The efficacy of assisted reproductive technologies has improved significantly over the past decades. The main indications for in vitro fertilisation include tubal obstruction, severe male-factor infertility, severe endometriosis, ovulatory dysfunction, diminished ovarian reserve, and infertility of unexplained cause. In vitro fertilisation has also become an effective treatment option for couples wishing to undergo pre-implantation genetic diagnosis or screening, and for those wishing to cryopreserve their oocytes or embryos for preservation of fertility. The management of women in late reproductive age poses a major challenge; the optimum in vitro fertilisation treatment for poor responders remains elusive. The success of in vitro fertilisation treatment can be optimised by taking an individualised, patient-centered approach to controlled ovarian hyperstimulation. Key components involve selection of an appropriate controlled ovarian protocol, close-cycle monitoring, adjustment of gonadotropin dosage to avoid hyper-response, and individualised timing of human chorionic gonadotropin injection. Future directions of assisted reproductive technologies include development of non-invasive embryo selection methods, use of transcriptomics, proteomics, metabolomics, and time-lapse imaging technologies.
Introduction
The efficacy of assisted reproductive technologies (ART) has improved significantly since the first reports of successful pregnancies and live births after in vitro fertilisation (IVF) by Steptoe and Edwards in 1978. In the USA, the live birth rate has increased from 38% to 48% among women under the age of 35 years treated with ART over the past decade. The European IVF-monitoring consortium and the International Committee for Monitoring Assisted Reproductive Technology also observed similar trends in ART success. Assisted reproductive technologies have also become more accessible. In 2003, the number of ART cycles carried out worldwide exceeded over 1 million. To date, an estimated 3.75 million babies have been born worldwide using ART.
The most common ART procedure is IVF, which involves controlled ovarian hyperstimulation (COH) with gonadotropin administration to stimulate ovarian follicle development, followed by transvaginal oocyte retrieval, fertilisation of the oocytes with sperm in vitro, culture of the resultant embryos, and transfer of the embryo(s) to the recipient. An important innovation in ART is assisted fertilisation by intracytoplasmic sperm injection (ICSI). Other modalities of ART include cryopreservation of gametes, embryos, and ovarian tissue, pre-implantation genetic diagnosis (PGD) or screening (PGS), the use of donor gametes, and gestational carriers.
In this chapter, the indications for IVF and factors affecting IVF success will be reviewed, along with IVF treatment options for women of advanced maternal age. We will also provide an update on the recent advancements in, and future directions of, ART.
Indications for in vitro fertilisation
In vitro fertilisation was first reported as a treatment option for women with severe tubal disease. With improved efficacy after the introduction of gonadotropin stimulation and ICSI, the indications for IVF have expanded to include infertility caused by severe male factor, diminished ovarian reserve, ovulatory dysfunction, severe endometriosis, and infertility of unexplained cause. In vitro fertilisation also provides a new means of preconception genetic diagnosis and preservation of fertility. In fact, IVF is the most effective treatment option for couples with multi-factorial infertility problems.
Tubal factor
Tubal-factor infertility accounts for 30% of cases of female infertility. Tubal damage has classically been associated with pelvic inflammatory disease, most often with Chlamydia trachomatis and gonorrhea infections. Other causes of tubal obstruction may be either intrinsic (e.g. ascending salpingitis and salpingitis isthmica nodosa) or extrinsic (e.g. surgical sterilisation, endometriosis and peritonitis) in origin.
Before the introduction of IVF, reconstructive tubal surgery was the only treatment option for women with tubal obstruction. At present, IVF is the treatment of choice for women over the age of 35 years with significant tubal disease and those with other co-existing infertility problems. In vitro fertilisation is also indicated in women who remain infertile for 1 year after tubal surgery.
Male factor
Abnormal semen parameters may be a contributing factor in up to 40% of infertile couples. In cases of severe oligospermia (fewer than 5 million motile sperm/ml), severe asthenospermia (less than 5% progressive motility), and severe teratospermia (less than 4% normal morphology based on strict Kruger criteria), IVF, or a combination of IVF and ICSI, should be offered, as these semen parameters are associated with poor success with artificial insemination. For women with obstructive or non-obstructive azoospermia, IVF with ICSI are indicated to achieve fertilisation using surgically retrieved spermatozoa from either microsurgical epididymal sperm aspiration or testicular sperm extraction.
