Ignite Creation Date:
2024-05-06 @ 1:24 AM
Last Modification Date:
2024-10-26 @ 11:04 AM
Study NCT ID:
NCT01809509
Status:
UNKNOWN
Last Update Posted:
2013-03-12
First Post:
2013-03-09
Brief Title:
Pilot Study on Molecular Quantitation and Sequencing of Endometrial Cytokines Gene Expression and Their Effect on the Outcome of In-vitro Fertilization IVF Cycle
Sponsor:
Cairo University
Organization:
Cairo University
Study Overview
Official Title:
A Prestudy of Impact of Cytokines and Gene Expression on Outcome of IVF
Status:
UNKNOWN
Status Verified Date:
2013-03
Last Known Status:
ENROLLING_BY_INVITATION
Delayed Posting:
No
If Stopped, Why?:
Not Stopped
Has Expanded Access:
False
If Expanded Access, NCT#:
N/A
Has Expanded Access, NCT# Status:
N/A
Acronym:
None
Brief Summary:
Pilot study on molecular quantitation and sequencing of endometrial cytokines gene expression and their effect on the outcome of in-vitro fertilization IVF Cycle
Detailed Description:
The study will be performed
in the Cairo IVE Unit Faculty of medicine Cairo University on 15 cases Inclusion criteria age 23 -35 FSH less than 10 no previous uterine operations no history of poor response in previous IVF no sever male factor no endometriosis and uterine factor are excluded no diabetes mellitus and antral follicle count AFC 5 undergoing IVF using standard long protocol
All patients will give their informed consent to participate in the study Endometrial tissue samples will be taken on the day of oocyte retrieval using soft suction plastic catheter The menstrual blood of 10 women with regular menstrual cycles and with no apparent endometrial dysfunction was taken as control samples The study protocol and informed consents were approved by the Human Ethics Committee of IVF department of Kasar Ini hospital
Total RNA isolation Endometrial biopsies and menstrual control blood will be lysed by RLT buffer QIAGEN Germantown MD The lysates further prepared for total RNA extraction using the RNeasy mini kit QIAGEN Germantown MD according to the manufacturers instructions The RNA extract stored at -80ºC until future use RNA purity yield and concentration will be determined through dual spectrophotometry Beckman USA and 1μg of RNA run on a 1 agarose gel Roche Castle Hill Australia to ensure integrity of the RNA Quantitative RT-PCR qRT-PCR Reverse transcriptase RT reaction mixture using High Capacity Reverse Transcriptase kit Applied Biosystems USA containing 1 μg total RNA from each sample for cDNA synthesis 05 μg random primer 5RT buffer 25 mmolL dNTP 20 U RNase inhibitor and 200 U MMLV reverse transcriptase in a total volume of 25 μl was incubated at 37ºC for 60 minutes then heated to 95 ºC for 5 minutes to inactivate MMLV RT will be followed by qPCR 50 ng of cDNA were added to 5 X Fast-Start SYBR green master mix with Rox Roche Diagnostics Indianapolis IN and 200 ng of primer mix Sigma The reaction will be carried out in micro optical plates Applied Biosystems and analyzed using StepOne real-time 203 PCR system Applied Biosystems The PCR running method was as follows 10 minutes at 950C for enzyme activation followed by 40 cycles of 15 seconds at 950C 20 seconds at 550C and 30 second at 720C for the amplification step The primers used in the qRT-PCR evaluation specific for target genes table 1 Relative mRNA expression will be calculated by the comparative cycle threshold method as outlined in the manufacturers user manual with GAPDH house-keeping gene The fluorescence was plotted versus PCR cycle number for reaction and each sample indicated Serum hormonal levels assay FSH LH and E2 were estimated by ELISA according to instructions of manufacturers DNA purification and sequencing analysis IL-11 and LIF genes analyzed by direct sequencing of the PCR products using SEQr kit Applied Biosystems according to manufacturers protocol PCR products will be purified using the QIAquick Gel Extraction Kit QIAGEN The relevant purified DNA samples of all the cases and controls will be amplified and sequenced using automated sequencing with the aid of a Big Dye Terminator Sequencing Kit PEApplied Biosystems Foster City CA The samples will be run in an automated sequencer ABI Prism 310 Avant PEApplied Biosystems All samples will be sequenced twice to ensure the results
