Art Protocols

Introduction

Infertility is defined as failure to conceive even after at least 12 months of unprotected intercourse. Couples who meet the criteria of infertility but are actually capable of conceiving are sometimes termed ‘subfertile’.

Among the methods used to treat infertility is assisted reproductive technology(ART). ART is a general term referring to methods used to achievepregnancyby artificial or partially artificial means. ART was initially developed for patients with tubal infertility, but now it is used for almost all kinds of infertility problems because of higher success rates compared to conventional treatments.¹

Indications of ART

• Tubal factor infertility
• Endometriosis
• Male factor infertility
• Unexplained infertility
• Ovarian failure and diminished ovarian reserve
• Pelvic malignancy
• Mullerian anomaly
• Genetic risk

Ovarian stimulation in ART

Many regimens are used for ovarian stimulation, e.g., natural cycle, clomiphene citrate, gonadotropins (follicle stimulating hormone, luteinizing hormone, human chorionic gonadotropin, human menopausal gonadotropin), and GnRH agonists and antagonists. Administration of exogenous sex steroids (estradiol and progesterone) can improve the efficacy of ovarian stimulation by reducing the dose of gonadotropin required to bring about the same response or improve response to gonadotropin stimulation, or by simplifying treatment protocols.²

Despite ongoing advances in ART, pregnancy rates remain around 20-30% per started cycle. In order to compensate for inefficiencies in IVF procedures, ovarian stimulation is done to induce the development of multiple dominant follicles and to mature many oocytes in a single cycle to improve chances of conception. The use of such ovarian stimulation protocols enables the selection of one or more embryos for transfer, while the spare embryos can be cryopreserved for transfer in a later cycle without repeating ovarian stimulation and oocyte retrieval.

Natural cycle

In 1978 the first successful IVF procedure was performed without the use of drugs. In this protocol (natural cycle IVF), the first birth resulting from IVF is derived from a single oocyte collected in a natural ovulatory cycle, spontaneous cycle is monitored and a single oocyte is retrieved before the midcycle LH surge occurs (Fig. 1).

This procedure is simple, less expensive, less invasive and less stressful than other protocols, and does not cause ovarian hyperstimulation syndrome (OHSS) as no drugs are used. However, the pregnancy rates are low and cancellation rates are high.

Stimulation with clomiphene citrate

Clomiphene citrate (CC) is considered the first line treatment strategy in most cases of anovulatory infertility and is the most used drug for infertility therapies worldwide. One reason for its popularity is that it is administered orally rather than by injection, which improves patient acceptance. Secondly, CC is highly effective, with a reported success rate of 80% in stimulating ovulation.³

CC is a non-steroidal anti-estrogen that stimulates the pituitary gland to produce more follicle stimulating hormone (FSH) which induces the development of one or two mature follicles, each containing an egg. Administration of CC from days 3 to 7 results in estrogen receptor (ER) blockage at the level of the pituitary and hypothalamus. As a result, estrogen-negative feedback is interrupted and FSH secretion increases from the anterior pituitary, leading to multiple follicular growth. After the drug is stopped, there is continuing secretion of estradiol, selection of the dominant follicle and, in successful cases, ovulation.⁴

Clomiphene citrate is most effective at inducing ovulation in women with anovulation or oligo-ovulation, a wide variety of menstrual disorders, relatively normal (or elevated) gonadotropin levels, and evidence of significant endogenous estrogen production (i.e., World Health Organization group II).⁵

In women who do not ovulate, CC is often recommended as an initial treatment, rather than gonadotropins. The advantages of CC over gonadotropins are shown in Table 1.⁶

CC administration is usually initiated on cycle day 2, 3, or 5, and is given daily for 5 subsequent days, with doses varying between 100 and 150 mg/day. In most applied regimens, exogenous gonadotropin medication (150 IU/day) is initiated after cessation of CC. It seems that CC alone induces a limited but dose-dependent increase in the number of developing follicles. However, the addition of gonadotropins elicits increased ovarian response as manifest by more follicles.

