Junko Otsuki

Damage of embryo development caused by peroxidized mineral oil and its association with albumin in culture

OBJECTIVES To examine the effect of free radicals from peroxidized oil and the role of albumin on the passage of radicals. DESIGN Prospective study. SETTING Clinical IVF laboratory and university department. PATIENT(S) Blood samples were donated by laboratory staff. INTERVENTION(S) Examination of the effects of mineral oil samples with various peroxide value (POV) on culture of erythrocytes and on the passage of a lipophilic tracer, DiI, into the zona pellucida. MAIN OUTCOME MEASURE(S) Time required for hemolysis of red blood cells by peroxidized oil, staining of zona pellucida from human oocytes and embryos by lipophilic tracer, and POV analysis of mineral oil samples in relation to various storage conditions. RESULT(S) The time for hemolysis was related to the POV levels of oil samples covering the culture medium. Albumin in the medium facilitated hemolysis and the passage of DiI into the zona. Peroxidized oil (POV >0.02 meq/kg) blocked the entry of DiI into the zona. CONCLUSION(S) The presence of albumin in the medium was associated with the entry into the human zona of agents present in peroxidized mineral oil. This process and variable oil peroxidation could be deleterious to embryos in culture.

A phase of chromosome aggregation during meiosis in human oocytes

A higher rate of chromosomal abnormality occurs in human oocytes compared with other animal oocytes. In this study, chromosome movement has been successfully observed during first and second meiosis using a time-lapse culture system and a differential interference contrast inverted microscope. In human oocytes, a specific sequence of early maturation changes was observed. Following the completion of nucleolar breakdown, chromosomes were assembled into a single aggregation that heralded the start of nuclear membrane breakdown. The chromosome aggregation phase (gere phase) persisted after germinal vesicle (GV) breakdown, lasting several hours, and a similar gere phase (chromosome gathering) occurred after the first polar body extrusion, lasting 1-4 h. In contrast, in mouse GV oocytes, nucleolar and nuclear membranes started to break down almost at the same time. A chromosome aggregation phase was not observed in mouse oocytes. The discovery of a gere phase during human oocyte maturation may provide important information related to the mechanism of abnormal chromosomal segregation, which often occurs during meiosis.

The influence of the redox state of follicular fluid albumin on the viability of aspirated human oocytes

A number of reports have suggested that the oxidative state of human albumin in serum and in some body fluids is associated with cell damage. However there are no reports on the redox state of human follicular fluid (FF) and its influence on oocyte viability. The aim of this study was to examine the relationship between the redox state of FF and serum on oocyte viability. The cytoplasmic condition of oocytes was evaluated microscopically at collection in 117 women. Deteriorating oocytes were recognized by degenerative changes in their cytoplasm. The redox state of FFs that yielded degenerated oocytes was evaluated and compared with fluids containing normal oocytes. The redox state of the corresponding FF and serum, at the time of oocyte retrieval, was analyzed by high performance liquid chromatography. The redox state of FF that contained degenerated oocytes was found to have a significantly elevated oxidized state compared with the FFs that yielded normal oocytes. Also the albumin in the FF of patients was found to be predominantly in the reduced state compared with that in their serum at the time of oocyte retrieval. In addition, increasing age and endometriosis were found to shift the redox of serum to the oxidative state. We propose that the reduced state of albumin in FF may play an important role in protecting oocytes from oxidative damage.

Timed IVM followed by ICSI in a patient with immature ovarian oocytes

A complete failure of meiotic maturation occasionally occurs following human chorionic gonadotrophin administration during IVF-intracytoplasmic sperm injection (ICSI) cycles. ICSI on day 1 is commonly used to allow maturation in culture. However, if the oocytes become mature in the evening soon after their recovery but ICSI is delayed until the next day, then subsequent ageing of matured oocytes may be unfavourable for fertilization and development. To avoid the deterioration associated with oocyte ageing, the timing of polar body extrusion was checked every 3 h and rescue in-vitro maturation (IVM)-ICSI was performed shortly after the polar body extrusion was confirmed. This report describes a successful pregnancy and birth of a healthy baby in a patient who had no mature oocytes at the time of oocyte retrieval, and illustrates the value of extra monitoring for IVM and ICSI in cases where only immature oocytes are available.

Symmetrical division of mouse oocytes during meiotic maturation can lead to the development of twin embryos that amalgamate to form a chimeric hermaphrodite

BACKGROUND Gentle compression of mouse oocytes during meiosis-1 prevented the usual extrusion of a small polar body and resulted in the symmetrical division of the ooplasm into two cells of similar size within the zona pellucida. The purpose of our study was to determine whether such cells, equivalent to two small oocytes, were capable of embryonic development and would result in birth following transfer to the uterus. METHODS IVF of the 2-celled oocytes was performed and the twin intra-zonal embryos were observed. In each case, the two embryos that originated from fertilized cells with two pronuclei were observed to amalgamate and form a single morula and subsequent blastocyst that was transferred to the uterus of a recipient of a different mouse strain. FISH analysis was performed on sectioned paraffin-embedded tissue of the offspring. RESULTS In symmetrically divided oocytes each cell contained a metaphase II spindle. Both cells were fertilizable and cleaved to form twin embryos within the same zona pellucida. Most twin embryos amalgamated to form a single compacted morula, which progressed to hatched blastocysts that contained a single inner cell mass. In total, 104 of these blastocysts were transferred to 19 mice, two of which became pregnant, resulting in the birth of three offspring. FISH analysis showed that one newborn contained both XX and XY cells. CONCLUSIONS We found that two small oocytes fertilized within the same zona pellucida to form twin embryos that amalgamate to establish a single chimeric embryo. This may be one mechanism that leads to the formation of a chimeric hermaphrodite when an embryo containing XX cells mixes with its intra-zonal twin containing XY cells.

