Clinical Outcomes of Rescue Intracytoplasmic Sperm Injection at Different Timings Following In Vitro Fertilization

Background: Although rescue intracytoplasmic sperm injection (r-ICSI) is extensively used worldwide, the indication of r-ICSI and its optimal timing remains obscure. This study aimed to assess the outcomes of r-ICSI following in vitro fertilization in different timings when fertilization is confirmed. Methods: This study included 5,156 cycles (47,785 eggs). Fertilization was confirmed by polar body analysis after 4 and 6 hr of coincubation of the sperm and oocyte. Oocytes that underwent IVF were divided into two groups based on the time when a second polar body was detected in more than 30% of all oocytes (Four-hr group and six-hr group). If the second polar body was not detected or was present in less than 30% of all oocytes after six hr of coincubation, rescue-ICSI (r-ICSI) was performed for oocytes without a second polar body (r-ICSI group). Results: The fertilization rates of two pronuclear (2PN) oocytes in the three groups (Four-hr group, six-hr group, and r-ICSI group) were 70.7%, 51.3%, and 58.0%, respectively. The blastocyst formation rates were 62.8%, 53.4%, and 42.9%, respectively. Conclusion: Performing r-ICSI after six hr of coincubation can salvage cases with fertilization failure in IVF. The higher fertilization rate of r-ICSI indicates that all oocytes without signs of fertilization after six hr of coincubation should undergo r-ICSI.

Parthenogenetic mosaicism: generation via second polar body retention and unmasking of a likely causative PER2 variant for hypersomnia

Background Parthenogenetic mosaicism is an extremely rare condition identified only in five subjects to date. The previous studies indicate that this condition is mediated by parthenogenetic activation and is free from a specific phenotype ascribed to unmaking of a maternally inherited recessive variant in the parthenogenetic cell lineage. Results We examined a 28-year-old Japanese 46,XX female with Silver-Russell syndrome and idiopathic hypersomnia. The results revealed (1) predominance of maternally derived alleles for all the differentially methylated regions examined; (2) no disease-related copy-number variant; (3) two types of regions for all chromosomes, i.e., four BAF (B-allele frequency) band regions with single major microsatellite peaks of maternal origin and single minor microsatellite peaks of non-maternal (paternal) origin, and six BAF band regions with single major microsatellite peaks of maternal origin and two minor microsatellite peaks of maternal and non-maternal (paternal) origin; (4) an unmasked extremely rare PER2 variant (c.1403G>A:p.(Arg468Gln)) with high predicted pathogenicity; (5) mildly affected local structure with altered hydrogen bonds of the p.Arg468Gln-PER2 protein; and (6) nucleus-dominant subcellular distribution of the p.Arg468Gln-PER2 protein. Conclusions The above findings imply that the second polar body retention occurred around fertilization, resulting in the generation of the parthenogenetic cell lineage by endoreplication of a female pronucleus and the normal cell lineage by fusion of male and female pronuclei, and that the homozygous PER2 variant in the parthenogenetic cells is the likely causative factor for idiopathic hypersomnia.

Early Rescue Intracytoplasmic Sperm Injection in Partial Fertilization Failure Is Necessary and Safe: A Retrospective Analysis of 1223 Patients

BACKGROUND: Total or near total fertilization failure remains inevitable in conventional in vitro fertilization (IVF). The experts searched for the release of the second polar body 6 h after initial insemination, and rescued those oocytes with unclear second polar body by ICSI (early R-ICSI). Studies found that early R-ICSI can effectively decrease complete IVF fertilization failure and excessive ICSI treatment.METHODS: We performed a retrospective study in 16,769 patients (short-term IVF, n=12,094; ICSI, n=3,452; early R-ICSI, n=1,223) who received IVF/ICSI treatment in our hospital from January 2009 to October 2018. Fertilization and clinical outcomes were compared among those three groups.RESULTS: When considering the R-ICSI embryos in the early R-ICSI group independently, the rates of fertilization and day-3 cleaved embryos in 2PN oocytes were comparable, the rates of fertilization (2PN) and high-quality embryos were lower, whereas the multi-PN fertilization rate (3.27%) was significantly higher than the ICSI group (1.26%). The difference of clinical pregnancy rate between the part of transferred R-ICSI embryos (40.81%) and the ICSI group (44.73) remained nonsignificant. Furthermore, the rate of congenital birth defects in the early R-ICSI group (0.99%) was not significantly different from those in the short-term IVF (0.76%) and ICSI groups (1.07%).CONCLUSIONS: Despite the multi-PN fertilization rate, our study highlights early R-ICSI as a safe and effective alternative in assisted reproduction to reduce ICSI utilization. Additional large amount and long-term follow-up studies are needed to further validate the use of early R-ICSI.