Use of CC usually leads to pregnancy rates that are higher compared to natural cycle IVF. An extensive summary of almost 40,000 cycles reported in the literature suggested pregnancy rates of 6% per started cycle and up to 20% per embryo transfer. Apart from hot flushes, which may occur in up to 10% of women taking CC, side effects are rare, dose-related and completely reversible once medication is stopped.²

CC is usually administered in the dose of 100 mg daily for 5-8 days, beginning on cycle day 3, and induces development of 2 or more follicles in most normally ovulating women. The egg yield of 1-3 is better than that with natural cycle, and can be improved with sequential treatment with CC, 100 mg daily for 5 days, and gonadotropins, 150-225 IU daily beginning on the last day of CC treatment or the day after. This treatment stimulates multifollicular development more effectively than with CC alone. The ensuing pregnancy rates are good and the risk of OHSS is less.⁷

CC appears to increase ovulation in women with oligo-ovulatory subfertility. It may also work in women with unexplained subfertility, perhaps by correcting an unidentifiable ovulatory dysfunction.

CC has been shown to be a sensible first choice treatment for women with unexplained infertility.^8,9   In doses between 50 and 150 mg/day, it appears to be effective in inducing ovulation and improving fertility in oligo-ovulatory women.

Over the years, CC has been shown to be successful in inducing ovulation in 50%-75% of cases. However, the pregnancy rate is much lower than expected. This can be ascribed to the prolonged antiestrogenic effects of CC, which negatively impact endometrial receptivity and cervical mucus. In addition, CC interferes with uterine blood flow. These negative effects can be overcome by the addition of estradiol.⁵

Stimulation with gonadotropins

Gonadotropin preparations have long been extensively used for ovarian stimulation in ovulatory women for empirical treatment of unexplained subfertility. The aim is to increase the number of oocytes available for fertilization in vivo.²   Women who have not been able to conceive with clomiphene may be given a trial of gonadotropins as a next step. In women who ovulate normally, gonadotropins may improve the chances of becoming pregnant (with IVF or IUI) by stimulating the ovaries to produce more than one follicle.⁶

The daily administration of gonadotropin preparations is usually efficacious in the maintenance of growth of multiple antral follicles, allowing for the retrieval of many oocytes for IVF. Preparations initially used were human menopausal gonadotropin (containing both LH and FSH bioactivity in a 1:1 ratio), followed by purified urinary FSH and, more recently, recombinant FSH and recombinant LH. Starting doses vary between 100 and 300 IU/day and are often adjusted depending on the observed individual ovarian response.²

In IVF treatment, stimulation with highly purified human menopausal gonadotropin (hMG) results in comparable or even higher pregnancy rates with lesser number of oocytes compared to recombinant FSH. Many of the advantages of highly purified hMG may be attributed to the LH activity it contains.¹⁰

hMG has been traditionally used, with or without the addition of CC, in normogonadotropic women to induce multiple follicular growth in order to maximize the yield of oocytes for fertilization in vitro. However, the frequent occurrence of an untimely, spontaneous LH surge led to the cancellation of a substantial number of treatment cycles with these stimulation methods. Moreover, increased levels of LH during the follicular phase of the menstrual cycle were found to be associated with reduced rates of fertilization and implantation and increased rates of miscarriage. In order to lower basal LH secretion during the follicular phase and prevent an untimely mid-cycle LH surge, addition of GnRH agonists to suppress the endogenous gonadotropin secretion during ovarian stimulation with FSH or hMG proved to be an effective treatment strategy.¹¹

The efficacy of this strategy in significantly improving clinical pregnancy rates in IVF was demonstrated in a meta-analysis¹²   of 10 trials comparing treatment cycle outcomes after GnRH agonist (n = 914) with other ovulation induction protocols (n = 722) and 7 trials comparing outcomes after short flare-up (n = 368) with longer suppression (n = 476) GnRH agonist protocols for IVF and gamete intrafallopian transfer (GIFT). GnRH agonist use resulted in:

• Significant improvement in clinical pregnancy rate (PR) per cycle commenced
• Significant improvement in clinical PR per embryo transfer
• Reduction in cycle cancellation

On the basis of the above results, it can be concluded that GnRH agonists can be used routinely for IVF and GIFT.

Stimulation with GnRH agonists

Long protocol

Use of long acting GnRH agonists, like leuprolide and buserelin, prevents LH surge in a large majority of cycles, hence stimulation can continue till full oocyte maturity is reached. This leads to higher egg yield and pregnancy rates than with exogenous gonadotropins alone.

In the long protocol, a GnRH agonist is started during the midluteal phase of the previous cycle, i.e., on day 21. Usually, patients will commence menses within 7 days, and the gonadotropin treatment will start on day 2 of the menstrual cycle if pituitary downregulation is achieved (Fig. 2). At this time, the levels of endogenous gonadotropins are at their lowest, and the flare effect will not affect follicular recruitment.

