| Background and objectiveIn human, the oocyte in the Graafian follicle is surrounded by tightly packed layers of cumulus cells, forming the cumulus-oocyte complex.In Assisted reproductive technology(ART),Women were stimulated with the long gonadotrophin-releasing hormone agonist (GnRHa) protocol combined with recombinant follicle-stimulating hormone (rFSH). Cumulus-oocyte-complexes were retrieved by transvaginal ultrasound When the follicles were maturity. The cumulus-oocyte-complexes retrieved were insemination with conventional in vitro fertilization (IVF)or intracytoplasmic sperm injection(ICSI).Conventional IVF technology be adopted when sperm concentration were at least20million spermatozoa per ml with a25%progressive motility rate and>4%morphologically normal spermatozoa,in which the cumulus-oocyte-complexes were cultured together in a large drop of fertilization medium with motile sperm at a concentration of10000sperm/ml.Otherwise,ICSI were performed,if the husbands haven’t a normal semen parameters.The ICSI technique has been widely used in ART,but it was argued that ICSI may increase potential genetic risks in the offspring through bypassing the natural selection and penetration of high quality spermatozoa into the cytoplasm of the oocyte.[1-4].So, In China,Indications for ICSI are strictly restrict for those couples who have had unexplained fertilization failure in a previous IVF cycle, obstructive azoospermia, severe oligospermia, asthenospermia, teratospermia and undescended testes and so on. In most clinic studies, human oocytes(cumulus-oocyte complex) intended for vitrification are usually denuded from cumulus cells and vitrified. Oocytes that survive the vitrification and thawing procedures are fertilized by intracytoplasmic sperm injection (ICSI). Because It has been suggested that freezing procedures may induce rupture and release of the content of the cortical granules, resulting in a hardening of the zona which impair or prevents the sperm from fertilizing the oocytes during conventional insemination[5-7].However, could do we use conventional IVF instead of ICSI for fertilization to vitrified-thawing oocytes when those couples have a normal semen parameters? could we use conventional IVF instead of ICSI for fertilization to vitrified-thawing oocytes with corona radiata?The objective of this study is to investigate the protective effects of corona radiata cell during fertilizaiton of human oocytes and if an corona radiata cell of vitrified-warmed oocytes retains their fertilization capacity in conventional IVF?Materials and methodsHuman cumulus-oocyte complex consist of oocyte, Corona radiata cells(CRCs), which directly lie on the zona pellucida,and cumulus oophorus cells(COCs), which is a dispersed structure of cells for the synthesis and deposition of a mucoid intercellular matrix.Before human oocytes are vitrified or ICSI, they are usually denuded from their COCs and CRCs with40IU/ml of hyaluronidase.In this study, we use oocyte, COCs and CRCs to demonstrate fallow as:1. To determine whether is there any difference in the zona pellucida solubility between vitrified-warmed oocytes with/without CRCs group and fresh oocyte. We record the dissolution time of the zona pellucida of human oocytes digested in0.1%(w/v) Pronase E solution to assessment of zona pellucida solubility (hardening). 2. To analysis of ultrastructure of vitrified-warmed or fresh oocytes with Transmission Electron microscopy(TEM).we want to elucidate that whether does freezing procedures induce rupture and release of the content of the cortical granules.3. To evaluate fertilization rate, pregnancy and implantation rates between vitrified-warmed oocytes with/without CRCs group and with/without CRCs fresh oocyte groups.4. To describes the clinical applications of vitrified-warmed oocytes with CRCs in ART medical centre.Results1.Zona pellucida solubility (hardening) As expected, the mean time required for zona pellucida digestion in the positive control group (polyspermic fertilized oocytes) increased significantly (P<0.001vs. control group). The results presented in Table1reveals the influence of vitrification upon the zona pellucida dissolution time. It appears that vitrification-warming process does not affect the solubility of the zona pellucida, whether the oocytes has an intact corona radiata or not (P>0.05)(Fig2).2.Oocyte ultrastructureBoth the control and the vitrified-warmed oocytes had a regular oolemma with numerous microvillus formations (Fig.3a,b,c). The area was rich in cortical granule aligned peripherally in a continuous sub-oolemma array. The mean number±SE of cortical granule per10μm in the control group, vitrified-warmed oocytes without corona radiata and vitrified-warmed oocytes with corona radiata was8.93±0.28,7.84±0.47and7.93±0.36, respectively (N=6, Fig.3d)(P>0.05vs. Control group).There were no differences in the morphology and electron density of zona pellucida, mitochondria, smooth endoplasmic reticulum and spindle between the different groups (data not shown).3.The survival, fertilization and developmental competence of the vitrified-warmed oocytesVitrified-warmed oocytes, with or without corona radiata, had the same survival rates (98%, Table2). These oocytes were subsequently inseminated via conventional IVF, and77%(N=37) were normally fertilized of the vitrified-warmed oocytes with an intact corona radiata, whereas only18.8%(N=9) of the completely denuded vitrified-warmed oocytes were fertilized (P<0.001). Also the fresh oocytes without a corona radiata showed a showed a low fertilization (12/53,22.6%), while the fresh oocytes with an intact corona radiata had a normal fertilization rate (40/54,75.9%)(P<0.001).All