Role of Metformin in Cases of (In Vitro Fertilization)

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Magdalene Grant
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Ch) Cairo University (2004) Supervised by Prof. Dr. Ismail Aboul Fetouh Dept.

1 Role of Metformin in Cases of (In Vitro Fertilization) Thesis Submitted for Partial Fulfillment of Master Degree In Obstetrics and Gynecology Presented by Shaimaa Sobh Mohamed (M.B., B. Ch) Cairo University (2004) Supervised by Prof. Dr. Ismail Aboul Fetouh Dept. of Gynecology and Obstetrics Faculty of Medicine, Cairo University Prof. Dr. Amal Darweish Dept. of Gynecology and Obstetrics Faculty of Medicine, Cairo University Cairo University (2012)

2 Abstract Background: Aim of study is to evaluate effect of metformin on controlled ovarian stimulation and outcome of IVF. Method: Randomized controlled study was carried out, total of 50 women undergoing IVF, were divided to two groups: group A: control group (n= 24), and group B: metformin group (n= 26), who received metformin 500 mg t.d.s starting the cycle before ovarian stimulation till the day of serum β- hcg. Both groups were subjected to controlled ovarian hyperstimulation according to long protocol; fertilization in all cases was done by ICSI. Results: Both groups were similar regarding demographic data and management. Metformin group showed no evidence of statistically significant difference regarding duration of stimulation (P=0.72), units of gonadotropins needed for stimulation (P=0.86), total number of follicles obtained (P= 0.59), fertilization rate (P= 0.40), β-hcg (P=0.60), OHSS incidence (P=0.36). Conclusion: metformin as co therapy with controlled ovarian hyperstimulation does not seem to facilitate IVF stimulation or to improve the outcome of IVF treatment. Key words: IVF, metformin, ICSI, OHSS, controlled ovarian hyperstimulation

3 List of Abbreviations IVF :In Vitro Fertilization ICSI :Intracytoplasm Sperm Injection PID :Pelvic Inflammatory Disease hmg :Human Menopausal Gonadotropins FSH :Follicle Stimulating Hormone GnRh :Gonadotropins Releasing Hormone OHSS :Ovarian Hyperstimulation Syndrome hcg :Human Chorionic Gonadotropin WHO :World Health Organization ART :Assisted Reproductive Techniques PCOS :Polycystic Ovarian Syndrome BMI :Body Mass Index VEGF :Vascular Endothelial Growth Factor E 2 :Estradiol PGE 2 :Prostaglandins E 2 PGI 2 :Prostaglandins I 2 LH :Luteinizing Hormone IGF-I :Insulin like Growth Factor-I EGF :Epidermal Growth Factor TGF :Transforming Growth Factor IL- 6 :Inter-Lukin -6 DVT :Deep Venous Thrombosis CBC :Complete Blood Count CXR :Chest X- Ray AMPK :Adenosine Monophosphate Protein Kinase SHP :Small Heterodimer Partner GLUT- 4 :Glucose Transporter -4 NAFLD :Non Alcoholic Fatty Liver Disease CC :Clomiphene Citrate SAB :Spontaneous Abortion IVM :In Vitro Maturation RCTs :Randomized Controlled Trials

4 Acknowledgement I wish to express my deepest gratitude and profound appreciation to Dr. I smail Aboul Fo utouh, Professor of Obstetrics and Gynecology, Faculty of Medicine, Cairo University, for his valuable advice, care, encouragement, guidance, great support and continuous help. I wish to convey my sincere appreciation to Dr. Amal Dar weish, Assistant professor of Obstetrics and Gynecology, Faculty of Medicine, Cairo University, for her kind supervision, support and confidence. I gratefully acknowledge the great effort and time spent by Dr. Mohamed Abdul Fattah, Teacher of Obstetrics and Gynecology, Faculty of Medicine, Cairo University, and his helpful discussions, sincere assistance and close observation through the whole work. Special thanks to my parents, for their tolerant support, encouragement and sacrifices they made for me.