In women with unpredictable cycles, the onset of menses can be controlled by oral contraceptives (OCs), and the GnRH agonist is started 1 week before OCs are stopped.

After pituitary downregulation has been confirmed (serum E₂ <30-40 pg/ml, no follicles >10 mm in diameter), gonadotropins are started in the dose of 150-300 IU of urinary FSH (uFSH) or urinary menotropins (hMG) daily. One can use either a ‘step-up’ or a ‘step-down’ approach, but the latter is generally preferred.

It has been observed that women who develop ovarian cysts or require longer duration of GnRH agonists to achieve suppression of endogenous gonadotropins usually respond poorly to gonadotropin stimulation and are less likely to become pregnant.⁷

Short/flare protocol

This protocol makes use of the brief initial agonistic phase of response to a GnRH agonist as well as the suppression resulting from longer term treatment. Here, leuprolide acetate is given in the dose of 1 mg/day on cycle days 2 to 4, and then reduced to 0.5 mg/day. Gonadotropin is begun on day 3 in the dose of 225-450 IU/day. The immediate stimulatory action of the GnRH agonist serves as the initial stimulus for follicular recruitment. Adequate follicular maturation is, on an average, reached in 12 days, which should allow enough time for sufficient pituitary desensitization to prevent any premature LH surges.

This protocol has been reported to improve the follicular response and lower the cycle cancellation rate in poor responders to the long protocol, but the pregnancy and live birth rates are low. Other disadvantages include decreased flexibility and significant increase in serum progesterone, which may affect oocyte quality, fertilization and pregnancy rates.⁷

Ultrashort protocol

The agonist is given for 3 days in the early follicular phase. On the second day of agonist administration, stimulation with gonadotropin is started. By this method, LH can be suppressed till the midcycle, and more oocytes can be retrieved with minimal risk of premature LH surge.¹³

Stimulation with GnRH antagonists

GnRH-antagonist protocols shorten the treatment period and reduce inconvenience for IVF patients. Unlike GnRH agonists, the antagonists block the GnRH receptors in a dose-dependent competitive manner and have no flare effect. Moreover, gonadotropin suppression occurs almost immediately.

So far, only two GnRH antagonists are available for IVF use: cetrorelix and ganirelix. There are two approaches:¹³

• Single-dose protocol: A single high dose (3 mg) of cetrorelix – not ganirelix as it is not available in depot formulation – is administered on day 8 or 9 of the stimulation cycle (Fig. 3). This is sufficient to prevent an LH surge, although in slow responders a repeat injection may be needed.

• Multiple-dose protocol: The antagonist (either cetrorelix or ganirelix) is given in small daily doses of 0.25 mg subcutaneously from the 6th or 7th day of gonadotropin stimulation, till and including the day of hCG administration (Fig. 4).

The advantages and disadvantages of GnRH antagonists vis-a-vis agonists are enumerated in Table 2.

Protocol for PCOS patients

Women with polycystic ovary syndrome (PCOS) are at high risk of developing ovarian hyperstimulation syndrome (OHSS) during ovarian stimulation. Use of GnRH antagonists in the general subfertile population is associated with lower incidence of OHSS than agonists and similar probability of live birth. Lainas and colleagues¹⁴   tried to compare the flexible GnRH antagonist and GnRH agonist long protocols in 220 patients with PCOS undergoing IVF by randomly assigning them to two groups: long GnRH agonist downregulation protocol (n = 110) and flexible GnRH antagonist protocol (n = 110).

Although the ongoing pregnancy rates were similar between the two protocols, the antagonist protocol was found to have an edge over the agonist protocol due to

• Lower incidence of OHSS Grade II (40.0 vs 60.0%)
• Shorter duration of stimulation (10 vs 12 days)
• Lesser total dose of gonadotrophin required (1575 vs 1850 IU)

Hence, GnRH antagonist might be the treatment of choice for patients with PCOS undergoing IVF.

Use of estrogen in ART

In GnRH antagonist protocols, there is a slight reduction in the number of retrieved oocytes and pregnancy rate compared with long GnRH agonist protocols. This may be partly due to the absence of synchronization of the follicular cohort before ovarian stimulation, reducing the number of viable oocytes and embryos and therefore the chance of pregnancy.