Grade and looseness of the inner cell mass may lead to the development of monochorionic diamniotic twins.

OBJECTIVE To examine the relationship between the inner cell mass (ICM) grade and its morphological configuration on the occurrence of monochorionic diamniotic (M-D) twinning. DESIGN Retrospective embryo cohort study. SETTING Private IVF clinic. PATIENT(S) Evaluation of frozen-thawed single blastocyst transfers with hormone replacement treatment in 8,435. This cohort included 71 blastocysts and their ICMs observed by time-lapse photography. INTERVENTION(S) Any changes in configuration of the ICMs observed by time-lapse photography were analyzed retrospectively. MAIN OUTCOME MEASURE(S) The amount of loosening of blastomeres within the ICM was evaluated by time-lapse observations. The number of cells that were involved in the loosening process was also assessed. Both of these parameters were correlated with the type of monozygotic twinning that eventuated. RESULT(S) The M-D twinning incidence resulting from blastocysts with a high grade ICM (grade A) were transferred was 0.38% (3/796), whereas it was significantly higher, 1.38% (34/2,463), when blastocysts with a poorer (B and C) grade ICM were transferred. Among 71 transferred frozen-thawed blastocysts that were studied with time-lapse photography, there were two dichorionic diamniotic and one M-D twins. Careful observations of the embryo that resulted in the one M-D case, revealed that the ICM acquired a looser appearance due to decompaction of at least eight cells. This type of decompaction was not observed in the ICMs of other transferred blastocysts. CONCLUSION(S) The occurrence of M-D twinning may be avoided by excluding blastocysts that contain decompacting ICMs.

The redox state of recombinant human serum albumin and its optimal concentration for mouse embryo culture

Albumin has multiple physiological functions in embryo culture, such as a chelator of heavy metals, free radical scavenger, pH and osmotic regulator, a stabilizer, growth factor carrier, a surfactant, and a nutrient. However, the commercially available human serum albumin (HSA) products may not be completely safe since they could be contaminated with viruses and prions. Recombinant human serum albumin (rHSA) has been reported to be as efficient as commercial HSA for fertilization and embryo development. Despite the possible benefits of rHSA, it has not been widely used for embryo culture due to its high cost of production. Our objective was to analyze the redox state of different types of HSA products and rHSA to define oxidative status batch variations of HSA and rHSA and to evaluate the optimal concentration of rHSA for mouse embryo culture. The redox state of the HSA and rHSA used in embryo culture media was found to vary widely. Redox variations were found among different HSA batches as well as among rHSA batches. The highest blastocyst development and hatching rates were obtained with rHSA used at a concentration of 0.05 mg/mL. We showed that very low concentrations of rHSA were most favorable for advanced mouse embryo development in culture.

Association of spindle midzone particles with polo-like kinase 1 during meiosis in mouse and human oocytes

Polo-like kinase 1 (Plk1) has been reported to localize to the spindle midzone during meiosis in mouse oocytes. However, it has not been reported in human oocytes. In this study, the interaction of the meiotic structures and chromosome segregation in mouse and human oocytes were studied by time-lapse differential interference contrast microscopy. Using immunocytochemical studies, the localization of polo-like kinase 1 and its association with microtubules were examined during the extrusion of first and second polar bodies. It was found that Plk1 was localized in the spindle midzone in human oocytes at anaphase I and telophase I. Also, three-dimensional confocal laser microscopy showed that the meiotic spindle midzone contained numerous dot-like particles that were stained by anti-Plk1 antibody. These particles were aligned in the plane of the meiotic midzone in mouse and human oocytes.

Aggregated chromosomes transfer in human oocytes.

Germinal-vesicle (GV) transfer, spindle-chromosome complex transfer in metaphase-II oocytes and two pronuclei transfer have been evaluated as possible treatments for patients who have mitochondrial diseases. However, GV transfers often lead to heteroplasmy while the other two methods are frequently associated with aneuploidy. The present study used a new method based on the transfer of aggregated chromosomes, which occurs in human oocytes, before the metaphase spindle is established.

A higher incidence of cleavage failure in oocytes containing smooth endoplasmic reticulum clusters

PurposeIn human oocytes, sERCs are one of the dysmorphic phenotypes that have been reported. Significantly reduced pregnancy rates and a comparatively higher number of abnormities in live births appear to be associated with the presence of sERCs in oocytes. However, some reports have shown that healthy babies can be born, without any reduced pregnancy rates, from oocytes observed to contain sERCs. Thus, the clinical and scientific significance of oocytes that harbor sERCs remains controversial.MethodsThe presence of sERCs was evaluated using a time-lapse system while studying the dynamic changes within oocytes and embryos. Logistic regression analysis was carried out to explore the independent variables for meiotic and mitotic cleavage failure..ResultsThe incidence of mitotic cleavage failure and the incidence of meiotic cleavage failure during the second polar body extrusion in oocytes with sERCs were found to be significantly higher than that in oocytes without sERCs. Furthermore, ICSI was found to have a greater frequency of meiotic failure than IVF.ConclusionsIn cases of cleavage failure, an embryonic cell could become tetraploid and may induce abnormal chromosomal configurations. Some cells exposed to cleavage failure may become trophectoderm cells and form placental abnormalities. Even if they develop into trophectoderm cells, the ICM can be susceptible to further cleavage failure and may in turn cause further aneuploidy. For these reasons, it is important to monitor pregnancies and births derived from oocytes that contained sERCs.