In vitro maturation of human immature oocytes for fertility preservation and research material

AimIn recent years, the importance of fertility preservation (FP) has increased. In vitro maturation (IVM), an important technique in FP, has started to be used in the clinic, but controversies persist regarding this technique. Here, a survey of IVM for FP is provided.MethodsBased on a literature review, the applications of FP, methods of FP, IVM of oocytes that had been collected in vivo and ex vivo, maturation of oocytes after IVM for FP, cryopreservation of oocytes for FP, explanation of the procedures to patients, and recent research on FP using IVM were investigated.ResultsAlthough IVM for FP remains controversial, the application of FP is expected to expand. Depending on the age and disease status of the patient, various methods of oocyte collection and ovarian stimulation, as well as various needle types and aspiration pressures, have been reported. The maturation rate of IVM in FP ranges widely and requires optimization in the future. In regard to cryopreservation for matured oocytes, the vitrification method is currently recommended.ConclusionRegarding FP for patients with cancer, the treatment of cancer is prioritized; thus, the time and use of medicines are often constrained. As several key points regarding IVM remain unclear, well-designed and specific counseling for patients is necessary

A case of ovarian mucinous cystadenoma in a child that recurred 1 year after surgery

Introduction and importanceIn children, mature cystic teratomas are the most common ovarian tumors. Mucinous cystadenomas are rarely seen. Further, the recurrence of mucinous cystadenomas is very rare. This report describes a case of ovarian mucous cystadenoma in an adolescent that recurred 1 year after surgery.Case presentationA 13-year-old patient, with a sizable ovarian tumor underwent laparoscopic-assisted cystectomy. On histopathology, the tumor was diagnosed to be an ovarian mucinous cystadenoma. The mucinous cystadenoma recurred 13 months after surgery and subsequently laparoscopic right adnexectomy was performed.Clinical discussionIt has been reported that intraoperative cyst rupture and cystectomy instead of adnexectomy are risk factors for mucinous cystadenoma recurrence. Close follow-up is required for post-cystectomy patients because of the possibility of recurrence.ConclusionThe risk of recurrence and the preservation of fertility should be carefully considered when deciding on treatment in young patients with a mucinous cystadenoma

Oocyte collection and in vitro maturation after train transportation of human follicular fluid aspirated from resected non-stimulated ovaries of patients with endometrial adenocarcinoma

PurposeImmature human oocytes from resected ovaries can be used for research and fertility preservation, though it is unknown whether it is feasible to transport oocytes for these purposes. This study examined in vitro maturation (IVM) outcomes after the transportation of human follicular fluid (HFF) containing oocytes. MethodsFourteen patients with endometrial adenocarcinoma were enrolled. Oocytes obtained from the resected ovaries of seven patients were transported with HFF by railway (transportation group). Samples of HFF from the other seven patients were not transported, and IVM was performed promptly (non-transportation group). The results of oocyte retrieval and IVM were compared. ResultsThe average ages in the transportation and non-transportation groups were 40.12.0 and 39.6 +/- 1.8years, respectively, and the average numbers of collected oocytes were 8.1 +/- 8.4 and 5.1 +/- 5.1, respectively. There was a significant negative correlation between the number of collected oocytes and age. The proportions of oocytes that reached meiosis II (maturation rate) after IVM were 38.6% and 69.2% in the transportation and non-transportation groups, respectively (P=0.013). ConclusionIn this preliminary study, the usefulness of the transportation of HFF was limited. Further studies on maintaining oocyte normality during transportation are necessary for becoming the effective method for research and clinical use

Human frozen-thawed blastocyst morphokinetics observed using time-lapse cinematography reflects the number of trophectoderm cells