In such situations, pre-treatment administration of steroid sex hormones may modify the hormonal environment in relation to the negative feedback exerted by steroid on endogenous gonadotropin secretion and therefore synchronize the follicular cohort before stimulation. The potential benefit of natural estradiol (E₂) pre-treatment has been assessed in GnRH antagonist cycles. This approach seems to be promising because endogenous estrogen secretion is actually the main factor involved in the negative regulation of FSH secretion during the luteal-follicular transition period. It has been reported that E₂pre-treatment prevents intercycle FSH rise, improves follicle synchronization effectively within the cohort and enhances the recovery of mature oocytes.¹⁵

Lukaszuk et al¹⁶   studied 166 women who were undergoing IVF with long protocol COH. Group 1 (progesterone only) included 80 cycles, group 2 (progesterone + 2 mg of E₂) included 73 cycles, and group 3 (progesterone + 6 mg of E₂) included 78 cycles. Supplementation was done in the luteal phase with different doses of E₂ (0, 2, or 6 mg/day).

In this study, significantly higher implantation rate and pregnancy rates were recorded in those who received low dose E₂ supplementation compared with no substitution. The best implantation and pregnancy results were found significantly in the group with high dose E₂supplementation (Fig. 5).

The researchers concluded that for women treated with a long GnRH analog protocol for COH, addition of a high dose of E₂ to daily progesterone supplementation significantly improved the IVF-embryo transfer results.

Later, Drakakis and colleagues¹⁷   also evaluated the effect of estradiol addition to progesterone supplementation during the luteal phase on pregnancy and implantation rates in patients undergoing IVF/ICSI-ET cycles, and noted higher pregnancy and implantation rates without any adverse effects.

Chang and colleagues¹⁸   conducted a retrospective analysis to find out if administering E₂ during the luteal phase of the preceding cycle and during ovarian stimulation in GnRH antagonist cycle could enhance follicular response and hence improve outcomes in poor responders.

For this purpose, they analyzed 155 poor responder patients subjected to IVF/ICSI. All the patients had a history of more than one prior IVF cycle failure with poor response (less than 5 oocytes retrieved and/or maximal E₂ level less than 500 pg/mL) by using conventional long agonist or antagonist protocol. In luteal E₂ treatment protocol (n = 86), oral estradiol valerate 4 mg/day was initiated on luteal day 21 and either stopped at menstrual cycle day 3 (Protocol A, n = 28) or continued during the period of ovarian stimulation until the day of hCG injection (Protocol B, n = 58). IVF parameters and pregnancy outcome of luteal E₂ treatments group were compared with a standard GnRH antagonist protocol (n = 69) in which the patients received no hormonal pretreatment.

Luteal E₂ was seen to have the following outcomes vis-a-vis standard GnRH antagonist protocol:

• Lower cancellation rate (15.1% vs 37.7%)
• Greater number of oocytes retrieved (4.5 ± 2.9 vs 3.2 ± 1.9)
• Greater number of normally fertilized embryos (2.9 ± 2.1vs 2.3 ± 1.9)
• Increased prevalence of good quality embryos (51.2% vs 25%)

Comparing protocol A and B, there were no significant difference between embryologic data, however there were slight increase in ongoing pregnancy rate in protocol B compared to A (27.1% vs 20%), although statistical significance was not achieved.

The investigators concluded that estrogen priming through luteal phase and stimulation phase improved ovarian responsiveness and this may lead to an increase in pregnancy rate in poor responders with failed cycle.

A meta-analysis was conducted by Torres et al¹⁹   to determine the effectiveness of adding estrogen during CC-induced ovulation cycles among infertile women in improving endometrial thickness, endometrial echogenicity and pregnancy rates. The investigators identified randomized controlled trials involving 354 women aged 21-37 years with unexplained/ anovulatory infertility to be included in this meta-analysis. The pooled weighted mean difference (WMD) for endometrial thickness was 1.71 mm [95% confidence interval (0.71, 2.69)] in favor of clomifene plus estrogen. This meta-analysis demonstrated that estrogen supplementation in clomiphene citrate ovulation induction increases endometrial thickness and serum estradiol levels with a trend towards increased chances of pregnancy among infertile women.

In another trial,²⁰   the effects of a timed sequence of hormone supplementation (HS) with oral estradiol (E₂) and vaginal progesterone (P) following clomiphene citrate (CC) therapy were examined to determine if this regimen can increase pregnancy rates in CC cycles. The patients were divided into two groups wherein one group received CC + E₂ + Progesterone (HS group) while the second group received CC alone. The protocol used was CC from days 3 to 7 for all patients; in the HS group, CC was followed by E₂ from day 8. E₂ was discontinued with LH surge and replaced with vaginal progesterone gel daily for 2 weeks starting 3 days after LH surge.

