A variety of predictors are available for ovarian stimulation cycles in assisted reproductive technology (ART) forecasting ovarian response and reproductive outcome in women including biomarkers such as anti- Müllerian hormone (AMH). The aim of our present study was to compare the relationship between AMH levels and pregnancy outcomes in patients undergoing intra-cytoplasmic sperm injection (ICSI). Overall, fifty patients (n = 50), aged 20–45 years were recruited for the present prospective study. Three AMH levels were presented with high often poly cystic ovarian syndrome (PCOS) amongst 52.4% patients, 40.5% in normal and 7.1% in low to normal, correspondingly. There was statistically significant relationship between AMH and day of embryo transfer (p < 0.05). The Pearson analysis between AMH, age, E2 and FSH displayed no statistically significant relationship between E2 and AMH (p < 0.05) and negative correlation between FSH and age (p > 0.05). The area under the receiver operating characteristic curve for E2 was 0.725 and for AMH levels as predictors of CPR was 0.497 indicating E2 as better predictor than AMH. The number of oocytes, mature oocytes and fertilized oocytes all presented a weak positive relationship to AMH. Our results confirm the clinical significance of AMH to accurately predict ovarian reserve as a marker and its limitations to use as predictor for a positive pregnancy outcome. Additional prospective studies should be conducted to validate the predictive capability of AMH levels for the outcome of clinical pregnancy.

Introduction ABSTRACT Woman are born with a lifetime supply of oocytes, these oocytes gradually decrease in both quantity and quality with age. Anti-Müllerian Hormone (AMH) is a hormone secreted by cells in the developing oocyte sacs (follicles). AMH has become an important predictor of ovarian reserve. Low AMH levels can forecast reduced response to gonadotropins in In-Vitro Fertilization (IVF) cycles a well as pregnancy rate. Accurate and detailed tests should be conducted that can aid in predicting the chance of a pregnancy through IVF. The clinician together with the patients can make an educated decision about whether to continue with the treatment or review other options that may be viable. Many medical studies have found that a woman’s AMH concentration in the blood can predict how many oocytes they can produce during IVF treatment. On this basis, tests have become available to measure AMH and other markers that indicate fertility changes and the state of advancement of ovarian ageing. Aim and Objectives The aim of the study was determine the relationship between Anti-Müllerian Hormone levels and pregnancy outcomes in patients undergoing in-vitro fertilization or intra- cytoplasmic sperm injection (ICSI). The objectives were as follows: (1) To determine whether there is a correlation in patients presenting with low AMH and low oocyte reserve; (2) To examine if AMH levels affect the oocyte quality; (3) To evaluate the correlation between AMH level and a positive pregnancy outcome. Methodology A total of 50 patients were recruited from C.A.R.E (Centre of Assisted Reproduction and Endocrinology) Clinic in Westville who were undergoing IVF treatment. The blood samples were taken at room temperature. Serum was used to determine estrogen (E2), progesterone (P4), luteinizing hormone (LH), anti-müllerian hormone (AMH), and follicle stimulating hormone (FSH) levels. Levels were determined using an ultra-sensitive enzyme-linked immunosorbent assays (ELISA) (Beckman Coulter). Two stimulation protocols were used to harvest a maximum number of oocytes. The long protocol used Lucrin Subcut 10 units daily and Lucrin Depo 3.75mg for down regulation. Stimulation drugs were Gonal F®, Menopur®, Puregon®, Clomid®, Fertomid® and Fostimon® The short protocol consisted of GnRH antagonist with Cetrotide 0.25mg primed with Logynon ED. No down regulation was required. The patient started on day 2 or day 3 of the menstrual cycle, Cetrotide 0.25mg is given to suppress the LH. The same drugs were used for stimulation as per the long protocol. The patients’ blood serum was tested to measure the amount of Estrogen in her body prior to the collection of oocytes. In all patient’s ovulation was induced using 5000 – 10 000 IU hCG (Pregnyl®, Ovitrel®) trigger injection, provided the lead follicle had reached a diameter of 19mm. Oocyte retrieval was performed trans-vaginally under ultra sound guidance 36 hours after the administration of the hCG. The fluid containing the oocytes was aspirated using an oocyte recovery needle and forwarded to the IVF laboratory where the oocytes were identified, rinsed in culture media and were incubated at 37°C in IVF incubators. After 2-4 hours incubation period the cumulus complex was removed from the oocyte using the enzyme hyaluronidase and glass pipettes. Following denudation, the oocytes were placed back into the incubator until the ICSI was performed. Maturation and morphological features of the oocytes were noted before the ICSI. The features of each oocyte were evaluated using an inverted microscope. Fertilization was assessed 