Production of triploid, doubled haploid (DH) and meiogynogenetic brook trout (Salvelinus fontinalis) – efficiency and development of body deformities

In the present research we produced triploid, mitogynogenetic (doubled haploid; DH) and meiogynogenetic brook trout (Salvelinus fontinalis) to examine efficiency of these technologies and potential susceptibility of chromosome set manipulated individuals for the spinal disorders. Triploidy was induced by shocking (High Hydrostatic Pressure – HHP) of fertilized eggs 30 min. after insemination. In turn, gynogenetic development was induced by activation of eggs with UV-irradiated sperm. Activated eggs were then exposed to HHP shock applied 30 and 420 minutes after insemination to provide meiogynogenotes and gynogenetic DHs, respectively. When compared to non-manipulated diploids, the highest survival rates was observed among triploid brook trout while DHs showed the highest mortality. Malformation rates in the diploid larvae from the control groups did not exceed 7.0% while percentage of malformed triploid individuals equaled 19.1%. Drastically increased number of deformed larvae (> 30%) was observed in both, DH and meiogynogenetic individuals. Intensification of kyphosis and scoliosis was clearly demonstrated in the gynogenetic and triploid brook trout. Genetic factors such as increased number of sets of chromosomes in triploids and expression of lethal alleles in the gynogenetic fish plus side effects of HHP shock utilized for retention of the second polar body or inhibition of the first cell cleavage when induced triploid and gynogenetic development have been discussed to affect survival rates and prevalence for the skeletal deformities in the chromosome set manipulated brook trout.

Effects of high calcium levels on the disturbed extrusion of the second polar body during in vitro fertilization in C3H/He mouse substrains

SummaryWe previously reported that high concentrations (≥3.42 mM) of calcium during in vitro fertilization (IVF) disturbed the extrusion of the second polar body (PBII) in C3H/He inbred mice. In this study, the substrain specificity of this phenomenon was examined under 1.71–6.84 mM calcium concentration in ova from six C3H/He mouse commercially available substrains in Japan. PBII extrusion in ova from J substrains was not affected by calcium concentrations (<10% at any calcium level), but was grossly disturbed at high calcium levels in the ova of other substrains. This result has practical applications for the efficient production of normal zygotes by IVF, therefore contributing to the reduction in the numbers of donor animals for further zygote or embryo manipulation. Care must be taken in choosing IVF medium for particular strains and substrains.

Increased mitochondrial distribution in early-cleaving embryos indicate successful pre-implantation development

The timing of the first zygotic cleavage is an accurate predictor of embryo quality. Embryos that cleaved early have higher developmental viability compared to their late counterparts. During embryonic development, cleavage is affected by cellular metabolic processes performed by mitochondria and its synergistic interaction with endoplasmic reticulum (ER). However, in depth study on differences of mitochondria and ER ultrastructures in early- cleaving (EC) versus late- cleaving (LC) embryos is limited. This study compares mitochondria and ER ultrastructures of EC versus LC embryos using Confocal Laser Scanning Microscopy (CLSM) and Transmission Electron Microscopy (TEM). Embryos were obtained from female ICR superovulated mice, 28-30 hours post hCG. Two-cell embryos were categorized as early-cleaving (EC), while zygotes with the second polar body and two pronuclei present were categorized as late-cleaving (LC). The LC embryos were cultured in vitro until the 2- cell stage. In EC embryos, mitochondria were mostly found at the perinuclear region and closely associated with dense ER. Meanwhile, mitochondria of LC embryos were distributed uniformly within the cytoplasm. Mitochondrial fluorescence intensity was significantly higher in EC versus LC [(18.7 ± 0.4) versus (14.6 ± 0.4)] x 105 pixel, (p<0.01). Development to the blastocyst stage was also significantly higher in EC compared to LC embryos (96.7% versus 60.9%) (p<0.01). Higher viability of EC embryos is attributed to the close association of their mitochondria to ER. This contributed to better mitochondrial fission, resulting in enhanced energy generating processes and preimplantation development.

Transcriptome Analysis of Rainbow Trout (Oncorhynchus mykiss) Eggs Subjected to the High Hydrostatic Pressure Treatment

High hydrostatic pressure (HHP) causes depolymerization of the spindle microtubules. HHP applied to fish eggs prevents extrusion of the second polar body and inhibits the first cell cleavage, and it is used to produce triploids and diploid gynogenetic and androgenetic individuals. HHP has been also found to affect biomolecules including nucleic acids, and it may be presumed that HHP administered to the rainbow trout (Oncorhynchus mykiss) eggs disturbs cytoplasmic maternal RNA indispensable for the early embryogenesis. To verify this assumption, quality and quantity of RNA extracted from the rainbow trout eggs subjected to the high hydrostatic pressure shock were analyzed. Provided results exhibited that maternal transcriptome was resistant to a three-minute exposure to 65.5 MPa of HHP treatment. Some trend showing increase of the RNA integrity was observed in the HHP-treated eggs; however, the difference was not statistically significant. Alterations in the expression profiles in the rainbow trout eggs subjected to HHP were also negligible. Greater differences in the maternal gene expression were observed between eggs from different clutches than between HHP-treated and untreated eggs from the same clutch. It may be assumed that exposure to HHP shock was too short to modify significantly maternal transcripts in the rainbow trout eggs.