Laser-assisted zona pellucida thinning does not facilitate hatching and may disrupt the in vitro hatching process: a morphokinetic study in the mouse

STUDY QUESTION: Does laser-assisted zona thinning of cleavage stage mouse embryos facilitate hatching in vitro? SUMMARY ANSWER: No, unlike laser zona opening, zona thinning does not facilitate embryo hatching. WHAT IS KNOWN ALREADY: Artificial opening of the zona pellucida facilitates hatching of mouse and human embryos. Laser-assisted zona thinning has also been used for the purpose of assisted hatching of human embryos but it has not been properly investigated in an animal model; thinning methods have produced inconsistent clinical results. STUDY DESIGN, SIZE, DURATION: Time-lapse microscopy was used to study the hatching process in the mouse after zona opening and zona thinning; a control group of embryos was not zona-manipulated but exposed to the same laser energy. PARTICIPANTS/MATERIALS, SETTING, METHODS: Eight-cell CB6F1/J mouse embryos were pooled and allocated to three groups (n = 56 per group): A control group of embryos that were exposed to a dose of laser energy focused outside the zona pellucida (zona intact); one experimental group of embryos in which the zona pellucida was opened by complete ablation using the same total number of pulses as the control group; a second experimental group of embryos in which the zona pellucida was thinned to establish a smooth lased area using the same number of pulses as used in the other two groups. The width of the zona opening was 25 mum and width of the thinned area was 35 mum. Development was monitored by time-lapse microscopy. Overall treatment differences for continuous variables were analyzed by analysis of variance and pairwise comparisons using the Student t-test allowing for unequal variances, while for categorical data, a standard chi-squared test was utilized for all pairwise comparisons. MAIN RESULTS AND THE ROLE OF CHANCE: The frequency of complete hatching was 33.9% in the control group, 94.4% after zona opening, and 39.3% after zona thinning (overall group comparison, P \u3c 0.0001). Overall, 60.7% of the zona-thinned embryos did not complete the hatching process and remained trapped within the zona; when they did hatch, they did not necessarily hatch from the zona-thinned area. Hatching in about one-third of the zona-intact embryos began with breaches at multiple sites by small groups of cells. Likewise, 53.6% of zona-thinned embryos had multiple breaches, always involving an area outside the thinned zone. Zona opening decreased multiple breaching and led to blastocyst escape an average of 14 h earlier than zona-thinned embryos and 5.5 h before control embryos (P = 0.0003). LIMITATIONS, REASONS FOR CAUTION: The experiments presented here were limited to in vitro experiments performed in the mouse. Whether human embryos would behave the same way under similar circumstances is unknown. We postulate that zona thinning is not beneficial in human embryos. WIDER IMPLICATIONS OF THE FINDINGS: The experiments demonstrate that zona thinning is not equivalent to zona opening for assisted hatching. The study provides reason for systematic reviews of assisted hatching trials to take the method of assisted hatching into consideration and not combine the results of zona thinning and zona opening procedures. STUDY FUNDING/COMPETING INTERESTS: Institutional funds were used for the study. No competing interests are declared

Monochorionic-triamniotic triplet pregnancy after intracytoplasmic sperm injection, assisted hatching, and two-embryo transfer: first reported case following IVF

BACKGROUND: We present a case of monochorionic-triamniotic pregnancy that developed after embryo transfer following in vitro fertilization (IVF). METHODS: After controlled ovarian hyperstimulation and transvaginal retrieval of 22 metaphase II oocytes, fertilization was accomplished with intracytoplasmic sperm injection (ICSI). Assisted embryo hatching was performed, and two embryos were transferred in utero. One non-transferred blastocyst was cryopreserved. RESULTS: Fourteen days post-transfer, serum hCG level was 423 mIU/ml and subsequent transvaginal ultrasound revealed a single intrauterine gestational sac with three separate amnion compartments. Three distinct foci of cardiac motion were detected and the diagnosis was revised to monochorionic-triamniotic triplet pregnancy. Antenatal management included cerclage placement at 19 weeks gestation and hospital admission at 28 weeks gestation due to mild preeclampsia. Three viable female infants were delivered via cesarean at 30 5/7 weeks gestation. CONCLUSIONS: The incidence of triplet delivery in humans is approximately 1:6400, and such pregnancies are classified as high-risk for reasons described in this report. We also outline an obstetric management strategy designed to optimize outcomes. The roles of IVF, ICSI, assisted embryo hatching and associated laboratory culture conditions on the subsequent development of monozygotic/monochorionic pregnancy remain controversial. As demonstrated here, even when two-embryo transfer is employed after IVF the statistical probability of monozygotic multiple gestation cannot be reduced to zero. We encourage discussion of this possibility during informed consent for the advanced reproductive technologies

Monozygotic multiple gestation following in vitro fertilization: analysis of seven cases from Japan

We present a series of monozygous multiple gestations achieved following in vitro fertilization (IVF): one case of monochorionic triplet pregnancy and six cases of dizygotic triplet pregnancy. From September 2000 to December 2006, all patients achieving clinical pregnancy by ART were reviewed (n = 2433). A 37 year-old woman who delivered a healthy singleton after IVF returned two years later for FET, and a single blastocyst was transferred. This also resulted in pregnancy, but TV-USG revealed a single gestational sac with three distinct amniotic sacs, each containing a distinct fetal pole with cardiac activity. This pregnancy was electively terminated at nine weeks' gestation. An additional six cases of dizygotic triplets established after fresh embryo transfer (no ICSI or assisted hatching) are also described. Of these, one resulted in a miscarriage at eight weeks' gestation and five patients have an ongoing pregnancy. This case series suggests the incidence of dizygotic/monochorionic triplets following IVF is approximately 10 times higher than the expected rate in unassisted conceptions, and underscores the importance of a conservative approach to lower the number of embryos at transfer. The role of embryo transfer technique and in vitro culture media in the twinning process requires further study

Reverse genetic studies of mitochondrial DNA-based diseases using a mouse model

In the situation that it would not be able to produce model animals for mitochondrial diseases caused by mitochondrial DNA (mtDNA) with pathogenic mutations, we succeeded in generating mice with pathogenic deletion mutant mtDNA (ΔmtDNA), named “mito-mice”, by direct introduction of mitochondria with ΔmtDNA into mouse zygotes. In the mito-mice, accumulation of ΔmtDNA induced mitochondrial respiration defects in various tissues, resulting in mitochondrial disease phenotypes, such as low body weight, lactic acidosis, ischemia, myopathy, heart block, deafness, male infertility, and renal failure. Thus, mito-mice are the first model animal for mtDNA-based diseases, and the mice could be valuable for understanding precise pathogeneses and testing therapies of mitochondrial diseases. In the present review, we summarized reverse genetic studies using the mito-mice