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

Inhibition of Uterine Sarcoma Cell Growth through Suppression of Endogenous Tyrosine Kinase B Signaling

Uterine leiomyosarcoma is an aggressive tumor typically found at advanced stages due to difficulties with early diagnosis. Because uterine leiomyosarcoma is resistant to conventional radiation and chemotherapy, the development of more potent medical therapeutics is anticipated. Using quantitative real-time RT-PCR and immunostaining, we found the expression of brain-derived neurotrophic factor (BDNF) and neurotropin-4/5, together with their receptor, tyrosine kinase B (TrkB), in different uterine sarcoma cell lines and primary tumor samples from uterine leiomyosarcoma patients. We noted that levels of BDNF were more abundant than those of neurotropin-4/5. Moreover, the expression of TrkB and its ligands was elevated in a multidrug-resistant cell line and samples obtained from patients with leiomyosarcoma. In cultured uterine sarcoma cells, inhibition of endogenous TrkB signaling by treatment with either the soluble TrkB ectodomain or the Trk receptor inhibitor, K252a, suppressed cell proliferation and increased apoptosis based on cell viability and proliferation, in situ terminal deoxynucleotidyl transferase-mediated 2\u27-deoxyuridine 5\u27-triphosphate nick end-labeling and caspase-3/7 assays, whereas an inactive plasma membrane nonpermeable K252b was ineffective. Correspondingly, treatment with exogenous BDNF increased cell proliferation. In in vivo studies in athymic nude mice bearing multidrug-resistant uterine sarcoma cell tumors, we demonstrate suppression of tumor growth by treatment with K252a, but not K252b, as reflected by decreased cell proliferation and increased levels of apoptosis and caspase-3/7 activities without obvious side effects. Our findings indicated that endogenous signaling of the TrkB pathway contributed to uterine sarcoma cell growth, and inhibition of TrkB signaling in these tumors could provide a novel medical therapy for patients with uterine sarcomas

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

Neutrophil elastase in amniotic fluid as a predictor of preterm birth after emergent cervical cerclage

Introduction. The aim of this study was to investigate neutrophil elastase (NE) in amniotic fluid as a potential marker for predicting pregnancy continuation. Material and methods. We enrolled 34 pregnant women with bulging fetal membrane during the second trimester who underwent emergent cerclage after confirming the absence of intrauterine infection (amniotic fluid glucose >= 15 mg/dL). Amniotic fluid NE levels were compared between women who completed and did not complete 30, 34, and 36 weeks of gestation, and the optimal cut-off value for predicting pregnancy continuation was estimated. Moreover, the differences in the duration of continued pregnancy were compared between women with NE levels above and below the optimal cut-off value. Results. The optimal cut-off value for NE in amniotic fluid that predicted pregnancy continuation beyond 30, 34, and 36 weeks of gestation was 180 ng/mL; this cut-off value had a sensitivity, specificity, positive predictive value, and negative predictive value of 84.0, 77.8, 91.3, and 63.7% beyond 30 weeks of gestation; 87.5, 80.0, 91.5, and 72.3% beyond 34 weeks of gestation; and 85.0, 71.4, 80.9, and 76.9% beyond 36 weeks of gestation, respectively. The duration of continued pregnancy from emergent cerclage to delivery was significantly longer in women with amniotic fluid NE = 180 ng/mL (44.8 +/- 14.3 days). Conclusion. The NE levels in amniotic fluid may serve as a useful marker for predicting the duration of continued pregnancy after cervical cerclage

Expression of Cell Competition Markers at the Interface between p53 Signature and Normal Epithelium in the Human Fallopian Tube

There is a growing body of evidence regarding cell competition between normal and mutant mammalian cells, which suggest that it may play a defensive role in the early phase of carcinogenesis. In vitro study in the past has shown that overexpression of vimentin in normal epithelial cells at the contact surface with transformed cells is essential for the cell competition involved in epithelial defense against cancer. In this study, we attempted to examine cell competition in human tissue in vivo by investigating surgically resected human fallopian tubes that contain p53 signatures and serous tubal intraepithelial lesions (STILs), a linear expansion of p53-immunopositive/TP53 mutant tubal epithelial cells that are considered as precursors of pelvic high grade serous carcinoma. Immunofluorescence double staining for p53 and the cell competition marker vimentin was performed in 21 sections of human fallopian tube tissue containing 17 p53 signatures and 4 STILs. The intensities of vimentin expression at the interface between p53-positive cells at the end of the p53 signature/STIL and adjacent p53-negative normal tubal epithelial cells were compared with the background tubal epithelium. As a result, the average vimentin intensity at the interfaces relative to the background intensity was 1.076 (95% CI, 0.9412 - 1.211 for p53 signature and 0.9790 (95% CI, 0.7206 - 1.237) for STIL. Thus, it can be concluded that overexpression of the cell competition marker vimentin are not observed in human tissue with TP53 alterations

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

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