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Infertility and Women's Age by Zohreh Nazemian a Thesis Title: Infertility and Women’s Age by Zohreh Nazemian A thesis submitted in conformity with the requirements for the degree of Master of Science Institute of Medical Science (IMS) University of Toronto © Copyright by Zohreh Nazemian (2011) Infertility and women's age Zohreh Nazemian Master of Science, IMS University of Toronto 2011 Abstract: In the first part of study, our objective was to determine the effect of CoQ10 supplementation of culture media on preimplantation mouse and human embryo development. CoQ10 supplementation of culture media did not improve mouse or human embryo development in vitro. Since the results appeared to be negative, we decided to move on to research the effect of age on female infertility. In the second part, we investigated the effect of female age and ovarian stimulation protocols on IUI outcome in 411 infertile women. We found that the ongoing/live birth rate per cycle in women ≤ 37 years was significantly higher than in older patients. In the third section, we determined if very young age (≤25 yrs) has an impact on pregnancy outcome in women undergoing IVF-ET. Our results demonstrating lower pregnancy rates in very young patients and egg donors compared to the patients in their early thirties were surprising. ii AKNOWLEDGEMENTS There are so many people involved in making my graduate studies a rewarding experience. I will take this opportunity to thank them all. I would like to express sincere appreciation to my supervisor Dr. Robert Casper for allowing me to do research in his clinic and for his continuous support and direction. I am also Thankful to the members of my PAC committee, Dr. Julia Knight, Dr. Andrea Jurisicova, and Dr Ian Rogers for their support and constructive suggestions. I am obliged to the IVF and Andrology Laboratory staff of TCART who has contributed to this project over the last few years. I would like to thank Murid Javed, PhD for his technical assistance. This project would not have been proceed without the support of Dr. Navid Esfandiari from IVF laboratory, TCART for providing scientific and technical support that have made the studies presented in this thesis possible. Finally, I would like to thank my family, specifically my husband and my daughter for their support and encouragement. iii Table of Contents Abstract ii Acknowledgements iv Table of Contents iv List of Abbreviations iv List of Tables iv List of Figures iv Chapter 1: Introduction and Literature Review 1 1.1 Cogenesis and Oocyte Biology 2 1.2 Oocyte Fertilization 3 1.3 Basics of Infertility 3 1.3.1 Male Infertility 4 1.3.2 Female Infertility 5 1.3.2.1 Age 5 1.3.2.2 Ovulation Disorder 7 1.3.2.3 Tubal Factor 8 1.3.2.4 Endometriosis 8 1.3.2.5 Unexplained Infertility 9 1.4 Infertility Treatment 9 1.4.1 Assisted Reproductive Technology 9 1.4.2 Intrauterine Insemination 10 1.4.3 In Vitro Fertilization 11 1.4.4 Intracytoplasmic Sperm Injection 14 1.4.4.1 Indication of ICSI 14 1.4.5 Human Embryo Culture and Development In Vitro 15 1.4.6 Embryo Cryopreservation 18 1.4.7 Children Born After IVF 19 1.5 Oocyte Maturation and Chromosomal Status 21 1.6 Mitochondria and Oocytes 22 iv 1.7 Mitochondria and Reactive Oxygen Species 26 1.8 Mitochondrial Nutrients 26 1.8.1 Coenzyme Q10 27 Hypothesis 31 Objectives 31 Chapter 2: Effects of Mitochondrial Nutrients, CoQ10 On Human Early Embryo Development In Vitro 33 2.1 Abstract 34 2.2 Introduction 35 2.3 Materials and Methods 37 2.3.1 Preparation of CoQ10 Solution 37 2.3.2 Preparation 0f Culture Dishes 37 2.3.3 Mouse Embryo Culture 37 2.3.4 Experimental Groups 38 2.3.5 Human Embryo Culture 39 2.3.6 Experimental Groups 41 2.3.7 Pronuclear Embryo Thawing 41 2.3.8 Blastocyst Culture and Scores 43 2.3.9 Different Alphanumeric Scores 45 2.3.10 Different Scores of Inner Cell Mass 46 2.3.11 Trophectoderm Grades 46 2.3.12 Blastocyst Vitrification 46 2.3.13 Blastocyst Thawing 47 2.4 Statistical Analysis 47 2.5 Results 49 2.5 1 Mouse Embryo Development Rate 49 2.5.2 Blastocyst Total Cell Number 49 2.5.3 Human Embryo Development Rates and Scores 49 2.5.4 Blastocyst Vitrification Rates 50 2.6 Discussion 51 2.7 Conclusion 56 2.8 Figures and Tables 57 v Chapter 3: Outcome of Intrauterine Insemination Cycles Using Various Ovarian Stimulation Protocols 67 3.1 Abstract 68 3.2 Introduction 69 3.3 Materials and Methods 70 3.4 Statistical Analysis 73 3.5 Results 74 3.6 Discussion 77 3.7 Conclusion 81 3.8 Figures and Tables 82 Chapter 4: The Effect of Age on In Vitro Fertilization Outcome 91 4.1 Abstract 92 4.2 Introduction 93 4.3 Materials and Methods 94 4.3.1 Patients 94 4.3.2 Semen Analysis and Sperm Preparation 94 4.3.3 Ovarian Stimulation and Oocyte Retrieval 95 4.3.4 IVF and ICSI Procedures 95 4.4 Primary and Secondary Outcome Measures 