[Retracted] MicroRNA­202 inhibits endometrial stromal cell migration and invasion by suppressing the K­Ras/Raf1/MEK/ERK signaling pathway.

Following the publication of this paper, it was drawn to the Editor's attention by a concerned reader that certain of the Transwell invasion assay data shown in Figs. 2C and 4B were strikingly similar to data appearing in different form in a paper by different authors at a different research institute that had already been submitted for publication. Owing to the fact that the contentious data in the above article had already been submitted for publication prior to its submission to International Journal of Molecular Medicine, the Editor has decided that this paper should be retracted from the Journal. The authors were asked for an explanation to account for these concerns, but the Editorial Office did not receive a satisfactory reply. The Editor apologizes to the readership for any inconvenience caused. [International Journal of Molecular Medicine 46: 2078­2088, 2020; DOI: 10.3892/ijmm.2020.4749].

IVM of human immature oocytes for infertility treatment and fertility preservation.

Background: Thousands of healthy babies are born from in vitro maturation (IVM) procedures, but the rate of efficiency differs with the source of immature oocytes obtained. Recently, there are different IVM protocols proposed for infertility treatment and fertility preservation. Methods: Based on the literature, the clinical application for IVM of immature oocytes was summarized. Main findings Results: Immature oocytes may be retrieved from women after priming with or without the use of follicular stimulation hormone (FSH), human chorionic gonadotrophin (hCG) or a combination of both FSH and hCG. Successful pregnancy rates with IVM technology seem to be correlated with the number of immature oocytes obtained. With the source and culture course of immature oocytes, there are various IVM protocols. IVM of immature oocytes is profoundly affected by the culture conditions, but no breakthrough has been made by improving the IVM medium itself. Thus, the clinical application of IVM technology continues to evolve. Conclusion: IVM technology is a useful technique for infertile women and fertility preservation. Mild stimulation IVF combined with IVM of immature oocytes is a viable alternative to the conventional stimulation IVF cycle treatment as it may prove to be an optimal first-line treatment approach.

Effects of different sperm sources on the clinical outcomes of in vitro oocyte maturation cycles combined with intracytoplasmic sperm injection.

Objectives: To evaluate the embryonic developments and clinical outcomes of different sperm sources with cycles of intracytoplasmic sperm injection (ICSI) and in vitro maturation (IVM). Methods: This retrospective study was approved by the hospital ethics committee and conducted in the hospital in vitro fertilization (IVF) clinic. From January 2005 to December 2018, 239 infertile couples underwent IVM-ICSI cycles and were divided into three groups according to different sperm sources. Group 1 comprised patients with percutaneous epididymal sperm aspiration (PESA; n = 62, 62 cycles), group 2 comprised patients with testicular sperm aspiration (TESA; n = 51, 51 cycles), and group 3 comprised patients with ejaculated sperm (n = 126, 126 cycles). We calculated the following outcomes: 1) outcomes per IVM-ICSI cycle: fertilization rate, cleavage rate, and embryo quality; 2) outcomes per embryo transfer cycle: endometrial thickness, implantation rate, biochemical pregnancy rate, clinical pregnancy rate, and live birth rate. Results: There was no difference in basic characteristics among the three groups, such as the female partner's age, basal follicle-stimulating hormone (FSH), basal luteinizing hormone (LH), and antral follicle count (p > 0.1). There were no statistically significant differences according to the IVM-ICSI cycle among the three groups in fertilization rate, cleavage rate, and rate of good-quality embryos (p > 0.05). The results were similar among cycles regarding the number of transfer embryos and endometrial thickness per embryo transfer cycle among the three groups (p > 0.05). There were also similar clinical outcomes per embryo transfer cycle among the three groups, such as the biochemical pregnancy rate, clinical pregnancy rate, and live birth rate (p > 0.05). Conclusions: Different sperm sources, percutaneous epididymal sperm aspiration, testicular sperm aspiration, and ejaculated sperm, do not affect the embryo and clinical outcomes after IVM-ICSI cycles.

Role of pyroglutamic acid in cumulus cells of women with polycystic ovary syndrome.