Endometriosis
The incidence of endometriosis is reported to be in the range of 9–50% among women who underwent laparoscopy for infertility evaluation. The exact pathophysiology of endometriosis and its effects on fertility remain enigmatic. Proposed mechanisms of damage include distortion of adnexal anatomy and adverse peritoneal environment characterised by increased inflammatory cytokines and oxidative stress. This may interfere with follicular development, ovum pick up, fertilisation, and embryo development.
Women with known or suspected moderate and severe endometriosis (stage III–IV) may be treated with either surgery or IVF. No prospective, randomised-controlled trial has compared the efficacy of the two treatment modalities. Surgical treatment is preferred in women who are symptomatic and those with endometriomas greater than 4 cm. In vitro fertilisation is indicated in women with co-existing causes of infertility, such as tubal obstruction, advanced maternal age, and abnormal semen parameters. A 3-month course of gonadotropin releasing hormone (GnRH) agonist administered before starting IVF has been shown to improve the ongoing pregnancy rate.
Ovulatory dysfunction
Ovulatory dysfunction is a very common cause of female infertility, accounting for 25% of diagnoses. In this category, polycystic ovary syndrome (PCOS), characterised by the triad of polycystic ovaries, oligo- or amenorrhoea, and clinical and biochemical signs of androgen excess, is most common. Ovulatory dysfunction may also be a manifestation of other endocrinopathies, such as thyroid disorders and hyperprolactinaemia. Other rare causes include congenital adrenal hyperplasia, adrenal tumours, and androgen-secreting ovarian tumours.
Women with normogonadotropic–normogonadal ovulatory disorders (World Health Organization group 2), including those with PCOS, can be successfully treated with ovulation induction combined with timed intercourse or intrauterine insemination (IUI). Ovulation induction is usually successful after treatment with clomiphene citrate, exogenous gonadotropins, aromatase inhibitors (e.g. letrozole, anastrazole), or selective oestrogen receptor modulators (e.g. tamoxifen).
Women with polycystic ovaries seen on ultrasound, irrespective of whether they harbor other clinical features of PCOS, have an increased risk of over-responding to gonadotropin stimulation and for developing ovarian hyperstimulation syndrome (OHSS). The risk of high-order multiple gestations is also significantly higher in women who have a high response to gonadotropin stimulation and who undergo an IUI. In these settings, conversion to IVF represents a safe and effective alternative to an IUI or to cycle cancellation. In vitro fertilisation is indicated for women who do not conceive after conventional ovarian-induction treatments, and especially for couples with other co-existing infertility factors.
Unexplained infertility
Unexplained infertility is defined as the absence of an identifiable cause of infertility despite a thorough investigation demonstrating tubal patency, normal semen parameters, ovulation, normal ovarian reserve, and a normal endometrial cavity. The incidence of unexplained infertility ranges from 10–30%.
Treatment options include expectant management, IUI, empiric treatment with clomiphene citrate and IUI, clomiphene citrate combined with gonadotropins and IUI, and IVF. In vitro fertilisation is the most effective treatment option for couples with unexplained infertility, resulting in the highest per cycle pregnancy rate in the shortest time interval.
An empirical treatment algorithm for couples with unexplained infertility typically involves three cycles of clomiphene citrate and IUI, followed by three cycles of gonadotropin and IUI, and by IVF if the patients remain unsuccessful. In a recent, prospective, randomised-controlled trial involving women with unexplained infertility between the ages of 21 and 39 years, participants were randomised to either an accelerated treatment algorithm (IVF after three unsuccessful clomiphene citrate and IUI treatment cycles) or the conventional treatment algorithm (IVF after three unsuccessful clomiphene citrate and IUI, and three unsuccessful gonadotropin and IUI treatment cycles). The time to pregnancy was significantly shorter in the accelerated arm compared with the conventional arm (hazard ratio 1.25; 95% CI, 1.00 to 1.56). The accelerated treatment algorithm was also more cost-effective, compared with the conventional treatment group. Age seems to be the single most important determining factor of success.
