Expression Of A Oocyte Secreted Factors In Human Individual Mâ…¡cumulus Cells And Its Relationship With Oocyte Development Potency

Objective:Cumulus cells(CCs), which are somatic cells that surround the oocyte, that catabolish of cholesterol, glucose,provide the necessary energy via gap junctions as oocytes,and play a crucial role in oocyte development, maturation,ovulation until to fertilization process. Recent evidence suggests that the expression of some candidate genes in cumulus cells(CC) have the potential to serve as markers of oocyte quality and development potency. GDF-9, BMP-15 and TGF-β1 are members of the TGFβ superfamily. Smad3 is known to serve as a signaling molecules downstream for the TGF-β, however, the functions of Smad3 in the human CCs remain unidentified. GDF-9 can start antral follicles, promote CCs growth and regulating cumulus expansion,stimulate hyaluronidase synthesis, regulate the oocyte mitosis,and to improve oocyte developmental potential. BMP15 induces CCs mitosis and proliferation and which promote the CCs meiosis is not dependent on the role of FSH. TGF-β1and the receptor of CCs affect the growth of oocytes and subsequent fertilization split potential, and is essential for oocyte and early embryo development. TGFβ/Smads can promote the proliferation and differentiation of CCs, and regulate follicle growth with gonadotropin. GDF-9 can stimulate the Smads protein phosphorylation levels of CCs, combinate with type I and type II receptor, which in turn activates the intracellularsignal-mediated(Smad) proteins3, that can promote CCs expansion.The aims of the present study were to detect the expression levels of GDF9,BMP15,TGF-β1 and Smad3 m RNAs in cumulus cells and to analyze the correlation between their expression levels and the oocyte quality and developmental potential. To research the effects of GDF9,BMP15,TGF-β1 and Smad3 m RNAs in cumulus cells and to explore the correlation between two ovulation induction protocols( standard long protocol and mild stimulation protocol) and the oocyte quality and developmental potential.Methods:1 Patients:A total of 332 individual CCs from 41 women were taken from shijiazhuang Bethune International Peace Hospital from October,2013 to December,2014 undergoing the treatment of l CSI. Inclusion criteria: All women were <35 years old, Ovaries without surgery,and exhibited normal basal plasma hormone levels(2-3 days of the menstrual cycle, FSH <10IU / L, E2 <80pg/m L, PRL in the normal range), regular 26-32 day menstrual cycles,the women recruited had a BMI of 18-25, and were free of any chronic diseases and were not on any form of medication recently 3 month. Exclusion criteria: signs of androgenicity, PCOS or endometriosis. The causes for infertility were mainly of male origin, whereas those without male factor infertility received conventional IVF treatment.Sign the informed consent form all patients should be approved by the hospital ethics committee.2 Controlled ovarian hyperstimulation protocol(1) Controlled ovarian stimulation was achieved using a standard long protocol of Gn RH-agonist downregulation in the middle of the corpus luteum in all cases, followed by ovarian hyperstimulation using daily injections of FSH(Gonal-f, Merck Serono,Switzerland). The standard starting dose of r FSH was 150-300IU/day. Patients who were judged to be at risk for high or low response to ovarian stimulation and antral follicle count had their starting dose adjusted, the ovarian response to stimulation was evaluated by ultrasound and the dose of gonadotrophins adjusted if necessary. Ovulation induction was initiated by subcutaneous injection of recombinant human chorionic gonadotrophin 250μg(Rhcg, Ovidre, Merck Serono Switzerland) when ovarian follicles diameter according to the 18 mm or greater /14 mm or greater(60% ~ 70%) and E2/14mm(200 ~ 300pg/m L). Oocytes were retrieved after 36 h, under transvaginal ultrasound guidance.(2) Controlled ovarian stimulation was achieved using a mild stimulation protocol: On the third day of the menstrual cycle, follicle stimulating human menopausal gonadotropin(HMG) are typically administered at a dose of 225 IU/day, in combination with oral medications of letrozole 2.5mg/day, five days later, clomiphene 50mg/day instead of letrozole. Ovulation induction was initiated by Diphereline 0.1mg subcutaneous injection of when ovarian follicles diameter according to the 18 mm or greater /14 mm or greater(60% ~ 70%) and E2/14mm(200 ~ 300pg/m L). Oocytes were retrieved after 36 h, under transvaginal ultrasound guidance.3 Collection of cumulus cells:COCs were collected 36 h after the administration of an ovulation trigger, each sample of cumulus cells were mechanically separated from the oocyte by microdissection using two needles, washed repeatly in PBS fluid of the PH value of 7.4, then individually placed in cryotubes and stored in – 80 ℃ refrigerator.4 Cumulus Cells RNA Extraction and Reverse transcription(RT)The TRizol Reagent was used to extract total RNA from each CCs sample(n =332) according to the instructions, and fully dissolved adding 20μL DEPC water. Take 1μL RNA was extracted immediately subjected to 2% agarose gel electrophoresis, after the determination of compliance with standards, c DNA was reverse transcribed(RT) following the manufacturer’s instructions of Reverse Transcription Reaction kit using 8μL of amplified RNA in a 20μL reaction volume.The reaction conditions are set as follows: at 70℃ for 10 min, at 42℃ for 50 min and at 95℃ for 5min.5 RT-PCR:The purpose and internal(RPL- 15) genes sequence primers were designed by Primer Premier 5.0 software. Sequence primers are shown in table 1. RT-PCR was performed following the manufacturer’s instructions of Quanti Fast® SYBR® Green PCR kit. The specific reaction conditions are set as follows:The program comprised an initial denaturation step at 95℃ for 2min, denaturation step at 95℃for 15 s, annealing at 60℃ for 30 s. Each PCR was performed for 40 cycles for each sample.6 Statistical analysis:Relative m RNA