Recent studies reported morphokinetic indices for optimal selection of embryos in assisted reproductive technology (ART). The morphokinetics in blastocyst stage include the collapse and re-expansion rates after thawing. However, evaluation methods using these morphokinetics have not been established, mainly because the underlying molecular mechanisms remain unclarified. In this study, we focused on the relationship between these morphokinetic observation of the blastocyst behaviour and the number of cells constituting the blastocyst. We evaluated 38 surplus human frozen-thawed blastocysts using time-lapse cinematography and recorded their expansion, contraction, and hatching. A total of 28 blastocysts expanded in culture (cross-sectional area >= 5,000 Pi mu m(2)). In comparison to the ones that did not, the expanded group presented significantly more number of inner cell mass (ICM) and trophectoderm (TE) cells, which eventually develop into the fetus and placenta, respectively (ICM: Expanded 10.2 +/- 6.3 vs. Non-Expanded 6.0 +/- 12.3, p<0.05; TE: Expanded 165.7 +/- 74.8 vs. Non-Expanded 57.0 +/- 29.4, p<0.05). Moreover, a positive correlation was found between the expansion rate (up to 4 h) and the number of TE cells (r=0.558, p=0.0021). Additionally, blastocysts that hatched had a significantly higher number of TE cells than those that did not (hatching 225.2 +/- 61.2 vs. no hatching 121.1 +/- 48.6, p<0.0001). The number of TE cells per unit of cross-sectional area correlated negatively with the contraction time (r=-0.601, p=0.0007). No correlation between the number of ICM cells and these morphokinetics was detected. In conclusion, our study demonstrates that different morphokinetics of frozen-thawed blastocysts reflect the number of TE cells. The differentiation of blastocysts containing sufficient TE cells would be beneficial for implantation and prognosis of a subsequent pregnancy. Thus, evaluation of these morphokinetics can be an effective method to screen good embryos for ART

Novel method for immunofluorescence staining of mammalian eggs using non-contact alternating-current electric-field mixing of microdroplets

Recently, a new technique was developed for non-catalytically mixing microdroplets. In this method, an alternating-current (AC) electric field is used to promote the antigen-antibody reaction within the microdroplet. Previously, this technique has only been applied to histological examinations of flat structures, such as surgical specimens. In this study, we applied this technique for the first time to immunofluorescence staining of three-dimensional structures, specifically, mammalian eggs. We diluted an antibody against microtubules from 1:1,000 to 1:16,000, and compared the chromatic degree and extent of fading across dilutions. In addition, we varied the frequency of AC electricfield mixing from 5 Hz to 46 Hz and evaluated the effect on microtubule staining. Microtubules were more strongly stained after AC electric-field mixing for only 5 minutes, even when the concentration of primary antibody was 10 times lower than in conventional methods. AC electric-field mixing also alleviated microtubule fading. At all frequencies tested, AC electric-field mixing resulted in stronger microtubule staining than in controls. There was no clear difference in a microtubule staining between frequencies. These results suggest that the novel method could reduce antibody consumption and shorten immunofluorescence staining time

The location of 8-shaped hatching influences inner cell mass formation in mouse blastocysts

The hatching of a blastocyst where the blastocyst portions on the inside and the outside of the zona pellucida feature a figure-of-eight shape is termed 8-shaped hatching; this type of hatching has been reported to affect the proper presentation of the inner cell mass (ICM) in both human and mouse embryos. Here, our aim was to investigate the factors that affect ICM presentation during 8-shaped hatching. We performed IVF by using B6D2F1 female mice and ICR male mice, and used the 104 captured blastocysts. Embryos were maintained in KSOM at 37 degrees C in a 5% CO2, 5% O-2, and 90% N-2 environment, and their growth behavior was monitored individually and continuously using time-lapse cinematography. At 120 h after insemination, embryos were immunostained and examined under a confocal microscope. We used the hatching form to identify 8-shaped hatching, and we classified the 8 shaped- hatching blastocysts into two groups, one in which the hatching site was near the ICM center, and the other in which the hatching site was far from the ICM center. We measured each group for ICM size and the number of Oct3/4-positive cells. Of the 95 hatching or hatched embryos, 74 were 8-shaped-hatching blastocysts, and in these embryos, the ICM was significantly wider when the hatching site was near the ICM than when the hatching site was far from the ICM (P = 0.0091). Moreover, in the 8-shaped-hatching blastocysts in which the ICM was included in the blastocyst portion outside the zona pellucida. the portion defined as the outside blastocyst. after the collapse of this outside blastocyst, the ICM adhered to the trophectoderm of the outside blastocyst, opposite the hatching site. Our results indicate that in 8-shaped-hatching blastocysts, the hatching site and the collapse of outside blastocyst affect ICM formation. Thus, the assessment of 8-shaped hatching behaviors could yield indices for accurately evaluating embryo quality

Cell-free DNA in spent culture medium effectively reflects the chromosomal status of embryos following culturing beyond implantation compared to trophectoderm biopsy