Fertility preservation
In recent years, individuals with cancer and oncologists have been increasingly aware of the effect of cancer treatment on fertility. Women at risk of premature ovarian failure owing to gonadotoxic chemotherapy or radiation treatment should be offered the possibility of fertility preservation. Women receiving gonadotoxic treatment for autoimmune disorders, such as systemic lupus erythematosus, and those at risk of premature ovarian failure caused by chromosomal anomalies, such as mosaic Turner syndrome, may also benefit from fertility preservation.
The only strategy of female fertility preservation recognised by the American Society of Clinical Oncology and the American Society of Reproductive Medicine is cryopreservation of embryos. Other fertility-preservation procedures, such as cryopreservation of oocytes and ovarian tissues and in vitro maturation, have enjoyed increasing success.
Pre-implantation genetic diagnosis and screening
Pre-implantation stage embryos can be screened for aneuploidy, genetic diseases, and inherited chromosome abnormalities, enabling transfer of genetically normal, euploid embryo(s). A more in-depth discussion on pre-implantation genetic diagnosis and pre-genetic screening will be provided in Chapter 8.
Indications for in vitro fertilisation
In vitro fertilisation was first reported as a treatment option for women with severe tubal disease. With improved efficacy after the introduction of gonadotropin stimulation and ICSI, the indications for IVF have expanded to include infertility caused by severe male factor, diminished ovarian reserve, ovulatory dysfunction, severe endometriosis, and infertility of unexplained cause. In vitro fertilisation also provides a new means of preconception genetic diagnosis and preservation of fertility. In fact, IVF is the most effective treatment option for couples with multi-factorial infertility problems.
Tubal factor
Tubal-factor infertility accounts for 30% of cases of female infertility. Tubal damage has classically been associated with pelvic inflammatory disease, most often with Chlamydia trachomatis and gonorrhea infections. Other causes of tubal obstruction may be either intrinsic (e.g. ascending salpingitis and salpingitis isthmica nodosa) or extrinsic (e.g. surgical sterilisation, endometriosis and peritonitis) in origin.
Before the introduction of IVF, reconstructive tubal surgery was the only treatment option for women with tubal obstruction. At present, IVF is the treatment of choice for women over the age of 35 years with significant tubal disease and those with other co-existing infertility problems. In vitro fertilisation is also indicated in women who remain infertile for 1 year after tubal surgery.
Male factor
Abnormal semen parameters may be a contributing factor in up to 40% of infertile couples. In cases of severe oligospermia (fewer than 5 million motile sperm/ml), severe asthenospermia (less than 5% progressive motility), and severe teratospermia (less than 4% normal morphology based on strict Kruger criteria), IVF, or a combination of IVF and ICSI, should be offered, as these semen parameters are associated with poor success with artificial insemination. For women with obstructive or non-obstructive azoospermia, IVF with ICSI are indicated to achieve fertilisation using surgically retrieved spermatozoa from either microsurgical epididymal sperm aspiration or testicular sperm extraction.
Endometriosis
The incidence of endometriosis is reported to be in the range of 9–50% among women who underwent laparoscopy for infertility evaluation. The exact pathophysiology of endometriosis and its effects on fertility remain enigmatic. Proposed mechanisms of damage include distortion of adnexal anatomy and adverse peritoneal environment characterised by increased inflammatory cytokines and oxidative stress. This may interfere with follicular development, ovum pick up, fertilisation, and embryo development.
Women with known or suspected moderate and severe endometriosis (stage III–IV) may be treated with either surgery or IVF. No prospective, randomised-controlled trial has compared the efficacy of the two treatment modalities. Surgical treatment is preferred in women who are symptomatic and those with endometriomas greater than 4 cm. In vitro fertilisation is indicated in women with co-existing causes of infertility, such as tubal obstruction, advanced maternal age, and abnormal semen parameters. A 3-month course of gonadotropin releasing hormone (GnRH) agonist administered before starting IVF has been shown to improve the ongoing pregnancy rate.