5 Contents Introduction 1 Aim of work 4 Chapter - 1: Ovarian hyperstimulation syndrome 5 Chapter - 2: Metformin 47 Patients and methodology 69 Statistical analysis 74 Results 75 Discussion 98 Conclusion 108 Recommendations 109 Summary 110 References 112 Arabic summary 114

6 Table List of Tables Page Table.1 Age distribution among study groups 76 Table.2 BMI distribution among study groups 77 Table.3 Cause of infertility among study groups 78 Table.4 Hormonal Profile among study groups 80 Table.5 Down Regulation Protocol 83 Table.6 Ovarian Stimulation: Duration and Units of 83 Gonadotropins Table.7 Total Number of Follicles 86 Table.8 Number of MII oocytes 87 Table.9 Fertilization Rate 88 Table.10 Total Number of Embryos and Embryos 90 Transferred Table.11 Cycle Cancellation 92 Table.12 Day of Embryo Transfer 92 Table.13 Serum β- HCG 94 Table.14 Number of sacs 94 Table.15 Implantation Rate 95 Table.16 Ovarian Hyperstimulation Syndrome 96

7 List of Figures Figure Page Fig.1 First step in the pathophysiology of OHSS 11 Fig.2 OHSS pathophysiology: VEGF 16 Fig.3 Pathogenesis of OHSS developing clinical manifestations 18 Fig.4 G. officinalis, a natural source of Galegine 47 Fig.5 Metformin structure 49 Fig.6 Potential mechanism of action of metformin 50 Fig.7 Effect of insulin receptor stimulation on GLUT4 distribution 52 Fig.8 Age distribution among study groups 76 Fig.9 BMI distribution among study groups 77 Fig.10 The mean of Basal FSH in study groups 80 Fig.11 The mean of Basal LH in study groups 81 Fig.12 The mean of Basal E 2 in study groups 81 Fig.13 Mean duration of stimulation among study groups 84 Fig.14 The mean total number of follicles in study groups 86 Fig.15 The mean number of MII oocytes in study groups 87 Fig.16 The mean number of fertilized follicles 88 Fig.17 The mean total number of embryos 90 Fig.18 The mean number of embryos transferred 91 Fig.19 Implantation rate among study groups 95

8 Introduction Worldwide it is estimated that one in seven couples have problems conceiving, with the incidence similar in most countries independent of the level of the country's development. (Marcus et al 2011), meanwhile; IVF technology has been impressively developed to vanquish the ghost of infertility. In vitro fertilization (IVF) is a process by which oocytes are fertilized by sperms outside the body, It is considered a major treatment in infertility when other methods of assisted reproductive technology have failed. The process involves hormonally controlling the ovulatory process, oocytes retrieval and letting sperms fertilize them either by incubation in a fluid medium or by intra-cytoplasm sperm injection (ICSI). The first successful birth of a "test tube baby", Louise Brown, occurred in Robert G. Edwards, the physiologist who developed the treatment, was awarded the Nobel Prize in Physiology or Medicine in Before that, there was a transient biochemical pregnancy reported by Australian Foxton School researchers in 1953 and an ectopic pregnancy reported by Patrick Steptoe and Edwards in (Schulman et al., 2010) IVF was first indicated to overcome female infertility due to tubal disease secondary to pelvic inflammatory disease (PID) or adhesions, endometriosis or premature ovarian failure. It may also assist in male infertility, and in such cases intra-cytoplasm sperm injection (ICSI) may be used, where a sperm cell is injected directly into the egg cell. Nowadays, unexplained infertility is also an indication for IVF, including immunological infertility. (Geoffrey et al., 2000).IVF 1

