Evaluation of pelvis

Ultrasound evaluation of the pelvis is often performed prior to ART to rule out any adnexal and uterine pathologies, such as polycystic ovary syndrome (PCOS), endometriosis, tubal disease, fibroids and endometrial polyps, which are clearly associated with aberrant and compromised outcomes during IVF.

The diagnosis of PCOS is currently based on the definition derived by the Rotterdam Consensus Workshop, which suggested the presence of 12 or more follicles measuring 2–9 mm in diameter and/or an ovarian volume of more than 10 cm³ for the ultrasound diagnosis (Balen et al 2003). Stromal vascularity and blood flow velocities are higher in women with polycystic ovaries (Agrawal et al 1998), and this may account for their tendency towards ovarian hyperstimulation in response to gonadotrophin therapy.

Endometriosis has been shown to have a negative effect on the outcome of ART, with affected women demonstrating a reduced response to ovarian stimulation (Gupta et al 2006); however, reports on its effects on embryo quality and implantation potential are conflicting. Moderate and severe degrees of endometriosis, often characterized by rectovaginal disease and the presence of ovarian endometrioma, can usually be diagnosed by transvaginal ultrasound (Moore et al 2002).

The presence of a hydrosalpinx is associated with poor implantation and pregnancy rates, and early pregnancy loss. The exact mechanism is unknown but substances toxic to the endometrium are thought to be produced that have a negative effect on endometrial receptivity (Strandell 2007). Salpingectomy, ideally performed laparoscopically, prior to IVF has been shown to be beneficial and is recommended. The diagnosis of hydrosalpinx can generally be made using transvaginal ultrasound with a high degree of confidence.

Ultrasound assessment of the uterus is generally restricted to measurement of the endometrial thickness and a description of the locality and size of any fibroids. Conventional ultrasound may also detect uterine anomalies, although these are more readily identified and correctly qualified with three-dimensional ultrasound. The diagnosis of uterine anomalies is important as they are associated with lower implantation rates and increased rates of early miscarriage, preterm labour and malpresentations (Lin 2004). The outcome appears to relate more to the length of the remaining uterine cavity than the degree of septum, and this may be measured reliably with three-dimensional ultrasound (Salim et al 2003).

The diagnosis and classification of fibroids into subserosal, intramural and submucosal is usually straightforward, and this is important as these have a progressively negative effect on pregnancy rates. Whilst intramural fibroids measuring more than 4 cm in diameter and submucous fibroids of any size are thought to have a negative effect on the outcome of fertility treatment and to be associated with lower pregnancy rates, the effect of smaller intramural fibroids on reproductive outcome is unclear (Oliveira et al 2004). There is also doubt about the exact impact of endometrial polyps, the other commonly encountered intrauterine pathology, on treatment outcome and early pregnancy. Some authors suggest conservative management, especially if the polyp is small (Isikoglu et al 2006), but this can be difficult in patients undergoing fertility treatment or with a history of miscarriage when a polyp is identified before treatment begins. Polyps are usually evident with conventional transvaginal imaging, although false-positive diagnoses are common. Saline infusion sonohysterography can reduce this and facilitate appropriate operative planning (Bartkowiak et al 2006). Delineation of the polyp with saline ensures that the size and position of the polyp can be defined accurately, allowing surgeons to modify their approach and resect the larger, more broad-based polyps rather than planning for simple polypectomy. Hysteroscopy is the gold standard technique for uterine cavity assessment, and may be necessary if diagnosis of intrauterine pathologies is in doubt.

Assessment of ovarian reserve

Evaluation of ovarian reserve has become an integral part of the pretreatment assessment of a woman about to undergo ART, and is recommended for all women planning ART (Speroff and Fritz 2005). The aim is to identify women likely to respond poorly, those who have a low chance of success and who are more likely to have their treatment cycle cancelled, and those prone to ovarian hyperstimulation which is associated with significant morbidity and even mortality. Accurate assessment of ovarian reserve and prediction of response therefore facilitates pretreatment counselling of couples of their potential risks, and allows treatment to be tailored to the individual, potentially increasing the number of oocytes retrieved without risking an exaggerated response. Ovarian reserve, defined by the size and quality of the remaining ovarian follicular pool at any given time, reflects the fertility potential of a woman (Broekmans et al 2006).

