The Role of DNA Damage of Granulosa Cell on Oocyte Quality and in Vitro Fertilization Outcome

Study Overview
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Brief Summary
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Deoxyribonucleic acid (DNA) damage of granulosa cells obtained during oocyte retrieval will be evaluated by comet assay in unexplained infertile patients undergoing in vitro fertilization (IVF) treatment. The oocytes will be graded by particular criteria. Fertilization, embryo quality, transfer rate, implantation, clinical pregnancy, pregnancy outcomes (gestational age at delivery, route of delivery, and birthweight etc.) will be recorded as well as demographic data. DNA damage of granulosa cells will be compared between unexplained infertile and control groups. The effect of DNA damage of granulosa cells on fertilization, quality of oocyte and embryo, implantation, and clinical pregnancy will be also evaluated.

Detailed Description
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Granulosa cells surrounding the oocytes will be mechanically obtained during the oocyte pick-up procedure in women undergoing in vitro fertilization (IVF) treatment due to unexplained infertility. Deoxyribonucleic acid (DNA) damage in these cells will be evaluated by comet assay. The quality of oocytes retrieved during the oocyte pick-up procedure will be graded by particular criteria (zona pellucida thickness, granulation, vacuolization, etc). Fertilization rates, embryo quality by grading, and transfer rates will also be assessed. Implantation and clinical pregnancy rates, and pregnancy outcomes including gestational age at delivery, route of delivery, and birthweight 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, and hormone levels on day 3. Implantation will be evaluated by determination of serum human chorionic gonadotropin (hCG) at day 12 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 unexplained infertile 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.

Official Title
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The Role of DNA Damage of Granulosa Cell on Oocyte Quality and in Vitro Fertilization Outcome

Conditions
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in Vitro Fertilization

Participation Criteria
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Eligibility Criteria
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Inclusion Criteria: Unexplained infertile patients undergoing in vitro fertilization (IVF) treatment Exclusion Criteria: Chronic systemic disease (rheumatoid arthritis, hypertension, diabetes..) Endocrinopathy (Thyroid, prolactin... abnormalities) Chemotherapy or radiotherapy history Cigarette, alcohol, chronic medication use Diminished ovarian reserve Endometriosis Having any medication use History of any surgical procedure on ovaries and uterus Smoking or alcohol consumption Severe oligoasthenospermia Tubal infertility associated with hydrosalpinx, severe pelvic adhesions, endometriosis or pelvic inflammatory disease

Ages Eligible for Study
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Minimum Age: 19 Years
Maximum Age: 44 Years

Sexes Eligible for Study
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Female

Accepts Healthy Volunteers
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Accepts Healthy Volunteers

Study Plan
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How is the study designed?
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Arms and Interventions

| Participant Group/Arm | Intervention/Treatment |
| --- | --- |
| Unexplained infertile group<br>Patients diagnosed as unexplained infertility (UI) were recruited to the study. 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. If the results of all this tests were normal, patients were accepted as UI. | |
| Control group<br>The control group received in vitro fertilization (IVF) for tubal factor and included only those women who had salpingectomy for ectopic pregnancy or proximal tubal obstruction because of low-grade infection or fimbrial occlusion with or without mild peritubal adhesions. Tubal infertility associated with hydrosalpinx, severe pelvic adhesions, endometriosis or pelvic inflammatory disease were excluded. Patients with male factor except oligoasthenospermia were also recruited for the study. | |

What is the study measuring?
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Primary Outcome Measures

| Outcome Measure | Measure Description | Time Frame |
| --- | --- | --- |
| Clinical pregnancy 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 after embryo transfer | 

Secondary Outcome Measures

| Outcome Measure | 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 intracytoplasmic sperm injection procedure | 
| Oocyte grade assessed by four parameters (cytoplasmic granulation, properties of the polar body, the perivitelline space and properties of the zona pellucida) under invert microscope 36 hours after human chorionic gonadotropin administration | Oocytes were graded by particular criteria including cytoplasmic granulation, properties of polar body, perivitellin space and zona pellucida. Central cytoplasmic granulation=0, homogenous cytoplasmic granulation=1; large fragmented polar bodies=0, non-fragmented polar bodies with normal size=1; presence of debris in perivitellin space=0, absence of debris in perivitelline space=1; >15 mm thickness and rough surfaced zona pellucida=0, <15 mm thickness and smooth surface zona pellucida=1. A total of used 3-4 points were considered as Grade 1 oocytes (best quality), 2-3 points were grade 2 (moderate quality), 0-1 points were considered as grade 3 (poor quality). | 36 hours after human chorionic gonadotropin administration | 
| Embryo grade assessed under invert microscope 3 days after intracytoplasmic sperm injection procedure | The quality of embryos was graded from 1 to 3 under inverted microscope 3 days after intracytoplasmic sperm injection procedure. Embryos with even-sized blastomers and/or <5% fragments were classified as Grade 1 (good quality). Grade 2 embryos (moderate quality) had blastomeres with slightly-moderate size differences and/or 5-50% fragments. Grade 3 embryos (poor quality) had markedly different-sized blastomers and/or >50% fragments. | 3 days after intracytoplasmic sperm injection procedure | 
| Implantation defined as positive serum human chorionic gonadotropin levels 12 days after embryo transfer | Positive human chorionic gonadotropin levels | 12 days after embryo transfer | 

 Terms related to the study
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Keywords Provided by Centre Hospitalier Valida
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comet assay, DNA damage, granulosa, IVF outcome