9 Introduction can also be combined with pre-implantation genetic diagnosis (PGD) to rule out presence of genetic disorders or for gender selection. (Handyside et al., 2010) A typical IVF cycle is composed of five basic steps, first is induction of ovulation also called controlled ovarian hyperstimulation, and second is egg retrieval which is done using ultrasound as a guide, with insertion of a thin needle through the vagina into the ovary and follicles containing the oocytes. The third step is fertilization mostly by (ICSI). The next step will be embryo transfer which is usually done three to five days after egg retrieval, and the last step is pregnancy confirmation by serum β-hcg (Lobo et al., 2007). As any medical procedure, IVF is carrying the risk of complications. The major complication of IVF is the risk of multiple births. This is directly related to the practice of transferring multiple embryos at embryo transfer. Multiple births are related to increased risk of pregnancy loss, obstetrical complications as preeclampsia, gestational diabetes and postpartum hemorrhage. Growth restriction, prematurity, and neonatal morbidity were also reported with multiple pregnancy. Strict limits on the number of embryos that may be transferred have been enacted in some countries (e.g. Britain, Belgium) to reduce the risk of highorder multiples (triplets or more), but are not universally followed or accepted. A double blind, randomized study followed IVF pregnancies has reached that the mean gestational age was 39.4 weeks for singleton pregnancies, and 36.6 weeks for multiple pregnancies. In total, 73 infants (33 boys, 40 girls) were born. A birth weight <2500 g was reported for 8.7% and 54.2% of the infants resulting from 2

10 Introduction singleton and twins delivery respectively. (Olivennes et al., 2001).Fortunately, embryo reduction is offering a necessary evil to reduce fetal and maternal risks associated with high order multi-fetal pregnancies. Very few patients experience side effects with the use of fertility drugs, include local reactions at the site of injections which is more common with hmg preparations than purified or recombinant FSH. There is also a small risk of a generalized allergic reaction (Marcus S. 2011). Other drugs such as GnRh analogues may cause headaches, mood changes, hot flushes and vaginal dryness in some women; these are usually short-lived and are no cause for concern (Derman et al., 1995). Controversy surrounds the relations among infertility, fertility drug use, and the risk of ovarian cancer, a pooled analysis of case-control studies suggest a role for specific biologic causes of infertility, but not for fertility drugs in overall risk for ovarian cancer (Roberta et al,. 2002). Another long-term follow-up study had reached a conclusion that no confirmed association between the use of fertility drugs and an increased risk of breast and ovarian cancers (Potashnik et al,. 1999) and same result was reached by Danish population based cohort study (Jensen et al,. 2008) Another risk of ovarian stimulation is the development of ovarian hyperstimulation syndrome (OHSS) which will be handled in details being the core of the study together with metformin drug. 3

11 Aim of the Work Aim of the Work To study the effect of metformin on number of oocytes, fertilization rate and prevention of ovarian hyperstimulation syndrome during induction of ovulation in cases subjected to in vitro fertilization. 4

12 Chapter 1 OHSS Ovarian Hyperstimulation Syndrome (OHSS) Ovarian hyperstimulation syndrome (OHSS) is an iatrogenic complication of ovarian stimulation for assisted reproduction technology and other infertility treatments, following gonadotropin therapy. OHSS usually develops several days after oocyte retrieval or assisted ovulation. This syndrome is characterized by ovarian enlargement due to multiple ovarian cysts and an acute fluid shift into the extravascular space. Results include ascites, hemoconcentration, hypovolemia, and electrolyte imbalance (Horwitz et al., 2011). It is the most serious complication of infertility treatment and particularly associated with injection of human chorionic gonadotropin (hcg) which is used for triggering oocyte release. The risk is further increased by multiple doses of hcg after ovulation and if the procedure results in pregnancy (Vorvick et al., 2009). Using GnRH agonist instead of hcg for triggering oocyte release results in an elimination of the risk of ovarian hyperstimulation syndrome, but with a slight decrease of the delivery rate of approximately 6 % (Humaidan et al., 2011). 5

13 Chapter 1 OHSS Incidence of OHSS: Despite careful monitoring, up to 33% of IVF cases have been reported to develop mild forms of OHSS. Severe OHSS has been reported in 3-8% of IVF cycles (Delvigne and Rosenberg 2002). According to the World Health Organization (WHO), severe OHSS develops in 0.2 1% of all stimulated ART cycles (Binder et al., 2007). Diagnosis and classification of OHSS: The symptoms usually begin within a week after the oocyte retrieval (early OHSS) or after a week from oocyte retrieval (late OHSS). The early OHSS pattern is associated with exogenously administered HCG and a higher risk of preclinical miscarriage, whereas late OHSS may be closely associated with the conception cycles, especially multiple pregnancies, and is more likely to be severe and to last longer than early OHSS (Papanikolaou et al., 2004). It s classified as mild, moderate or severe by symptoms, signs, biochemistry, blood tests and ultrasound scan findings. Most of the cases will develop mild or moderate form of the syndrome and invariably resolve within a few days unless pregnancy occurs, that may delay recovery. The worst cases tend to be associated with pregnancy. 6