PURPOSE: Polycystic ovary syndrome is a complex heterogeneous endocrine disorder associated with established metabolic abnormalities and is a common cause of infertility in females. Glutathione metabolism in the cumulus cells (CCs) of women with PCOS may be correlated to the quality of oocytes for infertility treatment; therefore, we used a metabolomics approach to examine changes in CCs from women with PCOS and oocyte quality. METHODS: Among 135 women undergoing fertility treatment in the present study, there were 43 women with PCOS and 92 without. CCs were collected from the two groups and levels of pyroglutamic acid were measured using LC-MS/MS followed by qPCR and Western blot analysis to examine genes and proteins involved in pyroglutamic acid metabolism related to glutathione synthesis. RESULTS: Women with PCOS showed increased levels of L-pyroglutamic acid, L-glutamate, and L-phenylalanine and decreased levels of Cys-Gly and N-acetyl-L-methionine. Gene expression of OPLAH, involved in pyroglutamic synthesis, was significantly increased in women with PCOS compared with those without. Gene expression of GSS was significantly decreased in women with PCOS and synthesis of glutathione synthetase protein was decreased. Expression of nuclear factor erythroid 2-related factor 2, involved in resistance to oxidative stress, was significantly increased in women with PCOS. CONCLUSIONS: CCs of women with PCOS showed high concentrations of pyroglutamic acid and reduced glutathione synthesis, which causes oxidative stress in CCs, suggesting that decreased glutathione synthesis due to high levels of pyroglutamic acid in CCs may be related to the quality of oocytes in women with PCOS.

The case for mild stimulation for IVF: recommendations from The International Society for Mild Approaches in Assisted Reproduction.

The practice of ovarian stimulation for IVF is undergoing a fundamental re-evaluation as recent data begin to successfully challenge the traditional paradigm that ovarian stimulation should be aimed at the retrieval of as many oocytes as possible, in the belief that this will increase pregnancy rates. An opposing view is that live birth rate should not be the only end-point in evaluating the success of IVF treatment and that equal emphasis should be placed on safety and affordability. The International Society for Mild Approaches in Assisted Reproduction (ISMAAR) committee has carried out an up-to-date literature search, with the evidence being graded according to the University of Oxford's Centre for Evidence-Based Medicine. The recommendations were formulated taking into account the quality of evidence on the efficacy, risk and cost of each intervention. ISMAAR recommends adopting a mild approach to ovarian stimulation in all clinical settings as an increasing body of evidence suggests that mild stimulation is as effective as conventional stimulation, while being safer and less expensive. Mild ovarian stimulation could replace conventional stimulation, thus making IVF safer and more accessible worldwide.

Dysregulated sphingolipid metabolism and autophagy in granulosa cells of women with endometriosis.

We evaluated metabolic profiles between cumulus cells (CCs) and mural granulosa cells (MGCs) derived from women with endometriosis to identify their correlations with oocyte quality. CCs and MGCs were collected from women with and without endometriosis undergoing in vitro fertilization/intracytoplasmic sperm injection treatment. The metabolomics of CCs and MGCs were measured by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) followed by a quantitative polymerase chain reaction to further confirm the genes involved in the metabolic results. LC-MS/MS analysis revealed differences in 24 metabolites of CCs and 71 metabolites of MGCs between groups. Among them, five metabolites were upregulated and 19 metabolites were downregulated in CCs with endometriosis, whereas three metabolites were upregulated and 68 metabolites were downregulated in MGCs with endometriosis. Metabolites related to sphingolipid metabolism, which included palmitic acid (PA) and docosahexaenoic acid, increased significantly only in CCs with endometriosis, whereas sphingosine and PA were significantly downregulated in MGCs with endometriosis compared with CCs and MGCs without endometriosis. Gene expression involved in ceramide synthesis (CERS1, SPTL1, and SMPD1) and autophagy (BECN1, LAMP, and PC3) were significantly higher in CCs with endometriosis according to FASN, BECN1, and LAMP protein expressions. However, gene expression involved in ceramide synthesis (SPHK1, ASAH1, and SGPP1) and autophagy (BECN1, LAMP, and PC3) were significantly lower in MGCs with endometriosis, whereas CERS1 and UGCG expression increased. There are differences in sphingolipid metabolites in CCs and MGCs with endometriosis compared with women without endometriosis. These differences seem to be involved in the regulation of autophagic cell death in preovulatory follicles.

Effect of dominant follicle status at the time of retrieval on the clinical outcomes in natural cycle IVF combined with immature oocyte treatment.

