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Deoxyribonucleic acid (DNA) damage of granulosa cells obtained during oocyte retrieval will be evaluated by flow cytometry with detection of Histone H2A.X and Phosphorylated Gamma H2A.X protein levels in patients with low ovarian reserve and unexplained infertile patients as a control group undergoing intracytoplasmic sperm injection (ICSI) treatment. Fertilization, embryo quality, transfer rate, implantation, clinical pregnancy will be recorded as well as demographic data. DNA damage of granulosa cells will be compared between two groups. The effect of DNA damage of granulosa cells on fertilization, quality of oocyte and embryo, implantation, and clinical pregnancy will be also evaluated.
Granulosa cells surrounding the oocytes will be mechanically obtained during the oocyte pick-up procedure in women undergoing intracytoplasmic sperm injection (ICSI) treatment due to unexplained infertility and low ovarian reserve. Deoxyribonucleic acid (DNA) damage in these cells will be evaluated by flow cytometry. Fertilization rates, embryo quality by grading, and transfer rates will also be assessed. Implantation and clinical pregnancy rates will be recorded as well as demographic data such as age, body-mass index, smoking, alcohol use, employment, coexisting chronic disease, infertility duration, etiology of infertility, treatment protocol. Implantation will be evaluated by determination of serum human chorionic gonadotropin (hCG) at day 15 following an embryo transfer. Clinical pregnancy will be diagnosed upon presence of gestational sac on ultrasound examination. DNA damage of granulosa cells will be compared between low ovarian reserve group and control group. The effect of DNA damage of granulosa cells on fertilization, quality of oocyte and embryo, implantation, and clinical pregnancy will be also evaluated.
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| Label | Type | Description | Intervention Names |
|---|---|---|---|
| Low Ovarian Reserve | Patients diagnosed as low ovarian reserve were recruited to the study who have Antimüllerian Hormone level of 1 ng/ml or lower. Patients with low ovarian reserve were diagnosed after normal standard infertility evaluation according to the guideline of The Practice Committee of the American Society for Reproductive Medicine which consist of the assesment of spermiogram, ovulation, hysterosalpingogram and if indicated ovarian reserve tests and laparoscopy. Male infertility is an exclusion. | ||
| Unexplained Infertile (Control group) | Patients diagnosed as unexplained infertility (UI) were recruited to the study who have Antimüllerian Hormone level of 1.5 ng/ml or higher as a control group. UI was diagnosed after normal standard infertility evaluation according to the guideline of The Practice Committee of the American Society for Reproductive Medicine which consist of the assesment of spermiogram, ovulation, hysterosalpingogram and if indicated ovarian reserve tests and laparoscopy. |
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| Measure | Description | Time Frame |
|---|---|---|
| Determining the correlation of ovarian reserve and age factor with DNA Damage markers in granulosa cells. | Demographic data will be noted for each patient at the day of oocyte retrieval, and DNA damage markers will be shown by flowcytometry at the same day. | 1 day |
| Measure | Description | Time Frame |
|---|---|---|
| Fertilization defined as the presence of two pronuclei under light microscope one day after intracytoplasmic sperm injection procedure | Presence of two pronuclei under light microscope | 1 day after ICSI procedure |
| Implantation defined as positive serum human chorionic gonadotropin levels 12 days after embryo transfer |
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Inclusion Criteria:
Exclusion Criteria:
Female patients ages between 18 years to 35 years
Low ovarian reserve group will be included as Antimullerian hormone level results as lower than 1 ng/ml without any other infertility evidence.The control group received IVF for Unexplained infertile (UI) group UI was diagnosed after normal standard infertility evaluation according to the guideline of The Practice Committee of the American Society for Reproductive Medicine which consist of the assesment of spermiogram, ovulation, hysterosalpingogram and if indicated ovarian reserve tests and laparoscopy as well as Antimullerian hormone level results as greater than 1,5 ng/ml. If the results of all this tests were normal, patients were accepted as UI.
| Name | Role | Phone | Extension | |
|---|---|---|---|---|
| Erman Ciftci, MD | Contact | +905074422414 | ermanciftci1@gmail.com |
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Granulosa cells will be isolated by using hyaluronidase. A single cell suspension will be obtained by filtering through 70 micron pores. The cells will be centrifuged remove the supernatants and the cells will be suspended in phosphate buffered saline (PBS). CD45+ cells which will be suspended in PBS will be labeled with Pacific Blue fluorochrome-conjugated anti-CD45 antibody. The cells will be fixed with 1% paraformaldehyde, the fixative solution will be removed and permeabilized with PBS containing 0.1% Triton X-100 and 1% bovine serum albumin (BSA) block solution. Fixation and permeabilization stages, both processes will be carried out at 4°C. Permeabilized cells will be suspended in PBS containing 1% BSA, with Alexa Fluor 700 fluorochrome-conjugated Histone H2A.X and Alexa Fluor 488 fluorochrome-conjugated Gamma H2A.X antibodies at 4°C. The cells will be read using the DxFLEX flow cell meter system. The protein levels of Histone H2A.X and its phosphorylated form will be determined.
Positive human chorionic gonadotropin levels |
| 12 days after embryo transfer |
| Clinical pregnancy will be defined as the presence of gestational sac in transvaginal ultrasonographic examination 5 weeks after embryo transfer | Presence of a gestational sac in transvaginal ultrasonographic examination | 5 weeks |