14 Chapter 1 OHSS The first classification of OHSS, which was prepared in 1967(Rabau et al., 1967), combined both laboratory and clinical findings, but others (Schenker and Weinstein, 1978) later reorganized and modified the classification into three main clinical categories and six grades according to the severity of symptoms and signs and laboratory findings. In 1989, a new classification of three categories and five grades was introduced (Golan et al., 1989) which was modified by A.Golan in 2009 to reduce the importance of gastrointestinal symptoms. Another classification with further dividing the severe form into two subgroups was made by Navot et al., (1992). A more recent classification with further modifications was introduced in 1999 by Rizk and Aboulghar (Aboulghar et al., 2003). The Royal College of Obstetricians and Gynecologists had approved Mathur et al (2007) modification of Navot et al (1992). Grade and associated clinical features Mild OHSS Abdominal bloating. Mild abdominal pain. Ovarian size usually 8 cm*. Moderate OHSS Moderate abdominal pain. Nausea ± vomiting. Ultrasound evidence of ascites. 7

15 Chapter 1 OHSS Ovarian size usually 8-12 cm*. Severe OHSS Clinical ascites (occasionally hydrothorax). Oliguria. Haemoconcentration, haematocrit 45%. Hypoproteinaemia. Ovarian size usually 12 cm*. Critical OHSS Tense ascites or large hydrothorax. Haematocrit 55%. White cell count 25 x 10 9 /L. Oligo-anuria. Thromboembolism. Acute respiratory distress syndrome. The Royal College of Obstetricians and Gynecologists had approved Mathur et al (2007) modification of Navot et al (1992). 8

16 Chapter 1 OHSS Risk factors: Polycystic ovarian syndrome (PCOS) greatly increases the risk. (Nader et al.,2010) Younger women are at greater risk since they have a larger number of recruitable follicles and higher density of gonadotropin receptors. (Lewis et al., 1990). High estrogen levels and a large number of follicles though the arbitrary level of serum estradiol above which one could be considered at high risk for OHSS is debatable. (Chenette et al., 1990 and Asch et al., 1991) The use of hcg for luteal phase support. (Forman et al., 1990) Administration of gonadotrophin-releasing hormone (GnRH) agonist. GnRH antagonists can be used within the treatment cycle to suppress the production of gonadotrophins and, in doing so, they shorten the treatment cycle.( Aboulghar et al., 2006) Low body mass index (BMI). (Whelan et al., 2000) Oligomenorrohea.( Kaushal et al., 2010) History of allergy was identified in 50% of the patients who ultimately developed severe OHSS (Enskong et al., 1999) Previous history of OHSS.(Kaushal et al., 2010) (Rizk et al., 1993) found OHSS to be much more frequent in conception cycles. (Tulandi and associates 1984) found the pregnancy rate in hyperstimulation cycles to be three times that in non-hyperstimulated 9

17 Chapter 1 OHSS cycles. A high incidence of multiple pregnancies (10-42 %) was also observed. (Rizk et al., 1991 and Reyad et al., 1988). Genetic or familial susceptibility is a considerable risk factor which is precipitated by mutation of FSH receptors gene (Smits et al., 2003). Pathophysiology: OHSS is an iatrogenic complication of pharmacological ovarian stimulation. Its pathophysiology is not completely understood but the hallmark of OHSS is an increase in capillary permeability resulting in a fluid shift from the intravascular compartment into the third space. OHSS is marked by massive bilateral cystic ovarian enlargement with significant degree of stromal edema, interspersed with multiple hemorrhagic follicular and theca lutein cysts, areas of cortical necrosis and neovascularization (Aboulghar et al., 1996). Initially, estrogens, histamine and prostaglandins were thought to cause OHSS. The ovarian reninangiotensin system was then proposed as the most possible mediator of OHSS (Morris et al., 1995), but more recently, the vascular endothelial growth factor (VEGF) as well as cytokines, were proposed as protagonists in OHSS pathogenesis (Neulen et al., 1995). Nevertheless, whatever the responsible factor is, it seems that human chorionic gonadotropin (hcg), exogenously administered or endogenously produced, play a key role in the development of OHSS. It usually occurs a several days after follicular rupture or oocyte retrival following hcg administration, which promotes the 10

