Poor Ovarian Stimulation Response in In Vitro Fertilization (IVF) Program

Assessing the Relation Between Hormone Receptors Gene Polymorphism and Ovarian Stimulation Response in In Vitro Fertilization (IVF) Program

The aim of this study is to assess the role of AMH in prediction of poor ovarian response as well as the relation between ESR2 (+1730G>A) (rs4986938), FSHR p.Thr307Ala (c.919A>G, rs6165) and FSHR p.Asn680Ser (c.2039A>G, rs6166) SNPs and the poor response in Egyptian women undergoing IVF procedure. Discovering the genetic variants associated with ovarian response is an important step towards individualized pharmacogenetic protocols of ovarian stimulation.

In-vitro fertilization treatment safety is highly challenged by the erratic individual variability to controlled ovarian hyperstimulation (COH). One of the diverse predictive factors is the AMH, which is considered to be the most sensitive marker. Nowadays, practices of pharmacogenetics could predict the stimulation success and thus tailoring the treatment reaching advancement in patient care. Since the efficiency of the Follicle stimulating hormone (FSH) dose is greatly related to the success of COH, the FSHR gene is treated as the primary candidate in explaining the difference in COH results.Furthermore, the estrogen receptors are important genes for improvements in the diagnosis and treatment of infertility.

Materials and Methods: Starting June 2013, couples with unexplained infertility, seeking the first attempt IVF/ICSI treatment cycle will be recruited in "AL shark Al-Awsat fertility center".In this prospective study, 216 Couples fulfilling our inclusion criteria will enroll in this study after informed consent. Ovarian stimulation will be performed according to the GnRH antagonist protocol with a fixed daily morning dose of Human menopausal gonadotrophin (HMG) [Merional® 75 IU ampoules, IBSA institut] intra-muscular injection starting on cycle day 2. Based on the patient's body mass index and hormonal profile, the daily dose of HMG will be adjusted to (225 IU) for participants with AMH levels > 1.5 ng/ml and/or FSH levels ≤ 8 mIU/ml and to (300 IU) for participants with AMH levels < 1.5 ng/ml and/or FSH levels > 8 mIU/ml, doses will be increased by 75 IU in cases with BMI > 30 Kg/m2. On day 2 of the menstrual cycle, the dose of HMG will be commenced for all patients and will be maintained for 9-11 days according to each participant individual ovarian response assessed by trans-vaginal ultrasound folliculometry starting on cycle day 7 and continued every other day until the day of Human chorionic gonadotrophin (hCG) injection. On cycle day 7, the GnRH antagonist Cetrorelix 0.25 mg [Cetrotide® 0.25 mg syringes, Merck Serono] will be introduced as daily subcutaneous injections and continued till the day of ovulation triggering. Finally, when at least 3 follicles will reach 17 mm in diameter, ovulation will be triggered by a single intra-muscular injection of 10,000 IU of hCG [Choriomon® 5000 IU ampoules, IBSA institut]. 36 hours later, oocyte retrieval will be performed and will be guided by transvaginal ultrasound. According to our primary outcome, the number of oocytes collected, patients will be classified as poor responders (4 oocytes or less) and will be considered as good responders if they produced 5 or more oocytes. The good responders group will serve as the control group.

Genotyping:Peripheral blood will be collected from each patient in an EDTA-containing tube. Genomic DNA will be extracted from lymphocytes of peripheral blood by fully automated system of QIAcube using QIAamp DNA Blood Mini Kit (250) plus QIAamp DNA Blood Mini Accessory Set B, cat. no. 1043369. Detection of the polymorphisms ESR2 (+1730G>A) (rs4986938), FSHR p.Thr307Ala (c.919A>G, rs6165) and FSHR p.Asn680Ser (c.2039A>G, rs6166) SNPs will be performed using the TaqMan system by real-time polymerase chain reaction (PCR). Primers and probes will be provided by applied biosystem, Life Technologies. Assays will be performed with TaqMan Universal Master Mix.

Statistical analysis: Data will be statistically described in terms of mean and standard deviation, for quantitative data and frequencies (number of cases) and relative frequencies (percentages) for qualitative data. Hardy-Weinberg (H-W) Equilibrium for each polymorphism will be tested using the Chi-square test. Comparison of quantitative variables will be done using unpaired t test when comparing 2 categories and one way analysis of variance (ANOVA) with post hoc test when comparing more than 2 categories.

For comparing categorical data, Chi square test will be performed. Exact test will be used instead when the expected frequency is less than 5.

Genotype and allele frequencies will be compared between the disease and the control groups using chi-square tests. Odds ratio (OR) with 95% confidence intervals will be calculated. A probability value (P value) less than 0.05 will be considered statistically significant. All statistical calculations will be done using SPSS (Statistical Package for the Social Science; SPSS Inc., Chicago, IL, USA) version 22.

Inclusion Criteria:

• Women at or under the age of 35 years.
• Normal thyroid stimulating hormone and prolactin levels.
• Normal ovulatory cycles (25-35 interval days), together with proven patent fallopian tubes at hysterosalpingography or laparoscopy done within six cycles preceding the ICSI cycle.
• Presence of both ovaries with normal findings as assessed by trans-vaginal ultrasound and laparoscopy.
• All male partners had a normal semen analysis according to WHO criteria (WHO, 2010), done within 6 months preceding the ICSI cycle

Exclusion Criteria

• Chronic medical disorders such as diabetes.
• Previous inadequate response to ovulation induction.
• Polycystic ovary syndrome.
• Women who performed ovarian surgery and cases of endometriosis diagnosed by laparoscopy or suspected by ultrasound or CA-125 assay.
• Abnormal pelvic pathology or congenital anomalies.