OBJECTIVE: It is commonly believed that the oocytes from small follicles are unhealthy when a dominant follicle (DF) is recruited in the ovaries, especially when the DF is ovulated. This study aims to confirm whether the presence or ovulation of DF at the time of retrieval affects the clinical outcome of the natural cycle IVF with in vitro maturation (NC-IVF/M) treatment. METHODS: Data were collected from 446 women with regular menstrual cycle and 536 retrieval cycles using NC-IVF/M treatment. The cycles were divided into three groups based on the results of the oocyte retrieval cycle. Group A covers the collection of oocytes from the DF and small follicles; Group B incorporates failed oocyte retrieval from DF and then the oocytes are retrieved only from small follicles; and Group C includes the retrieval of oocytes only from small follicles accompanied with an ovulated DF. Furthermore, Group B and C have subgroups to include whether in vivo matured oocytes were obtained from small follicles. Following aspiration of DF and small follicles, mature oocytes were inseminated on the date of retrieval by intracytoplasmic sperm injection (ICSI) and the immature oocytes were matured in vitro. If the immature oocytes were matured in vitro, they were inseminated using ICSI, and then the embryos obtained from in vivo and in vitro matured oocytes were transferred accordingly. RESULTS: The oocytes from DF were successfully retrieved in 445 cycles (83.0%), failed to be retrieved in 54 cycles (10.1%) and ovulated in 37 cycles (6.9%). In Group A, an average of 2.0 ± 1.7 mature oocytes were retrieved, which was significantly higher than the average of Group B, with 1.3 ± 1.3 matured oocytes and Group C, with an average of 1.1 ± 1.5 matured oocytes (P < 0.01). However, the average number of immature oocytes retrieved from each group show no difference among the three groups. There was no significant difference in maturation rates of immature oocytes, fertilization rates among the three groups. The clinical pregnancy rate per transfer cycle is 34.5%, 34.6% and 25.7% in Group A, B and C, respectively. No significant differences were observed in embryonic development and implantation capacity in Group B and C in comparison to Group A. And there was no significant difference in clinical pregnancy, implantation, live birth and miscarriage rates among the three groups. No significant differences were observed in the developmental and implantation capacity according to with or without in vivo matured oocytes were retrieved in Group B and Group C. CONCLUSION: The presence or ovulation of the dominant follicle from the ovaries does not significantly influence the developmental and implantation capacity of immature oocytes retrieved from small follicles, suggesting that NC-IVF/M is a promising treatment option for women without ovarian stimulation.

Changes in the Mitochondria-Related Nuclear Gene Expression Profile during Human Oocyte Maturation by the IVM Technique.

To address which mitochondria-related nuclear differentially expressed genes (DEGs) and related pathways are altered during human oocyte maturation, single-cell analysis was performed in three oocyte states: in vivo matured (M-IVO), in vitro matured (M-IVT), and failed to mature in vitro (IM-IVT). There were 691 DEGs and 16 mitochondria-related DEGs in the comparison of M-IVT vs. IM-IVT oocytes, and 2281 DEGs and 160 mitochondria-related DEGs in the comparison of M-IVT vs. M-IVO oocytes, respectively. The GO and KEGG analyses showed that most of them were involved in pathways such as oxidative phosphorylation, pyruvate metabolism, peroxisome, and amino acid metabolism, i.e., valine, leucine, isoleucine, glycine, serine, and threonine metabolism or degradation. During the progress of oocyte maturation, the metabolic pathway, which derives the main source of ATP, shifted from glucose metabolism to pyruvate and fatty acid oxidation in order to maintain a low level of damaging reactive oxygen species (ROS) production. Although the immature oocytes could be cultured to a mature stage by an in vitro technique (IVM), there were still some differences in mitochondria-related regulations, which showed that the mitochondria were regulated by nuclear genes to compensate for their developmental needs. Meanwhile, the results indicated that the current IVM culture medium should be optimized to compensate for the special need for further development according to this disclosure, as it was a latent strategy to improve the effectiveness of the IVM procedure.

The Differential Metabolomes in Cumulus and Mural Granulosa Cells from Human Preovulatory Follicles.