Ovulatory dysfunction
Ovulatory dysfunction is a very common cause of female infertility, accounting for 25% of diagnoses. In this category, polycystic ovary syndrome (PCOS), characterised by the triad of polycystic ovaries, oligo- or amenorrhoea, and clinical and biochemical signs of androgen excess, is most common. Ovulatory dysfunction may also be a manifestation of other endocrinopathies, such as thyroid disorders and hyperprolactinaemia. Other rare causes include congenital adrenal hyperplasia, adrenal tumours, and androgen-secreting ovarian tumours.
Women with normogonadotropic–normogonadal ovulatory disorders (World Health Organization group 2), including those with PCOS, can be successfully treated with ovulation induction combined with timed intercourse or intrauterine insemination (IUI). Ovulation induction is usually successful after treatment with clomiphene citrate, exogenous gonadotropins, aromatase inhibitors (e.g. letrozole, anastrazole), or selective oestrogen receptor modulators (e.g. tamoxifen).
Women with polycystic ovaries seen on ultrasound, irrespective of whether they harbor other clinical features of PCOS, have an increased risk of over-responding to gonadotropin stimulation and for developing ovarian hyperstimulation syndrome (OHSS). The risk of high-order multiple gestations is also significantly higher in women who have a high response to gonadotropin stimulation and who undergo an IUI. In these settings, conversion to IVF represents a safe and effective alternative to an IUI or to cycle cancellation. In vitro fertilisation is indicated for women who do not conceive after conventional ovarian-induction treatments, and especially for couples with other co-existing infertility factors.
Unexplained infertility
Unexplained infertility is defined as the absence of an identifiable cause of infertility despite a thorough investigation demonstrating tubal patency, normal semen parameters, ovulation, normal ovarian reserve, and a normal endometrial cavity. The incidence of unexplained infertility ranges from 10–30%.
Treatment options include expectant management, IUI, empiric treatment with clomiphene citrate and IUI, clomiphene citrate combined with gonadotropins and IUI, and IVF. In vitro fertilisation is the most effective treatment option for couples with unexplained infertility, resulting in the highest per cycle pregnancy rate in the shortest time interval.
An empirical treatment algorithm for couples with unexplained infertility typically involves three cycles of clomiphene citrate and IUI, followed by three cycles of gonadotropin and IUI, and by IVF if the patients remain unsuccessful. In a recent, prospective, randomised-controlled trial involving women with unexplained infertility between the ages of 21 and 39 years, participants were randomised to either an accelerated treatment algorithm (IVF after three unsuccessful clomiphene citrate and IUI treatment cycles) or the conventional treatment algorithm (IVF after three unsuccessful clomiphene citrate and IUI, and three unsuccessful gonadotropin and IUI treatment cycles). The time to pregnancy was significantly shorter in the accelerated arm compared with the conventional arm (hazard ratio 1.25; 95% CI, 1.00 to 1.56). The accelerated treatment algorithm was also more cost-effective, compared with the conventional treatment group. Age seems to be the single most important determining factor of success.
Fertility preservation
In recent years, individuals with cancer and oncologists have been increasingly aware of the effect of cancer treatment on fertility. Women at risk of premature ovarian failure owing to gonadotoxic chemotherapy or radiation treatment should be offered the possibility of fertility preservation. Women receiving gonadotoxic treatment for autoimmune disorders, such as systemic lupus erythematosus, and those at risk of premature ovarian failure caused by chromosomal anomalies, such as mosaic Turner syndrome, may also benefit from fertility preservation.
The only strategy of female fertility preservation recognised by the American Society of Clinical Oncology and the American Society of Reproductive Medicine is cryopreservation of embryos. Other fertility-preservation procedures, such as cryopreservation of oocytes and ovarian tissues and in vitro maturation, have enjoyed increasing success.