Chapter 1 OHSS release of vasoactive substances that affect the endothelial adherence junctions and result in trans-endothelial permeability leading to

18 Chapter 1 OHSS release of vasoactive substances that affect the endothelial adherence junctions and result in trans-endothelial permeability leading to development of clinical manifestations. (Forman et al., 1990) Fig.1. First steps in the pathophysiology of OHSS. (Hum. Reprod. Update (2008) 14 (4): ) 11

19 Chapter 1 OHSS On the other hand, it was recently identified a mutation in the FSH receptor gene in a patient presenting spontaneous OHSS during each of her four pregnancies (Smits et al., 2003). The mutation consisted of a substitution of an adenine for a guanine at the first base of codon 567 in exon 10 of the follitropin receptor gene, resulting in the replacement of an aspartic acid with an asparagine. When tested in vitro, the functional response of the mutant receptor displayed an enhanced basal activity and an increased sensitivity to hcg. A distinct mutation in the FSH receptor gene was concomitantly reported in a patient who developed OHSS during all of her four pregnancies that went beyond 6 weeks of gestation (Vasseur et al., 2003). Mediators of increased capillary permeability: 1- Estrogen: It induces increased capillary permeability of the uterine and ovarian circulation. Levy et al. (1996) reported development of severe OHSS in a woman with hypogonadotropic hypogonadism during ovulation induction with urinary human FSH/hCG in the presence of low circulating estradiol concentration. Hence, questioning the role of estradiol in the pathogenesis of OHSS. The use of E 2 levels to differentiate poor, normal and high responders, and to predict IVF outcome and OHSS, has been a subject of debate. In most studies, cut-off levels were chosen arbitrarily and depend on various factors. Percentile curves offer a more objective definition of these three categories of patients. To our knowledge, only one study has used percentile curves to adjust the doses of 12

20 Chapter 1 OHSS gonadotrophins to the ovarian response and to evaluate IVF outcome in stimulation cycles (Forman et al., 1988) and (Theocharis et al., 2002). Another retrospective case-control study of a cohort of women undergoing assisted reproduction treatment (ART) over 12 months to assess the value of different serum E 2 cut-off levels for predicting women at risk for OHSS reported that serum E 2 level of 12,315 pmol/l (3,354 pg/ml) on day 11 of ovarian stimulation gives a sensitivity and specificity of 85% for the detection of women at risk for OHSS. (D'Angelo et al., 2004) 2- Prostaglandins: They were proposed as possible mediators, and prostaglandins synthetase inhibitors were used to prevent the fluid shift responsible for the clinical picture of OHSS. However, Pride et al. (1986) found that indomethacin, in pharmacological dose, did not influence the clinical features of OHSS. Balasch et al. (1991) suggested that renal prostaglandin PGE2 and PGI2, by antagonizing the renal vasoconstrictor effect of angiotensin II and noradrenaline, play a major role in the maintenance of renal function in severe OHSS. 3- Histamine: With its well known effects on vascular permeability, it was proposed to play a role in OHSS. However, Erlik et al. (1979) could not show any changes in peripheral plasma histamine level or in ovarian mast cell content in OHSS. 13

Ovarian hyperstimulation syndrome (OHSS)

Ovarian hyperstimulation syndrome (OHSS) OHSS OHSS: exaggerated response to gonadotropins and hcg Characterized by: ovarian enlargement increased vascular permeability fluid accumulation in abdomen Associated