This study evaluated the differences in metabolites between cumulus cells (CCs) and mural granulosa cells (MGCs) from human preovulatory follicles to understand the mechanism of oocyte maturation involving CCs and MGCs. CCs and MGCs were collected from women who were undergoing in vitro fertilization (IVF)/intracytoplasmic sperm injection (ICSI) treatment. The differences in morphology were determined by immunofluorescence. The metabolomics of CCs and MGCs was measured by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) followed by quantitative polymerase chain reaction (qPCR) and western blot analysis to further confirm the genes and proteins involved in oocyte maturation. CCs and MGCs were cultured for 48 h in vitro, and the medium was collected for detection of hormone levels. There were minor morphological differences between CCs and MGCs. LC-MS/MS analysis showed that there were differences in 101 metabolites between CCs and MGCs: 7 metabolites were upregulated in CCs, and 94 metabolites were upregulated in MGCs. The metabolites related to cholesterol transport and estradiol production were enriched in CCs, while metabolites related to antiapoptosis were enriched in MGCs. The expression of genes and proteins involved in cholesterol transport (ABCA1, LDLR, and SCARB1) and estradiol production (SULT2B1 and CYP19A1) was significantly higher in CCs, and the expression of genes and proteins involved in antiapoptosis (CRLS1, LPCAT3, and PLA2G4A) was significantly higher in MGCs. The level of estrogen in CCs was significantly higher than that in MGCs, while the progesterone level showed no significant differences. There are differences between the metabolomes of CCs and MGCs. These differences may be involved in the regulation of oocyte maturation.

Chromosome aneuploidy analysis in embryos derived from in vivo and in vitro matured human oocytes.

BACKGROUND: In vitro oocyte maturation (IVM) is being increasingly approached in assisted reproductive technology (ART). This study aimed to evaluate the quality of embryos generated by in-vitro matured immature follicles, as a guideline for further clinical decision-making. METHODS: A total of 52 couples with normal karyotypes underwent in vitro fertilization, and 162 embryos were donated for genetic screening. Embryos in IVF group were generated by mature follicles retrieved during gonadotrophin-stimulated in vitro fertilization (IVF) cycles. And embryos in IVM group were fertilized from IVM immature oocytes. RESULTS: The average age of the women was 30.50 ± 4.55 years (range 21-42 years) with 87 embryos from IVF group and 75 embryos from IVM group. The rate of aneuploid with 28 of the 87 (32.2%) embryos from IVF group and 21 of the 75 (28%) embryos from IVM group, with no significant difference. The frequency of aneuploid embryos was lowest in the youngest age and increased gradually with women's age, whether in IVF group or IVM group and risen significantly over 35 years old. The embryos with morphological grade 1 have the lowest aneuploidy frequency (16.6%), and increase by the grade, especially in IVF group. In grade 3, embryos in IVM group were more likely to be euploid than IVF group (60% vs 40%, respectively). CONCLUSIONS: IVM does not affect the quality of embryos and does not increase the aneuploidy rate of embryos. It is clinically recommended that women more than 35 years have a high aneuploidy rate and recommended to test by PGS (strongly recommended to screened by PGS for women more than 40 years). Women aged less than 35 years old for PGS according to their physical and economic conditions. Embryo with poor quality is also recommended to test by PGS, especially for grade III embryos.

The Function of Cumulus Cells in Oocyte Growth and Maturation and in Subsequent Ovulation and Fertilization.

Cumulus cells (CCs) originating from undifferentiated granulosa cells (GCs) differentiate in mural granulosa cells (MGCs) and CCs during antrum formation in the follicle by the distribution of location. CCs are supporting cells of the oocyte that protect the oocyte from the microenvironment, which helps oocyte growth and maturation in the follicles. Bi-directional communications between an oocyte and CCs are necessary for the oocyte for the acquisition of maturation and early embryonic developmental competence following fertilization. Follicle-stimulation hormone (FSH) and luteinizing hormone (LH) surges lead to the synthesis of an extracellular matrix in CCs, and CCs undergo expansion to assist meiotic resumption of the oocyte. The function of CCs is involved in the completion of oocyte meiotic maturation and ovulation, fertilization, and subsequent early embryo development. Therefore, understanding the function of CCs during follicular development may be helpful for predicting oocyte quality and subsequent embryonic development competence, as well as pregnancy outcomes in the field of reproductive medicine and assisted reproductive technology (ART) for infertility treatment.