The pregnancy rate in the HS group was 17% compared to 7% in the CC alone group, showing that CC plus oral estradiol and vaginal progesterone gel have a potential supportive effect on pregnancy rates compared to CC alone.

Use of natural micronized progesterone in ART

The introduction of GnRH analogs has led to fewer cycle cancellations in IVF, by the prevention of a premature LH surge. In cycles using GnRH agonists, a luteal phase defect has been described, necessitating some form of luteal phase support. hCG and progesterone are used as luteal phase support in ovulation induction.²¹

As natural progesterone is rapidly metabolized after oral ingestion, resulting in poor absorption, a number of techniques have been developed in order to improve its pharmacokinetic properties. One such technique is micronization, which increases the half-life of progesterone and reduces its destruction in the gastrointestinal tract. Micronization decreases particle size and enhances the dissolution of progesterone, thereby increasing its absorption and bioavailability. Maximal serum concentrations are achieved more rapidly with orally administered micronized progesterone than with the injected formulation. Absorption of micronized progesterone is enhanced twofold when the hormone is taken with food.²²

Since the discovery of micronization, oral absorption has been improved such that adequate blood levels are achieved. Studies indicate that orally administered natural micronized progesterone (NMP):²³

• Is effective in preventing endometrial hyperplasia associated with estrogen
• Does not negate the bone-sparing effects of estrogen
• Does not have negative effects on lipids
• May be used to treat infertility attributed to luteal phase defects
• Produces fewer side effects than synthetic forms of the hormone

A 2004 Cochrane systematic review²⁴   of 59 studies concluded that in IVF cycles involving down-regulation with a long-acting GnRH agonist, progesterone supplementation achieved higher ongoing pregnancy rates per embryo transfer compared with placebo or no treatment.

A study by Frishman et al²⁵   evaluated the efficacy of oral micronized progesterone for the treatment of luteal phase defects. In this study, 7 women with luteal phase defects previously corrected by vaginal suppositories were administered oral micronized progesterone (200 mg three times a day). Endometrial biopsies were taken to evaluate treatment efficacy. Questionnaires were used to assess side effects, including sedation. Oral micronized progesterone was noted to be as efficacious as vaginal suppositories in the treatment of luteal phase defects, and was preferred by most of the patients over the vaginal route.

One study²⁶   compared the efficacy of oral micronized progesterone when applied by the vaginal route in 60 female volunteers. The subjects were equally divided into two groups to receive the drug either via the oral or vaginal route. Results revealed that the serum progesterone levels achieved by oral administration (5.06 ± 2.95 ng/ml) differed significantly from those achieved by vaginal administration (8.26 ± 4.09 ng/ml). The data also revealed that the serum progesterone levels of the oral administration group (4.23 ± 2.68 ng/ml) did not differ significantly from the other group (4.15 ± 3.40 ng/ml) when the serum estrogen level was less than 30 pg/ml. On the contrary, when the serum estrogen level was at least 30 pg/ml, there was a significant difference in the serum progesterone levels between these two groups (6.32 ± 2.99 ng/ml for the oral route and 9.76 ± 3.23 ng/ml for the vaginal route).

A case-control study²⁷   was performed to compare the efficacy and satisfaction rate of combined therapy with oral micronized progesterone capsules and vaginal progesterone gel versus monotherapy with vaginal progesterone gel in luteal support. Three hundred and seventy women undergoing IVF-ET treatment were assigned to receive either a combination of vaginal gel and oral capsules, or vaginal gel alone. The combined progesterone therapy led to higher pregnancy rates and lower abortion rates than vaginal monotherapy (39.5% vs 33.5% and 6.4% vs. 15.6%, respectively). Vaginal monotherapy was also associated with a higher frequency of bleeding.

Conclusion

The introduction of assisted reproductive technology (ART) techniques has enabled many an infertile couple to have children of their own. Ovarian stimulation enhances the chances of success in ART procedures by increasing the number of eggs available for fertilization. Agents used for ovarian stimulation include clomiphene citrate, gonadotropins (FSH, LH, hCG, hMG) and GnRH agonists and antagonists. The efficacy of ovarian stimulation can be improved by giving exogenous sex steroids (estradiol and natural micronized progesterone).

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