Although women’s age is an important predictor of ovarian reserve and response, a great deal of interindividual variation exists, even among women of the same age (te Velde and Pearson 2002). This is indicative of a wide variation in the rate of age-related decline in the ovarian follicle population. Several endocrine and ultrasound markers have been reported, with the aim of estimating the number of gonadotrophin-responsive or ‘selectable follicles’ more accurately. The endocrine markers include factors produced by the developing follicles [oestradiol, inhibin B and anti-Müllerian hormone (AMH)] or hormones under the inhibitory control of these factors [follicule-stimulating hormone (FSH)]. The ultrasound markers are antral follicle count, ovarian volume and ovarian vascularity. The sensitivities, specificities and predictive values of these tests have been evaluated in many studies by correlating the test results with the number of oocytes retrieved, poor or exaggerated ovarian response, and pregnancy or livebirth rates during ART (Broekmans et al 2006).

Evaluation of male partner

The male partner should be evaluated carefully. Clinical examination, semen analysis including sperm function tests, genetic assessment and biochemical tests show that the aetiology of male subfertility can be divided into four main groups as follows.

Many causes of male infertility may have considerable congenital implications for the children of such patients. Sixty percent of men with congenital bilateral absence of the vas deferens were shown to have one or more of the cystic fibrosis genes. The use of ICSI in these circumstances should be considered carefully, and men must be screened with the appropriate tests before treatment.

In order to give adequate counselling to couples and their families, genetic tests should be performed before, during or after ART in cases such as women with Turner’s syndrome, men with 47XXY, men or women with structural chromosomal aberrations, and men with either Yq11 deletion or congenital bilateral absence of the vas deferens (ESHRE Capri Workshop Group 2000).

The couple must be well informed about the treatment options that are suitable for them, the indications for the tests they have to undergo, details of the proposed treatment and its success rate, and risks and complications.

Normal folliculogenesis

This involves several important and inter-related steps.

Follicular development beyond the antral stage depends on the concentration of FSH in the circulation. Once a threshold level has been attained, follicular growth beyond 4 mm occurs. The interval during which FSH remains elevated above the threshold level can be regarded as a window through which a follicle must pass to avoid atresia. The width of the window will therefore determine the number of follicles that can be selected for ovulation (Figure 22.2). The preovulatory luteinizing hormone (LH) surge is triggered by the positive feedback of oestradiol from the dominant follicle, as well as other follicular contributory factors. Ovulation occurs 24–36 h after the onset of the LH surge.

Figure 22.2 (A) Selection and maturation of the dominant follicle (DF) during a natural cycle. (B) Induced follicle maturation with gonadotrophin therapy over-riding selection of a single dominant follicle, as in the natural cycle. hCG, human chorionic gonadotrophin.

Pituitary downregulation

In the conventional ART protocol, it is standard practice to use a continuous and supraphysiological dose of GnRH agonist to obtain control over the hypothalamic–pituitary–ovarian axis. GnRH agonists cause an initial flare response, characterized by increased gonadotrophin secretion, due to upregulation of GnRH receptors. However, prolonged administration of GnRH agonists induces downregulation of GnRH receptors. This prevents a premature surge in LH and subsequent spontaneous ovulation with luteinization, which occurs in up to 23% of cycles when gonadotrophins alone are used and facilitates the timing of oocyte retrieval. The use of GnRH agonists in ART has been shown to increase the number of oocytes retrieved, improve the pregnancy rate and significantly reduce the chance of cycle cancellation (Hughes et al 1992).