The effect of lysophosphatidic acid-supplemented culture medium on human immature oocytes matured in vitro.

BACKGROUND: Lysophosphatidic acid-supplemented culture medium significantly increases the oocyte maturation rate in vitro. However, potential targets and pathways involved remain unknown. METHODS: A total of 43 women, who underwent cesarean section and aged between 18 and 35 years with good health, were included in this study. Immature oocytes were obtained and cultured with 10 µM lysophosphatidic acid. After culture, oocyte maturation was assessed and oocytes and cumulus cells were collected for RNA sequencing. Hierarchical indexing for spliced alignment of transcripts 2 method was used to align clean reads to the human genome. The featureCounts and edgeR package were used to calculate gene expression and analyze differences between groups respectively. ClusterProfiler program was used to perform Gene Ontology and Kyoto Encyclopedia of Genes and Genomes analysis. RESULTS: Oocyte maturation rate increased significantly following 48 h culture with lysophosphatidic acid. In cumulus cells, Gene Ontology analysis revealed the top 20 items enriched by upregulated genes and downregulated genes respectively; Kyoto Encyclopedia of Genes and Genomes analysis showed that upregulated genes in the treatment group were enriched in TNF signaling and insulin secretion pathways and downregulated genes were enriched in TNF signaling and cell adhesion molecules. In oocytes, Gene Ontology analysis revealed the top 20 items enriched by upregulated genes and downregulated genes respectively; Kyoto Encyclopedia of Genes and Genomes analysis showed that upregulated genes in the treatment group were enriched in MAPK signaling, gap junction, and cell cycle pathways and downregulated genes were enriched in MAPK signaling, estrogen signaling, RAP1 signaling, and gap junction pathways. CONCLUSIONS: Lysophosphatidic acid in culture medium enhances human oocyte maturation in vitro and the identified some potential pathways may associate with oocyte maturation.

MicroRNA­202 inhibits endometrial stromal cell migration and invasion by suppressing the K­Ras/Raf1/MEK/ERK signaling pathway.

The enhanced migratory ability of endometrial stromal cells (ESCs) is a key factor in the formation of functional endometrium­like tissues outside the uterine cavity during endometriosis (EMS). Although accumulating evidence has suggested the importance of microRNAs (miRNAs) in the pathogenesis of EMS, the role of particular miRNAs in the invasiveness of ESCs remain poorly understood. In the present study, the function of miRNAs in the invasiveness of ESCs, along with the associated underlying mechanism involved, were investigated. Initially, the expression patterns of miRNAs in the ectopic and eutopic endometrium isolated from patients with EMS were analyzed using microarray. MicroRNA­202­5p (miR­202) was selected for further study due to its previously reported suppressive effects on the invasion in various types of cancers. The expression of miR­202 and K­Ras in eutopic and ectopic endometrioma tissues were detected using reverse transcription­quantitative PCR, immunohistochemistry and western blotting. The migration and invasion ability of ESCs was determined using wound healing and Transwell invasion assays, respectively. Compared with that from healthy individuals, miR­202 expression was demonstrated to be lower in the eutopic endometrium from patients with EMS, which was even lower in ectopic endometrium. Functional experiments in primary ESCs revealed that enhanced miR­202 expression suppressed the cell invasion and migration abilities, which was also accompanied with increased E­cadherin and reduced N­cadherin expression in ESCs, suggesting its potentially suppressive role in epithelial­mesenchymal transition. K­Ras is a well­known regulator of the ERK signaling pathway that was shown to be directly targeted and negatively regulated by miR­202. In addition, K­Ras expression was found to be upregulated in the ectopic endometrium, where it correlated negatively with that of miR­202. Knocking down K­Ras expression mimicked the anti­invasive effects of miR­202 overexpression on ESCs, whilst K­Ras overexpression attenuated the inhibitory role of miR­202 overexpression in ESC invasion. The K­Ras/Raf1/MEK/ERK signaling pathway was also blocked by miR­202 overexpression. These findings suggested that miR­202 inhibited ESC migration and invasion by inhibiting the K­Ras/Raf1/MEK/ERK signaling pathway, rendering miR­202 a candidate for being a therapeutic target for EMS.

Corrigendum: Fertility Preservation for Child and Adolescent Cancer Patients in Asian Countries.