This prospective study evaluated the accuracy of non-invasive preimplantation genetic testing for aneuploidy (niPGT-A) using cell-free DNA in spent culture medium, as well as that of preimplantation genetic testing for aneuploidy (PGT-A) using trophectoderm (TE) biopsy after culturing beyond implantation. Twenty frozen blastocysts donated by 12 patients who underwent IVF at our institution were investigated. Of these, 10 were frozen on day 5 and 10 on day 6. Spent culture medium and TE cells were collected from each blastocyst after thawing, and the embryos were cultured in vitro for up to 10 days. The outgrowths after culturing beyond implantation were sampled and subjected to chromosome analysis using next-generation sequencing. Chromosomal concordance rate, sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV), false-positive rate (FPR), and false-negative rate (FNR) of niPGT-A and PGT-A against each outgrowth were analyzed. The concordance rate between the niPGT-A and outgrowth samples was 9/16 (56.3%), and the concordance rate between the PGT-A and outgrowth samples was 7/16 (43.8%). NiPGT-A exhibited 100% sensitivity, 87.5% specificity, 88.9% PPV, 100% NPV, 12.5% FPR, and 0% FNR. PGT-A exhibited 87.5% sensitivity, 77.8% specificity, 87.5% PPV, 75% NPV, 14.3% FPR, and 22.2% FNR. NiPGT-A may be more accurate than PGT-A in terms of ploidy diagnostic accuracy in outgrowths

Live visualisation of electrolytes during mouse embryonic development using electrolyte indicators

Studies have shown that some electrolytes, including Na+ and K+, play important roles in embryonic development. However, these studies evaluated these electrolytes by using inhibitors or knockout mice, with no mention on the changes in the intracellular electrolyte concentrations during embryogenesis. In this study, we used the electrolyte indicators CoroNa Green AM and ION Potassium Green-2 AM to directly visualise intracellular concentrations of Na+ and K+, respectively, at each embryonic developmental stage in mouse embryos. We directly observed intracellular electrolyte concentrations at the morula, blastocyst, and hatching stages. Our results revealed dynamic changes in intracellular electrolyte concentrations; we found that the intracellular Na+ concentration decreased, while K+ concentration increased during blastocoel formation. The degree of change in intensity in response to ouabain, an inhibitor of Na+/K+ ATPase, was considered to correspond to the degree of Na+/K+ ATPase activity at each developmental stage. Additionally, after the blastocyst stage, trophectoderm cells in direct contact with the blastocoel showed higher K+ concentrations than in direct contact with inner cell mass, indicating that Na+/K+ ATPase activity differs depending on the location in the trophectoderm. This is the first study to use CoroNa Green AM and ION Potassium Green-2 AM in mouse embryos and visualise electrolytes during embryonic development. The changes in electrolyte concentration observed in this study were consistent with the activity of Na+/K+ ATPase reported previously, and it was possible to image more detailed electrolyte behaviour in embryo cells. This method can be used to improve the understanding of cell physiology and is useful for future embryonic development studies

Consistency between chromosomal status analysis of biopsied human blastocyst trophectoderm cells and whole blastocyst cells

Purpose This study investigated the consistency between results of preimplantation genetic testing for aneuploidy performed on trophectoderm (TE) cells and remaining blastocyst cells. Methods TE biopsy was performed on 29 surplus cryopreserved human blastocysts. Biopsy samples and remaining blastocysts were processed using the VeriSeq PGS kit, and chromosomal statuses were compared by next-generation sequencing. Results Discordance was observed in the chromosomal status of 11 out of 29 blastocysts between the biopsied TE and remaining blastocysts. Concordance was observed in 11 of 12 blastocysts classified as euploid by TE biopsy and in 7 of 17 blastocysts classified as aneuploid. There was 100% concordance (7/7) in cases diagnosed as aneuploid with no mosaicism by TE biopsy. However, discordance was observed in all 10 cases showing mosaicism or partial chromosomal abnormality. Conclusion Chromosomal status analysis based on TE biopsy does not accurately reflect the chromosomal status of the whole blastocyst. The chromosomal status is usually the same between the TE and remaining blastocyst cells in cases diagnosed as euploid or aneuploid with no mosaicism. However, mosaic blastocysts and those with other types of structural rearrangements have a higher risk of inconsistency, warranting caution during embryo selection