Statistical analysis Data will be statistically described in terms of mean standard deviation median and range Comparison between women who could achieve pregnancy and those who didnt was done using Mann Whitney U test for independent samples Correlation between various variables was done using Spearman rank correlation equation for non-normal variables
in the Cairo IVE Unit Faculty of medicine Cairo University on 15 cases Inclusion criteria age 23 -35 FSH less than 10 no previous uterine operations no history of poor response in previous IVF no sever male factor no endometriosis and uterine factor are excluded no diabetes mellitus and antral follicle count AFC 5 undergoing IVF using standard long protocol
All patients will give their informed consent to participate in the study Endometrial tissue samples will be taken on the day of oocyte retrieval using soft suction plastic catheter The menstrual blood of 10 women with regular menstrual cycles and with no apparent endometrial dysfunction was taken as control samples The study protocol and informed consents were approved by the Human Ethics Committee of IVF department of Kasar Ini hospital
Total RNA isolation Endometrial biopsies and menstrual control blood will be lysed by RLT buffer QIAGEN Germantown MD The lysates further prepared for total RNA extraction using the RNeasy mini kit QIAGEN Germantown MD according to the manufacturers instructions The RNA extract stored at -80ºC until future use RNA purity yield and concentration will be determined through dual spectrophotometry Beckman USA and 1μg of RNA run on a 1 agarose gel Roche Castle Hill Australia to ensure integrity of the RNA Quantitative RT-PCR qRT-PCR Reverse transcriptase RT reaction mixture using High Capacity Reverse Transcriptase kit Applied Biosystems USA containing 1 μg total RNA from each sample for cDNA synthesis 05 μg random primer 5RT buffer 25 mmolL dNTP 20 U RNase inhibitor and 200 U MMLV reverse transcriptase in a total volume of 25 μl was incubated at 37ºC for 60 minutes then heated to 95 ºC for 5 minutes to inactivate MMLV RT will be followed by qPCR 50 ng of cDNA were added to 5 X Fast-Start SYBR green master mix with Rox Roche Diagnostics Indianapolis IN and 200 ng of primer mix Sigma The reaction will be carried out in micro optical plates Applied Biosystems and analyzed using StepOne real-time 203 PCR system Applied Biosystems The PCR running method was as follows 10 minutes at 950C for enzyme activation followed by 40 cycles of 15 seconds at 950C 20 seconds at 550C and 30 second at 720C for the amplification step The primers used in the qRT-PCR evaluation specific for target genes table 1 Relative mRNA expression will be calculated by the comparative cycle threshold method as outlined in the manufacturers user manual with GAPDH house-keeping gene The fluorescence was plotted versus PCR cycle number for reaction and each sample indicated Serum hormonal levels assay FSH LH and E2 were estimated by ELISA according to instructions of manufacturers DNA purification and sequencing analysis IL-11 and LIF genes analyzed by direct sequencing of the PCR products using SEQr kit Applied Biosystems according to manufacturers protocol PCR products will be purified using the QIAquick Gel Extraction Kit QIAGEN The relevant purified DNA samples of all the cases and controls will be amplified and sequenced using automated sequencing with the aid of a Big Dye Terminator Sequencing Kit PEApplied Biosystems Foster City CA The samples will be run in an automated sequencer ABI Prism 310 Avant PEApplied Biosystems All samples will be sequenced twice to ensure the results
Statistical analysis Data will be statistically described in terms of mean standard deviation median and range Comparison between women who could achieve pregnancy and those who didnt was done using Mann Whitney U test for independent samples Correlation between various variables was done using Spearman rank correlation equation for non-normal variables
Study Oversight
Has Oversight DMC:
None
Is a FDA Regulated Drug?:
None
Is a FDA Regulated Device?:
None
Is an Unapproved Device?:
None
Is a PPSD?:
None
Is a US Export?:
None
Is an FDA AA801 Violation?:
None