[This corrects the article DOI: 10.3389/fendo.2019.00655.].

Single-cell multiomic analysis of in vivo and in vitro matured human oocytes.

STUDY QUESTION: Are there any differences between in vivo (IVV) and in vitro (IVT) matured metaphase II (MII) oocytes at the molecular level? SUMMARY ANSWER: Between IVV and IVT oocytes, 507 differentially expressed genes (DEGs) were identified; the non-CpG methylomes were significantly different, but the CpG methylomes and genomic copy number variations (CNVs) were similar. WHAT IS KNOWN ALREADY: A previous study using microarray and single-cell RNA-seq analysis revealed that numerous genes were differentially expressed between IVV and IVT oocytes. Independent studies of DNA methylation profiling in human oocytes have revealed negative correlations between gene transcription and the DNA methylation level at gene promoter regions. No study has compared global CpG or non-CpG methylation between these two groups of oocytes. Although a high level of aneuploidy has been reported in MII oocytes, no direct comparison of IVV and IVT oocytes based on single-cell sequencing data has been performed. STUDY DESIGN, SIZE, DURATION: We collected eight IVV oocytes from six patients and seven IVT oocytes from seven patients and then analysed each oocyte using the previously established single-cell triple omics sequencing (scTrioseq) analysis to determine associations among the transcriptome, DNA methylome and chromosome ploidy in the oocytes. PARTICIPANTS/MATERIALS, SETTING, METHODS: All IVV oocytes were donated by patients who received 150 IU gonadotropin per day from the third day of their menstrual cycle, followed by GnRH antagonist after 5 days of gonadotropin stimulation. All IVT oocytes were from immature oocytes which were donated by volunteers undergoing delivery by caesarean section then cultured in oocyte maturation medium containing 75 mIU/ml hMG for 24 to 48 h. Every single oocyte was analysed using the previously established single-cell multiomic sequencing analysis. MAIN RESULTS AND THE ROLE OF CHANCE: There were 507 genes differentially expressed between the IVV (n = 8) and IVT (n = 7) oocytes, even though their global transcriptome profiles were similar. The enriched genes in IVV oocytes were related to the cell cycle process while those in IVT oocytes were related to mitochondrial respiration biogenesis. Although the global CpG methylation of the two groups of oocytes was similar, the non-CpG methylation level in IVV oocytes was higher than that in IVT oocytes. A high aneuploidy ratio was found in both groups, but the aneuploidy did not affect transcription according to the correlation analysis. LARGE-SCALE DATA: N/A. LIMITATIONS AND REASONS FOR CAUTION: Due to the difficulty in collecting MII oocytes, especially IVV matured oocytes, the sample size was limited. WIDER IMPLICATIONS OF THE FINDINGS: Our findings indicate that single-cell multiomic sequencing can be utilised to examine the similarity and differences between IVV and IVT matured MII oocytes. STUDY FUNDING/COMPETING INTEREST(S): This work was supported by the Ministry of Science and Technology of China, National Key R&D Program of China (No. 2017YFC1001601). The donated oocytes were collected by Shanghai Tenth People's Hospital. The authors declare no competing interests.

Fertility Preservation for Child and Adolescent Cancer Patients in Asian Countries.

Background: At present, fertility is one of the main concerns of young cancer patients. Following this trend, "fertility preservation (FP)" has been established and has become a new field of reproductive medicine. However, FP for child and adolescent (C-A) cancer patients is still developing, even in advanced countries. The aim of the present study was to assess the barriers to FP for C-A patients by investigating the current status of FP for C-A patients in Asian countries, which just have started FP activities. Method: A questionnaire survey of founding members of the Asian Society for Fertility Preservation (ASFP) was conducted in November 2018. Main findings: Of the 14 countries, 11 country representatives replied to this survey. FP for C-A patients is still developing in Asian countries, even in Australia, Japan, and Korea, which have organizations or academic societies specialized for FP. In all countries that replied to the present survey, the patients can receive embryo cryopreservation (EC), oocyte cryopreservation (OC), and sperm cryopreservation (SC) as FP. Compared with ovarian tissue cryopreservation (OTC), testicular tissue cryopreservation (TTC) is an uncommon FP treatment because of its still extremely experimental status (7 of 11 countries provide it). Most Asian countries can provide FP for C-A patients in terms of medical technology, but most have factors inhibiting to promote FP for C-A patients, due to lack of sufficient experience and an established system promoting FP for C-A patients. "Don't know how to provide FP treatment for C-A" is a major barrier. Also, low recognition in society and among medical staff is still a particularly major issue. There is also a problem with cooperative frameworks with pediatric departments. To achieve high-quality FP for C-A patients, a multidisciplinary approach is vital, but, according to the present study, few paramedical staff can participate in FP for C-A patients in Asia. Only Australia and Korea provide FP information by video and specific resources. Conclusion: The present study demonstrated the developing status of FP for C-A patients in Asian countries. More intensive consideration and discussion are needed to provide FP in Asian societies based on the local cultural and religious needs of patients.