Pre-implantation genetic diagnosis and screening
Pre-implantation stage embryos can be screened for aneuploidy, genetic diseases, and inherited chromosome abnormalities, enabling transfer of genetically normal, euploid embryo(s). A more in-depth discussion on pre-implantation genetic diagnosis and pre-genetic screening will be provided in Chapter 8.
Factors affecting in vitro fertilisation success
Although many attribute IVF success to innovations in the ART laboratory, we believe that the success of IVF treatment can be optimised by taking an individualised, patient-directed approach in the management of women undergoing ovarian hyperstimulation. Key components involve selection of the appropriate COH protocol and gonadotropin dosage, close monitoring of follicular growth and serum E 2 levels, adjustment of gonadotropin dosage to avoid hyper-response, and individualised timing of human chorionic gonadotropin (hCG) injection. We believe this approach to COH monitoring improves oocyte and embryo quality, pregnancy and implantation rates, and, most importantly, minimises the risk of complications, namely OHSS Figs. 1–3 .
Selection of an appropriate controlled ovarian hyperstimulation protocol
A significant clinical challenge in determining the optimal COH protocol is predicting and managing variability between women. The central question when designing an IVF protocol is whether the woman will likely have a good or poor response to exogenous gonadotropin stimulation. We believe the appropriate IVF protocol should be determined on the basis of an aggregate of patient characteristics and ovarian reserve assessment.
Predictive factors of ovarian response include patient characteristics, such as age, parity, reproductive history, body mass index, and prior response to ART treatment. Endocrine markers of ovarian reserve, including basal follicle stimulating hormone (FSH), oestradiol (E 2 ), inhibin B, and, more recently, anti-Müllerian hormone (AMH) levels, are also useful in distinguishing good and poor responders. Ultrasonographic assessments of the ovary, including antral follicle count (AFC), ovarian volume, and ovarian blood flow, have also been extensively evaluated as potential markers of ovarian reserve. Dynamic evaluation of ovarian reserve, including clomiphene citrate challenge testing, GnRH agonist stimulation testing, and exogenous FSH ovarian test, may be helpful. A recent meta-analysis concluded that all the dynamic ovarian reserve tests have limited predictive values.
Anti-Müllerian hormone and AFC seem to be the two most accurate predictors of ovarian response to COH. Advantages of AMH over AFC and other endocrine and sonographic markers of ovarian reserve include consistent serum levels throughout the menstrual cycle and minimal cycle-to-cycle variability. Anti-Müllerian hormone has been shown to be an accurate marker of both hyper- and poor ovarian response to gonadotropin stimulation. A recent meta-analysis found that AMH, using cut-offs ranging from over 1.59 ng/ml to over 7.00 ng/ml, has an estimated sensitivity of 82% and specificity of 76% in predicting excessive ovarian response. A recent prospective study of poor responders found an AMH level of less than 0.99 ug/L to be associated with 100% sensitivity and 73% specificity in predicting poor response. None of the ovarian reserve markers, however, possesses adequate sensitivity or specificity for predicting pregnancy outcomes.
Normal responders
Normal responders are characterised by favourable prognostic factors, including, but not limited to, young maternal age (less than 35 years), normal body mass index, adequate ovarian reserve (AFC between 6 and 10), normal basal FSH and E 2 levels (FSH < 10 mIU/ml, E 2 < 75 IUpg/ml), short duration of subfertility, a history of previous live birth, and previous successful IVF treatment.
Commonly used COH protocols include the mid-luteal, GnRH agonist down regulation or ‘long’ protocol and, more recently, the GnRH antagonist COH protocol, which is also known as the ‘short’ protocol.
High responders
The management of women who are at risk of developing an exaggerated response to COH represents a formidable challenge. An important consideration is the prevention of OHSS. Women with polycystic ovaries on ultrasound, even in the absence of other clinical features of PCOS, are at greater risk of developing OHSS ; the incidence of OHSS has been reported to be as high as 30% in this patient population. Other known risk factors for OHSS include young age, lean body weight, and a history of OHSS. In women undergoing COH treatment, high gonadotropin doses, high absolute levels (greater than 3000 pg/ml), and rapidly rising E 2 levels also represent risk factors for the development of OHSS.