Two different GnRH agonist protocols are used in most assisted conception units: long and short protocols. In the long protocol, GnRH agonists (buserelin or nafarelin) are started in the mid-luteal phase (day 21) of the previous menstrual cycle to achieve pituitary downregulation in approximately 8–21 days, after which gonadotrophins are commenced. GnRH agonists are administered for approximately 10–14 days in the short protocol. In both regimens, GnRH agonists are administered daily until the day of hCG or LH injection to trigger oocyte maturation. The short protocol takes advantage of the initial flare effect, which can be utilized to augment the ovarian response to gonadotrophin stimulation. However, a systematic review of 26 randomized-controlled trials found an increased clinical pregnancy rate per cycle and fewer cycle cancellations with the long protocol compared with the short protocol (Daya 2000). Therefore, the long GnRH agonist protocol is generally recommended in most ART cycles.

Use of the long GnRH agonist protocol often prolongs the stimulation phase, resulting in increased requirement for gonadotrophins and increased treatment cost in addition to causing undesirable side-effects of hypo-oestrogenism. It may also be associated with poor response in some patients, as well as introducing a higher risk of ovarian hyperstimulation syndrome (OHSS). Some of the disadvantages can be obviated with the use of GnRH antagonists, such as cetrorelix and ganirelix. GnRH antagonists induce immediate pituitary downregulation by competitively binding to GnRH receptors, and avoid the flare effects or the need for prolonged treatment associated with GnRH agonist protocols. Two different regimes have been described. The multiple-dose protocol involves the administration of 0.25 mg cetrorelix or ganirelix daily from day 6–7 of stimulation, or when the leading follicle is 14–15 mm, until hCG/LH administration. The single-dose protocol involves the single administration of 3 mg cetrorelix on day 7–8 of stimulation. GnRH antagonist protocols are therefore short and simple, but more importantly they reduce the incidence of severe OHSS. However, the clinical pregnancy rates following IVF treatment were significantly lower with GnRH antagonist use compared with the long GnRH agonist protocol [odds ratio (OR) 0.82, 95% confidence interval (CI) 0.68–0.97] (Al-Inany et al 2007), although data from units with experience of the GnRH antagonist protocol are encouraging. More recent trials of GnRH antagonists have reported better pregnancy rates than earlier trials, which used antagonist protocols that would now be recognized as suboptimal.

Controlled ovarian stimulation

The pregnancy rate with unstimulated IVF remains much lower than that in stimulated cycles, achieving a livebirth rate of 2.3% per initiated cycle and 4.7% per ET (Human Fertilisation and Embryology Authority 2000). These results have remained low over the past decade. Although an unstimulated cycle is simpler, faster, less painful, less expensive and has no risk of ovarian hyperstimulation, it requires relatively extensive monitoring which increases the cost and results in inconvenience in the timing of oocyte retrieval. Additionally, women with irregular cycles and/or hormonal imbalance or couples with male factor infertility, where fertilization may be a problem, are not suitable for natural-cycle IVF. The aims of superovulation regimens in assisted reproduction are to maximize the number of follicles that mature, to minimize the degree of asynchrony amongst developing follicles, and to minimize the deleterious effects of the abnormal follicular environment on luteal function and endometrial receptivity. Multiple follicular development and ovulation can be achieved by widening the FSH window. Several drug combinations have been used to achieve this.

Oocyte retrieval may be programmed to coincide with a working day by manipulating the onset of the menstrual cycle using norethisterone tablets, the combined oral contraceptive pill, or utilizing the hypogonadotrophic effect of GnRH agonists prior to commencing gonadotrophins on a predetermined day of the week. The downregulatory phase may be prolonged to allow a fixed number of patients to be treated each week. In the long GnRH agonist protocol, gonadotrophins are commenced following the confirmation of ovarian suppression, characterized by a thin endometrium of 5 mm less than transvaginal scan and a serum oestradiol level of less than 200 pmol/l.