expression levels of the studied genes were quantified using real-time PCR analysis with the SPSS13.0 Sequence Detection System.The formula 2ΔCt was used to calculate the levels of m RNAs in serum. Results were expressed as means ±SEM( x ±s). To compare the differences between the levels of cytokines and different groups for independent t-test, P < 0.05, the difference was statistically significant.Results:1 The CCs were divided into two groups of standard long protocol group(CCs=172) and mild stimulation protocol group(CCs=160) according to stimulate ovulation scheme. Because oocytes and cumulus cells is one-to-one, according to oocyte fertilization, embryo development and blastocyst formation, retrospectively divided the CCs into different groups. â…°)The CCs were divided into two groups of normal fertilization group(standard long protocol group CCs=127, mild stimulation protocol group CCs=136)and abnormal fertilization group(standard long protocol group CCs=45, mild stimulation protocol group CCs=24)according to fertilization case.â…±)Normal fertilization group were divided into two groups of good quality embryos(standard long protocol group CCs=58, mild stimulation protocol group CCs=76) and poor quality embryo(standard long protocol group CCs=69, mild stimulation protocol group CCs=60) on 3 day according to embryos development. â…²) poor quality embryos were divided into two groups of good quality blastocyst(standard long protocol group CCs=24, mild stimulation protocol group CCs=12) and poor quality blastocyst(standard long protocol group CCs=45, mild stimulation protocol group CCs=48) development according to blastocyst development on 5 day. GV and abnormal oocytes are not included in the group in the study. A total of 332 CCs from 41 patients(standard long protocol group CCs=69, mild stimulation protocol group CCs=60) are detected by RT-PCR.2 The relationship GDF-9 in cumulus cells with the oocyte developmental potentialGDF- 9 is favorable for oocyte fertilization, cleavage, and blastocyst formation. Through this experiment can be seen, in this two stimulation protocols, the expression level of GDF-9 gene m RNA of normal fertilization group were significantly higher than the abnormal fertilization group(P=0.020, P=0.001), good quality embryos group higher than poor quality embryos(P=0.001, P=0.043), and good quality blastocyst group higher than poor quality embryos(P=0.002, P=0.001).3 The relationship BMP-15 in cumulus cells with the oocyte developmental potentialThis experiment demonstrated that Whether standard long protocol or mild stimulation protocol, the expression level of BMP-15 gene m RNA has the significant difference in different result of oocytes development.In two stimulation protocols, the expression level of BMP-15 gene m RNA of normal fertilization group were significantly higher than the abnormal fertilization group(P=0.000, P=0.000), good quality embryos group higher than poor quality embryos(P=0.003, P=0.048), and good quality blastocyst group and poor quality embryos have significant difference(P=0.033, P=0.029).So we can see that the relative levels of BMP- 15 m RNA and oocyte development is closely relative to potential.4 The relationship TGF-β1 in cumulus cells with the oocyte developmental potentialTGF-β1 levels in the cumulus cells were significantly correlated with oocyte developmental potential. Whether standard long protocol or mild stimulation protocol, the expression level of TGF-β1 gene m RNA of normal fertilization group were significantly higher than the abnormal fertilization group(P=0.003, P=0.022), good quality embryos group higher than poor quality embryos(P=0.015, P=0.022),and good quality blastocyst group higher than poor quality embryos(P=0.001, P=0.025).5 The relationship Smad3 in cumulus cells with the oocyte developmental potentialIn standard long protocol and mild stimulation protocol group, the expression level of Smad3 gene m RNA of normal fertilization group were significantly higher than the abnormal fertilization group(P=0.006, P=0.012), good quality embryos group and poor quality embryos have significant difference(P=0.019, P=0.041),and good quality blastocyst group higher than poor quality embryos(P=0.000, P=0.028).It is revealed that the higher level of Smad3 gene m RNA, the more development potential of oocytes.6 The relationship the two ovulation induction protocols with the oocyte developmental potentialAge no statistical difference between standard long protocol and mild stimulation protocol group(P=0.861). The expression levels of GDF-9, BMP-15 and Smad3 m RNA of the two ovulation induction protocols were have significant difference(P=0.000, P=0.004, P=0.005),but the expression level of TGF-β1 m RNA was not significant between the two ovulation induction protocols(P=0.653).It is revealed that expression levels of GDF – 9, BMP-15 and Smad3 are correlated with stimulate ovary protocol, standard long protocol could stimulate the secretion of CCs more GDF-9 and Smad3 factor, and mild stimulation protocol could stimulate the secretion of CCs more BMP- 15 factor.But the expression level of TGF-β1 m RNA were not significant between the two ovulation induction protocols.Discussion:1 This study demonstrated that expression levels of GDF-9,BMP-15, TGF-β1 and Smad3 in CCs are positive correlation with the oocyte quality and developmental potential.It is revealed that human cumulus cells in GDF-9, BMP – 15, TGF- beta 1, Smad3 level could predict the development potential of oocyte, can be used as the objective index to evaluate the quality of the oocytes.2 It is detected that the expression levels of GDF- 9 and Smad3 m RNA of standard long protocol were significantly higher than mild stimulation protocol, the expression levels of BMP-15 m RNA of mild stimulation protocol were significantly higher than standard long protocol.It is revealed that different stimulate ovulation protocols can influence the expression patterns of the oocyte secreted factors, and related to M â…¡oocyte developmental potential.