normally fertilized oocytes cleaved (100%cleavage rate) and two embryos were transferred to each patient.In vitrified-warmed oocytes with an intact corona radiata, four patients had a positive test of which three continued. The implantation rate per embryo transfer was31.5%(5/16) and three babies were born.In fresh oocytes with an intact corona radiata, four patients concieved (50%) and one case miscarriaged. The implantation rate per embryo transfer was37.5%(6/16) and three babies were born.There were no differences seen between fresh or vitrified-warmed oocytes with CRCs, with respect to fertilization, cleavage, implantation and baby take home rates.4. Clinical applications of vitrified-warmed oocytes with CRCsThere was no significant difference in2PN fertilization rate(73.8%vs73.6%), clinical pregnancy rate(50.0%vs44.4%) respectively between the autologous follicular fluid (AFF) group and the control group.Conclusions1.The oocytes with CRCs have a higher fertilization rate than the oocytes without CRCs fertilized with conventional IVF.2. We could use conventional IVF instead of ICSI for fertilization to vitrified-thawing oocytes with CRCs.3.Zona pellucida hardening does not seem to occur in vitrified human oocytes. 4. There were no differences in fertilization rate and pregnancy rate between vitrified-warmed oocytes with CRCs and fresh oocytes fertilized with conventional Background and objectiveDuring folliculogenesis, cumulus cells surrounding the oocyte differentiate into corona radiata cells (CRCs) and cumulus oophorus cells (COCs), which are involved in gonadal steroidogenesis and the development of germ cells. Several studies suggested that microRNAs (miRNAs) play an important regulatory role at the post-transcriptional level in cumulus cells. However, comparative miRNA profiles and associated processes in human CRCs and COCs have not been reported before. Our work aim to extend the current knowledge of the regulatory role of miRNAs and their targeted pathways in CRCs and COCs. During folliculogenesis, and provides novel candidates for molecular biomarkers in the research of female infertility.Materials and methodsFive women from the Reproductive Medical Centre of Anhui Provincial Hospital, aged29.1±2.7(Mean±SD), undergoing ICSI-ET with a standard long stimulation protocol due to male factor infertility and achieving a clinical pregnancy were enrolled. All five patients were stimulated with the standard long gonadotropin-releasing hormone agonist protocol combined with the administration of recombinant FSH. For oocyte retrieval, all patients underwent ovarian puncture of follicles>15mm after36h of administration of10000IU human chorionic gonadotropin. The cumulus-oocyte complexes were retrieved36h after hCG treatment and washed in multiple dishes with flushing medium. The cumulus oophorus cells were collected in fertilisation medium using two disposable needles and two1-ml plastic disposable syringes without hyaluronidase. Library construction and sequencing was performed at BGI-Shenzhen. The small RNA NGS data were analysed according the previously published tools using CPSS. To predict the miRNA targets, the targeted mRNA of differentially expressed and selected miRNAs were predicted by miRanda, Targetscan, and MicroCosm. For GO and KEGG\pathway analysis of the predicted miRNA target genes from CRCs and COCs, the predicted target genes of differentially expressed and selected miRNAs were subjected to analysis of GO and KEGG. The miRNA quantification was performed by quantitative real-time PCR using an Applied Biosystems StepOne Real-Time PCR SystemResultsIn this study, A total of785and799annotated miRNAs were identified in CRCs and COCs, while high expression levels of six novel miRNAs were detected both in CRCs and in COCs. In addition, different expression patterns in CRCs and COCs were detected in72annotated miRNAs. To confirm the miRNA profile in COCs and CRCs, quantitative real-time PCR was used to validate the expression of annotated miRNAs, differentially expressed miRNAs, and novel miRNAs. The miRNAs in the let-7family were found to be involved in the regulation of a broad range of biological processes in both cumulus cell populations, which was accompanied by a large amount of miRNA editing. Bioinformatics analysis showed that amino acid and energy metabolism were targeted significantly by miRNAs that were differentially expressed between CRCs and COCs. Moreover, It has been corfimed by qRT-PCR, Western Blot and luciferase reporter system that miR-4286p highly expressing in GRCs could degrade the SLC2A1mRNA and reduce the protein expression though directly combining with MSLC2A1mRNA3’UTR.Conclusions In summary, for the first time we have analysed known and novel miRNAs in human stimulated preovulatory luteinizing CRCs and COCs by high-throughput Solexa sequencing. We have detected similarities and differences in the miRNA expression profile between CRCs and COCs, and confirmed their expression by quantitative real-time PCR analysis. The GO term annotation and KEGG pathway analysis for the predicted miRNA targets further indicate that these miRNAs are involved in various signalling pathways, such as amino acid and energy metabolism. Thus, the presence of a large number of miRNAs and the nature of their target genes suggested that miRNAs play important roles in the function of the follicular cumulus cells. Our work supports and further extends the knowledge of a regulatory role of miRNAs and their targeted pathways in folliculogenesis, which might facilitate the development of prophylactic strategies for the treatment of female infertility. |
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