The first commercially available gonadotrophin preparation used in superovulation protocols, human menopausal gonadotrophin (HMG), was extracted from the urine of menopausal women. The initial preparations were shown to be of low purity, and the major protein components were not gonadotrophins; only 5% constituted FSH and LH activity. The repeated injection of non-gonadotrophin proteins may be the cause of unwanted effects, such as pain and allergic reactions. The desirability for high-purity preparations led to the development of an immunopurified FSH product with more than 95% purity following the removal of LH activity from HMG. The introduction of highly purified urinary FSH was associated with much reduced hypersensitivity, enabling administration via the subcutaneous route. The currently available commercial preparation of HMG is also highly purified and contains both FSH and LH activity at a ratio of 1:1. The LH activity in HMG is partly due to the presence of exogenous hCG, which is added to correct imbalances in the FSH/LH ratio.

Genetic and molecular engineering has led to the production of recombinant human FSH (rFSH) with high purity (>99%), high specific bioactivity (>10,000 IU/mg protein) and absent intrinsic LH activity which is suitable for intramuscular or subcutaneous administration (Mannaerts et al 1991). Two different rFSH preparations are commercially available: follitropin alpha (Gonal F) and follitropin beta (Puregon). These two rFSH preparations differ in that follitropin alpha has a more acidic isoform profile than follitropin beta due to a slight difference in its carbohydrate component. Consequently, they differ in metabolic clearance, half-life and biological activity, but both are equally effective during ART cycles in terms of livebirth rates. A number of well-designed randomized-controlled trials have compared both preparations of rFSH against HMG in their clinical effectiveness during conventional IVF with the long GnRH agonist protocol. While none of them individually showed any statistically significant difference in livebirth rates between the two treatment arms, two recent systematic reviews have demonstrated a significant 4% increase in livebirth rate in the HMG arm (Al-Inany et al 2008, Coomarasamy et al 2008). However, the results of the meta-analysis need to be interpreted with caution because of the clinical heterogeneity of the trials; therefore, a large multicentre randomized double-blind trial is warranted. Moreover, such studies should consider reporting the more clinically relevant cumulative livebirth rates as the primary outcome.

Recombinant DNA technologies have also made it possible to produce LH (rLH). Due to the potential role of LH in follicular development according to the two-cell theory of gonadotrophin system, supplemental rLH has been suggested to improve IVF outcome if rFSH is used for ovarian stimulation. However, a recent systematic review of randomized trials failed to demonstrate any difference in livebirth rates with adding rLH to the rFSH protocol (Kolibianakis et al 2007). Although it is possible that rLH supplementation may be beneficial in a subset of the IVF population, such as poor responders (Mochtar et al 2007), its generalized use is currently limited.

The collective evidence from many studies suggests that 150–225 IU/day gonadotrophin is the appropriate starting dose for most women undergoing conventional IVF treatment. The majority of units use a daily dose according to women’s age and the presence or absence of polycystic ovaries: 150 IU in women under 30 years of age or with polycystic ovaries, 225 IU for those between 30 and 39 years of age, and 300 IU for those aged 40 years or over. The lowest possible dose to achieve the optimum ovarian response should be used to reduce the risks of ovarian hyperstimulation. Although there is no consensus on what constitutes optimum ovarian stimulation, a recent study has reported that the implantation rate is maximum when 10 oocytes are retrieved following conventional IVF using the long downregulation protocol (Verberg et al 2009a).

Recently, mild ovarian stimulation protocols have gained a lot of interest in the field of ART, with the current trend of limiting the number of embryos transferred reducing the need for large numbers of oocytes. The principal aim with this approach is to reduce the number of developing follicles and thereby minimize the adverse effects of ovarian stimulation, particularly OHSS and multiple pregnancies. Three reported mild stimulation protocols include clomiphene citrate alone or in combination with gonadotrophins, delayed commencement (day 5 of the cycle) of gonadotrophins with GnRH antagonist co-medication, and late follicular replacement of FSH with administration of hCG/LH (Verberg et al 2009b). All of these protocols have been shown to reduce the gonadotrophin dosage and have the potential to reduce the cost of treatment and patient distress associated with conventional ART. As expected, the ongoing pregnancy rates/livebirth rates per cycle have been lower compared with the conventional protocol, but similar effectiveness could be achieved with more treatment cycles.