19-21 hours after the ICSI was performed and was characterized by the presence of two pronuclei to show the result of the union between the male and female genetic material to form a zygote cell. Embryos were grown up to Day 3 (8 cell stage), Day 5 (blastocyst stage) or Day 6 (hatching blastocyst) and transferred into the patient. A pregnancy test was performed 14 days post transfer. Results 50 patients that met the inclusion criteria were recruited for the study. From the initial sample size of 50, 42 presented with data that could be analysed whilst 8 patients had oocytes that where abnormal and did not result in a transfer. The data from these 8 patients were not included in the study due to poor embryo development. According to the AMH levels, 52.4% of patients were in High Category, 40.5% were in the Normal and 7.1% were in the Low to Normal Category. A cross-tabulation of the number of oocytes retrieved; the number of oocytes mature, and the number of oocytes fertilized was done. Not all eggs obtained were at the metaphase 2 stages and had to be matured in the incubator overnight and injected with sperm the following day. A Chi-square test for Independence was performed to check whether there is an association between the number of oocytes fertilized and the AMH category. A Chi-squared value of 18.5, degrees of freedom = 12, with a p = 0.10 was found. Therefore, showing no statistically significant relationship between the numbers of embryo’s fertilized versus AMH category (p > 0.05). A Chi- squared test were done of AMH category and Number of embryos transferred was done resulting in a value of 6.384 with df = 4 and a cross tabulation ensued a p- value of 0.172. There was thus not a significant association between AMH category and No. of embryos transferred. A cross-tabulation and a Chi-square test were done of AMH category and the day of embryo transfer was done. A Chi-square value of 14.117, 6 degrees of freedom and p = 0.028 was observed. There was a statistically significant relationship between the AMH category and the day of embryo transfer (p< 0.05). A cross-tabulation of AMH category and Pregnancy outcome was calculated. It can also be seen that of the 22 cases reported in the High category, 6 resulted in a positive pregnancy, 17 cases where the AMH category was “Normal”, 6 resulted in a positive outcome (6/12 = 50.0%), while out of the 3 cases where the AMH category was “Low to Normal“ there were no pregnancies reported. The Chi-squared test for independence of AMH category and Pregnancy outcome gave a Chi-Squared value of 0.502, 2 degrees of freedom and p = 0.778. Race and pregnancy outcome were calculated using a cross tabulation and a Chi- square test for independence gave a Chi-squared value of 2.246, with 3 degrees of freedom and p = 0.532 (p > 0.05). To determine if a statistical significance exists between AMH and age, E2 and FSH a Pearson Correlations was performed. Table 12 shows the Pearson analysis between E2 and AMH. The Pearson Correlation coefficient of 0.151 with p = 0.341 (p<0.05) indicates a very weak/ no statistically significant relationship between E2 and AMH. AMH and age produced a coefficient of -0.028 thus showing a weak, negative correlation with p = 0.859 (p> 0.05). A stronger relationship between these two variables was expected as it is known that as age increase, AMH should decrease. Pearson Correlation between the AMH and FSH produced a coefficient of -0.185 thus showing a weak, negative correlation with p = 0.240 (p> 0.05). The Pearson Correlation between FSH and age also showed that there was no statistical significance, p = 0.583 (p > 0.05) but a very weak negative correlation (Pearson Correlation -0.087). Pearson correlation between the number of oocytes and age also did not show any statistically significant relationship (p = 0.082; p<0.05). Pearson Correlation value of -0.271 shows a weak negative relationship. No significant relationship was shown between AMH and number of oocytes using a Pearson Correlation test (p = 0.191), number of mature oocytes (p = 0.300) and number of oocytes fertilized (p = 0.146). The number of oocytes, mature oocytes and oocytes fertilized all showed a weak positive relationship to AMH (0.206, 0.164, and 0.228, respectively). Conclusion In conclusion, while appropriate reference values are being created per age category and until the consequences of having a low or high AMH for one’s age are being established, AMH should only be determined in the context of clinical studies. At present, the most important clinical role of AMH at this stage is to serve as a red-flag for reduced ovarian reserve in women of reproductive age who must undergo further diagnostics. As per the study conducted, we can deduce that AMH can accurately predict ovarian reserve but cannot predict the oocyte quality or a positive pregnancy outcome. The more oocytes obtained, increases a patient’s chance of more viable embryos and therefore, improving chances of a healthy pregnancy and ultimately a live birth. This thesis has established a definite role for AMH as a forecaster for both current and future individual fertility.