protocol (72.2%). Similarly, morphokinetic events from early cleavage until late blastocyst stages were statistically significantly equivalent between both groups. No differences were found either in clinical pregnancy rates when comparing pure transfers performed with embryos selected from the two-step (75.0%), one-step (70.0%, respectively), and mixed (57.1%) groups. A total of 55 out of 91 embryos transferred implanted successfully (60.4%), resulting in a total of 37 newborns with a comparable birth weight mean among groups. CONCLUSION(S): Our findings support the idea that in a TLI with a controlled air purification system, human embryos can be successfully cultured continuously from day 0 onward in single medium with no need to renew it on day-3. This strategy does not affect embryo morphokinetics or development to term and offers more stable culture conditions for embryos as well as practical advantages and reduced costs for the IVF laboratory.

Demecolcine- and nocodazole-induced enucleation in mouse and goat oocytes for the preparation of recipient cytoplasts in somatic cell nuclear transfer procedures.

Treatment of pre-activated oocytes with demecolcine (DEM) has been shown to induce the extrusion of all oocyte chromosomes within the second polar body (PB2). However, induced enucleation (IE) rates are generally low and the competence of these cytoplasts to support embryonic development following somatic cell nuclear transfer (SCNT) is impaired. Here, we explored whether short treatments with DEM or another antimitotic, nocodazole (NOC), improve IE efficiency, and determined the most appropriate timing for nuclear transfer in the cytoplasts produced. We show, for the first time, that IE can be accomplished in mouse and goat oocytes using NOC and that short treatments with DEM or NOC result in similar IE rates, which proved to be strain- and species-specific. Because enucleation induced by both antimitotic drugs is reversible, the IE protocol was combined with the mechanical aspiration of PB2s to increase permanent enucleation rates in mouse oocytes. None of the cloned mouse embryos produced from the resultant cytoplasts developed to the blastocyst stage. However, when they were reconstructed prior to the activation and antimitotic treatment, their in vitro embryonic development was similar to that of cloned embryos produced from mechanically-enucleated oocytes.

Comparison between the effects of valproic acid and trichostatin A on the in vitro development, blastocyst quality, and full-term development of mouse somatic cell nuclear transfer embryos.

Reprogramming of differentiated nuclei into a totipotent embryonic state following somatic cell nuclear transfer (SCNT) is not efficient. Previous studies in the hybrid B6D2F1 mouse strain revealed that a transient treatment of the SCNT embryos with the histone deacetylase inhibitor (HDACi) trichostatin A (TSA) significantly enhance the potential of the cloned embryos to develop in vitro and to term. Here, we compare two different SCNT protocols with TSA and explore, for the first time, the effect of another HDACi, valproic acid (VPA), on the in vitro development, blastocyst quality, and full-term development of mouse B6CBAF1 cloned embryos. Rates of blastocyst development in SCNT embryos treated with either 5 nM TSA during and after activation (31.8%) or with 100 nM TSA or 2 mM VPA before and during activation (34.5 and 38.3%, respectively) were clearly superior to those of nontreated SCNT embryos (22.9-25.1%). These increased in vitro development rates of the HDACi-treated embryos were correlated with an increased level of histone H3 lysine 14 acetylation and an improved blastocyst quality, as judged by the increased number of total and ICM cells in comparison to the nontreated embryos (30-35% increase). Treatment of SCNT embryos with TSA or VPA also allowed the obtention of viable cloned mice, whereas none could be produced from untreated SCNT embryos. In conclusion, we have demonstrated for the first time that VPA can improve the in vitro and full-term development of B6CBAF1 SCNT embryos, at a similar level as TSA. Our findings may open new opportunities to improve cloning efficiencies in other mouse strains or species.

Antimitotic treatments for chemically assisted oocyte enucleation in nuclear transfer procedures.

Chemically assisted enucleation has been successfully applied to porcine and bovine oocytes to prepare recipient cytoplasts for nuclear transfer procedures. In this study, the antimitotic drugs demecolcine, nocodazole, and vinblastine were first assessed for their ability to induce the formation of cortical membrane protrusions in mouse, goat, and human oocytes. While only 2% of the treated human oocytes were able to form a protrusion, high rates of protrusion formation were obtained both in mouse (84%) and goat oocytes (92%), once the treatment was optimized for each species. None of the antimitotics applied was superior to the others in terms of protrusion formation, but mouse oocytes treated with vinblastine were unable to restore normal spindle morphology after drug removal and their in vitro development after parthenogenetic activation was severely compromised, rendering this antimitotic useless for chemically assisted enucleation approaches. Aspiration of the protrusions in mouse oocytes treated with demecolcine or nocodazole yielded 90% of successfully enucleated oocytes and allowed the extraction of a smaller amount of cytoplasm than with mechanical enucleation, but both enucleation methods resulted in the depletion of spindle-associated gamma-tubulin from the prepared cytoplasts. Treatment of mouse oocytes with demecolcine or nocodazole had no effect on their in vitro development after parthenogenetic activation, or on their ability to repolymerize a new spindle after the removal of the drug or the reconstruction of the treated cytoplasts with a somatic nucleus. Therefore, demecolcine- and nocodazole-assisted enucleation appears as an efficient alternative to mechanical enucleation, which can simplify nuclear transfer procedures.

Collection and cryopreservation of hamster oocytes and mouse embryos.

Embryos and oocytes were first successfully cryopreserved more than 30 years ago, when Whittingham et al. and Wilmut separately described that mouse embryos could be frozen and stored at -196 degrees C and, a few years later, Parkening et al. reported the birth of live offspring resulting from in vitro fertilization (IVF) of cryopreserved oocytes. Since then, the use of cryopreservation techniques has rapidly spread to become an essential component in the practice of human and animal assisted reproduction and in the conservation of animal genetic resources. Currently, there are two main methods used to cryopreserve oocytes and embryos: slow freezing and vitrification. A wide variety of approaches have been used to try to improve both techniques and millions of animals and thousands of children have been born from cryopreserved embryos. However, important shortcomings associated to cryopreservation still have to be overcome, since ice-crystal formation, solution effects and osmotic shock seem to cause several cryoinjuries in post-thawed oocytes and embryos. Slow freezing with programmable freezers has the advantage of using low concentrations of cryoprotectants, which are usually associated with chemical toxicity and osmotic shock, but their ability to avoid ice-crystal formation at low concentrations is limited. Slow freezing also induces supercooling effects that must be avoided using manual or automatic seeding. In the vitrification process, high concentrations of cryoprotectants inhibit the formation of ice-crystals and lead to the formation of a glasslike vitrified state in which water is solidified, but not expanded. However, due to the toxicity of cyroprotectants at the concentrations used, oocytes/embryos can only be exposed to the cryoprotectant solution for a very short period of time and in a minimum volume solution, before submerging the samples directly in liquid nitrogen. In the last decade, vitrification has become more popular because it is a very quick method in which no expensive equipment (programmable freezer) is required. However, slow freezing continues to be the most widely used method for oocyte/embryo cryopreservation. In this video-article we show, step-by-step, how to collect and slowly freeze hamster oocytes with high post-thaw survival rates. The same procedure can also be applied to successfully freeze and thaw mouse embryos at different stages of preimplantation development.