96 4.5 Statistical Analysis 96 4.6 Results 98 4.7 Discussion 100 4.8 Figures and Tables 103 Chapter 5: Future Directions 108 Chapter 6: References 110 vi LIST OF ABBREVIATIONS AE Adverse event ART Assisted reproductive technologies CC Clomiphene citrate COH Controlled ovarian hyperstimulation CoQ10 Co-Enzyme Q10 E2 17--estradiol FSH Follicle stimulating hormone hCG Human chorionic gonadotropin HTF Human tubal fluid IVF in vitro fertilization ICSI intracytoplasmic sperm injection IU International units IUI Intrauterine insemination LH Luteinizing hormone OHSS Ovarian hyperstimulation syndrome P Progesterone SOF Synthetic oviduct fluid SSS Serum supplement substitute TVS Transvaginal sonography US Ultrasound/ultrasonography vii List of Tables Table I.a: Mouse embryo development after 48 and 72 hours Table II.a: Human embryo development on Day-2 and Day-3 Table I.b: Outcome of IUI treatment with reference to age Table II.b. Pregnancy rate per cycle with reference to infertility diagnosis Table III. Pregnancy rate per cycle with reference to ovarian stimulation protocol Table III.b: Pregnancy rate per cycle with reference to ovarian stimulation protocol Table IV.b: Semen characteristics in IUI patients Table V.b: Pregnancy rate as reference to total motile sperm Table VI.b: Pregnancy rate as reference to number of follicles bigger than 14 mm Table VII.b: Pregnancy rate as reference to E2 level at the day of hCG Table VIII.b: Pregnancy rate as reference to basal FSH level Table I.c: Patient demographics of study groups Table II.c: Semen characteristics in all groups Table III.c: Cycle characteristics of study groups viii List of Figures Figure 1a: Structure of Coenzyme Q 10 Figure 1b: intracytoiplasmic sperm injection Figure 2: Mouse embryos at morula stage Figure 3: Mouse embryos at early, expanding, expanded, and hatching blastocyst stage Figure 4: Flourescence image of mouse blastocyst Figure 5: Human embryo development in A) control, B) 10µL alcohol, C) 5µM CoQ10, D) 10 µM CoQ10 group. Figure 6. Mouse embryo development in vitro after 24 hours Figure 7. Mouse embryo development in vitro after 48 hours Figure 8. Mouse embryo development in vitro after 72 hours Figure 9. Human embryo development in culture media supplemented with CoQ10 Figure 10. Retrieved oocytes A) from a young infertility patient B) from a young egg donor, and C) embryo quality on day-2 post retrieval in a young patient ix Chapter 1: Introduction and Literature Review Fertility is the capacity to produce offspring, and a couple is considered to be infertile if they cannot conceive after 12 months of unprotected intercourse. A more Strict definition of infertility is failure to achieve a pregnancy in a 12 month period for patients under 35 years of age and failure to conceive in a 6 month period for the over 35 years (1). The prevalence of infertility worldwide is estimated to be one in seven to one in five (14-20%) couples in their reproductive age. The causes of infertility can be male, female or a combination of both and include ovulatory disorders, tubal disease, endometriosis, chromosomal abnormalities, sperm factors and unexplained infertility. The majority of infertility cases, both male and female factors, are overcome through surgical and medical infertility treatment. Medical treatment options include assisted reproductive techniques (ART) such as in vitro fertilization (IVF), intracytoplasmic sperm injection (ICSI), and intrauterine insemination (IUI). Infertility treatment has been dramatically advanced in recent years; however, age related infertility remains as one of the most difficult challenges. Age must be taken into account when couples are considering assisted reproductive technology. It is well known for years that the pregnancy rate is inversely related to the age of the female (2 Some explanations for this include diminished ovarian reserve, increased rate of aneuploidy, decreased frequency of sexual intercourse, diminished desire for childbearing and increased rate of spontaneous abortion. Among infertile women undergoing infertility treatment, advanced maternal age is also associated with lower fertilization rates and higher risk of chromosomal abnormalities (3). In humans, the age of the oocyte, not the age of the uterus, is the main cause of reproductive failure in IVF and embryo transfer techniques. 1 1.1: Oogenesis and Oocyte Biology: Oogenesis starts early in fetal life when primordial germ cells (oogonia) migrate to ovarian cortex. Oogonia undergo many cycles of mitotic divisions and their number increase to several million in the fetal ovary by 20 weeks of pregnancy. However, the majority of these oogonia are degenerated in the process of atresia while some others enlarge and undergo nuclear changes and called primary oocytes.
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