Strategies for the prevention of OHSS include identifying patients at risk, individualisation of COH protocols, and judicious use of gonadotropins. In this context, the aim of the COH is to decrease ovarian response, ideally to develop five to 15 follicles, while maintaining an E 2 level of less than 3000 pg/ml. Two effective COH protocols for high responders are the oral contraceptive pill GnRH agonist dual suppression protocol and the GnRH antagonist protocol in combination with GnRH agonist ovulatory trigger.
Poor responders
Diminished ovarian reserve is relatively common among women undergoing IVF, with reported prevalence ranging from 10–25%. The variations in the prevalence rate can be attributed to a lack of a universally accepted definition of what constitutes a poor response. Most definitions of poor response are based on arbitrary levels of peak serum E 2 or oocyte yield. Poor responders have also been defined by age over 40 years, elevated basal FSH levels (over 10 to 15 mIU/L), previous cycle cancellation, poor response to clomiphene citrate challenge testing, prolonged duration of COH, increased daily and total gonadotropin ampoules used (over 44), and when less than three to five oocytes are retrieved.
Recently, a European Society of Human Reproduction and Embryology consensus working group defined poor ovarian response as having at least two of the following three features: (1) advanced maternal age (≥ 40 years) or any other risk factor for diminished ovarian reserve; (2) previous history of poor ovarian response (fewer than three oocytes retrieved with a conventional COH protocol); and (3) an abnormal ovarian reserve test (AFC less than five to seven follicles or AMH more than 0.5–1.1 ng/ml).
Given the relationship between advanced maternal age and the decline in fertility, it is not surprising that age is the most important determining factor of success in women undergoing IVF. Although ART may overcome infertility in younger women, it does not reverse the age-dependent decline in fertility. Advanced maternal age is associated with a decline in the number of oocytes retrieved, embryos available for transfer, and embryo quality, ultimately resulting in lower implantation, pregnancy, and live birth rates. A strong correlation also exists between advanced maternal age and the incidence of chromosomal anomalies. The risk of miscarriage approaches 50% among women over the age of 40 years.
The management of women in late reproductive age remains a major challenge. In fact, among women undergoing IVF for various indications, patients with diminished ovarian reserve seem to have the worst prognosis. In vitro fertilisation treatment among older women is often associated with low fecundity and poor clinical outcomes. These women also have a high no-start rate owing to abnormally elevated baseline serum FSH levels and high cycle cancellation rates owing to poor response.
A variety of COH protocols have been published for treating poor responders. Commonly used COH protocols include Luteal E 2 patch and GnRH antagonist protocol, co-flare and micro-flare protocols, clomid and letrozole protocols, and modified natural cycles. The optimum IVF treatment strategy remains elusive.
Timing and dose of human chorionic gonadotropin
The timing of hCG injection should be individualised on the basis of several factors, including leading follicle diameter, E 2 level, prior cycle response and embryo quality, and the type of COH protocol. In normal responders who are undergoing an IVF cycle for the first time, hCG is usually administered when two or more follicles reach a size of 17 mm or larger in diameter. Ideally, the E 2 level should be greater than 400 pg/ml for 3 days.
In women with a history of retrieval of mostly immature oocytes, consideration should be given to continue COH until the follicles attain 19–20 mm in diameter before triggering with hCG. Similarly, in women who are treated with clomiphene citrate or letrozole COH protocols, hCG injection is usually given when follicles reach 19–20 mm. Each clinic, however, should establish their ideal criteria for hCG administration based on their methods of ultrasound follicular assessment. In women with poor oocyte or embryo quality, especially in the presence of a high proportion of polyspermic fertilisation after IVF, one should be wary of oocyte post-maturity. In such circumstances, the woman may benefit from administering the ovulatory trigger at smaller lead follicle diameters in the subsequent cycles. Another important parameter for administrating hCG is a plateau or doubling of E 2 levels on consecutive days once the leading follicle has exceeds 16 mm in average diameter.
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