Monitoring follicular development

The purpose of monitoring ovarian response is to ensure safe practice in reducing the incidence and severity of OHSS, and also to optimize the timing of luteinization before oocyte retrieval. Ultrasound forms an integral part of monitoring. Follicular response together with endometrial thickness is monitored by ultrasound scans commencing on day 6–8 of the treatment cycle. Some units also measure serum oestradiol levels. However, current evidence does not support the adjuvant use of oestradiol for monitoring, as it does not give additional information with regard to livebirth rate or OHSS rate. The gonadotrophin dosage is adjusted according to ovarian response, although the evidence for this practice is limited. Typically, follicles are noted to grow at a rate of 2–3 mm/day, accompanied by a steady increase in serum oestradiol levels. When three or more follicles are 18 mm or greater in diameter, 5000–10,000 U hCG or 250 µg choriogonadotropin alpha (recombinant preparation) are given subcutaneously to induce oocyte maturation before oocyte retrieval.

Ovarian response is poor when no follicles or less than three follicles develop after 14 days of gonadotrophin treatment; this generally results in cancellation of the stimulation cycle. This problem is frequently encountered in women with reduced predicted ovarian reserve. Conversely, excessive ovarian response may result in OHSS, which will be discussed later. Other problems such as a premature LH surge or preoperative ovulation have been largely eliminated by the introduction of GnRH analogues in superovulation protocols.

Equipment and preparation

Oocyte retrieval is performed 36 h after the trigger injection of hCG/LH. Methods of egg collection have improved considerably since the introduction of ART. At first, all egg collections were performed laparoscopically. This is now rarely used and is reserved for women whose ovaries are inaccessible through the transvaginal route. With the introduction of endovaginal transducers, the transvaginal-ultrasound-guided approach became the predominant method for egg collection. In this, the specially designed transducer is used to visualize the follicles and the aspirating needle is passed alongside it. This method is generally well tolerated when carried out under light intravenous sedation, can be learnt very quickly and is associated with minimal morbidity.

Technique

Several types of aspiration needle are available. They may have a single or double lumen to enable aspiration and flushing through different routes. The needle is usually 16-guage and must have a very sharp tip to enable easy puncture of mobile ovaries; the distal 2 cm should be roughened to enhance ultrasound visualization (Figure 22.3). The needle is connected to a test tube by tubing, and suction is applied either from a foot-operated pump (Figure 22.4) or manually. The ultrasound transducer (Figure 22.5) is enclosed in a special sterile condom and plastic sleeve prior to insertion into the vagina, and should be cleaned thoroughly with a damp cloth after each procedure.

Figure 22.3 Ultrasound picture during vaginal egg recovery. The tip of the needle (arrow) is seen within the follicle (F).

Figure 22.4 Follicle aspiration equipment. An assembled follicle aspiration needle with a single lumen is connected to a test tube. The test tube is, in turn, connected to a suction pump. The needle is attached to the transvaginal ultrasound transducer through needle guide and bracket.

Figure 22.5 Vaginal ultrasound transducer enclosed in a sterile condom with needle guide attached. The needle tip is protruding from the proximal end of the probe.

Generally, very few technical difficulties are encountered during vaginal egg collection. The main risks of transvaginal oocyte recovery are pelvic infection (0.6%) and bleeding (>100 ml in 0.8% cases) (Bennett et al 1993), which may be serious, sometimes even fatal. Appropriate preoperative vaginal preparation and minimizing the number of repeated vaginal penetrations may serve to lower the risk of infection. While there is no evidence that routine antibiotics reduce the risk of infection, the administration of an intravenous bolus of antibiotic is generally recommended for women with a history of severe pelvic inflammatory disease or if an endometrioma is punctured. Intestinal, vascular, uterine and tubal injuries with the aspiration needle have also been reported. Bleeding and infections may be serious, sometimes even fatal, complications.