Development of in vitro maturation techniques for clinical applications.

In vitro maturation (IVM) refers to maturation in culture of immature oocytes at different stages that may or may not have been exposed to short courses of gonadotropins. The source of immature oocytes is an important feature for subsequent embryonic development, pregnancy, and healthy live births. IVM is an effective treatment that has already achieved significant outcomes of acceptable pregnancy and implantation rates and has led to the births of several thousand healthy babies. As the development of IVM treatment continues, an attractive possibility for improving the already successful outcome is to combine a natural-cycle in vitro fertilization (IVF) treatment with immature-oocyte retrieval followed by IVM of those immature oocytes. If the treatment processes can be simplified for immature-oocyte retrieval, different types of infertile women may be able to take advantage of these treatments. Mild-stimulation IVF combined with IVM treatment may represent a viable alternative to the standard treatment. Although IVM treatment is still considered to be experimental, it is now time to reconsider the IVM technology and its development. Mild-stimulation IVF combined with IVM may prove to be not just alternatives to standard treatments, but potentially first-line treatment choices.

Gene expression profiles in mouse cumulus cells derived from in vitro matured oocytes with and without blastocyst formation.

Cumulus cells (CCs) are considered as an important source to predict oocyte quality. Despite numerous candidate genes in CCs have been identified for embryonic developmental competence, the results are inconsistent. The next generation RNA-sequencing was used to investigate the transcriptomic differences in CCs from in vitro matured oocytes did or did not develop to blastocyst stage following in vitro fertilization (IVF). In our study, the corresponding mouse oocytes were traced using a single-cell tracking system, and CCs were pooled into groups based on the embryonic developmental outcomes. In vivo matured oocytes with blastocyst development were set as a reference group. The transcriptomic differences in mouse CCs from in vitro maturated oocytes with or without blastocyst formation were tested by RNA-sequencing. Real-time PCR was used to verify the expression levels of those candidate genes. A total of 103 transcripts were significantly up-regulated, and 97 down-regulated, in the CCs with the oocytes developed to blastocyst stage. The bioinformatics study showed that those genes were involved in tube morphogenesis, cell-cell signaling and cell projection formation. Nine genes were selected from the most significantly changed transcripts after comparison with the reference group: Arrb1, Atp2c1, Cdh5, Cntnap1, Mkln1, Lgr4, Rhobtb1, Smc2 and Six2, as the candidate target genes. They were associated with the regulation of G-protein coupled receptors, Wnt and MAPK signaling, actin filaments and cell adhesion. Real-time PCR verified the up-regulation of all 9 genes, and significantly increased of Rhobtb1, Mkln1, Smc2, Arrb1, Atp2c1, Cdh5 and Lgr4. Based on RNA-sequencing, we found the changes in gene transcription of mouse CCs that were critical for the communication between CCs and oocytes. The results could provide novel insights on non-invasively predicting the oocyte quality and improving developmental competence.

Chapter 3 Current Challenges in Immature Oocyte Cryopreservation.

Current freezing technology, especially the vitrification method, has markedly improved oocyte survival rate after warming, and the pregnancy rate is comparable to that achieved with fresh oocytes. However, most groups report using oocytes matured in vivo for vitrification. Although immature oocytes can be vitrified successfully, clinical outcomes do not reach that of vitrification of matured oocytes. The current literature suggests that oocytes should be vitrified at mature metaphase II (M-II) stage following IVM rather than at the immature germinal vesicle (GV) stage, because the potential for oocyte maturation is reduced when vitrification is performed on immature oocytes at the GV stage.