Dual Stimulation Protocol
The increasing number of women who delay pregnancy and must undergo assisted reproductive technology (ART), poses the challenge of finding increasingly efficient ovarian stimulation protocols, since oocyte donation is not always accepted. It is known that 76% of the blastocysts produced from women older than 40 years are aneuploid.In these women, the ovarian response is below ideal and worsens with the interval between treatments.
The number of oocytes used in in vitro fertilization (IVF) is directly related to the reproductive outcome. Patients with few oocytes are less likely to get pregnant and pose a great challenge for specialists. Therefore, the shorter the time, the greater the number of eggs obtained and the higher the likelihood of reaching an embryo with potential for implantation and development of a full-term pregnancy. However, an aggressive stimulation, in addition to the risk of hyperstimulation, may recover lower quality oocytes due to the risk of premature luteinization.
Baerwald et al. (2003) demonstrated that, during the luteal phase, remaining small antral follicles could be in the early stages of follicular development, suggesting that the ovary could have been continuously stimulated during the menstrual cycle. That possibility has proved to be especially useful in fertility preservation for patients in a hurry to initiate cancer treatment. Aware of that, and excited about a patient’s outcome who accidentally had a luteal-phase ovarian stimulation (LPS), Kuang et al. (2014) studied the efficacy of initiating ovarian stimulation in the luteal phase, so they could extend the concept to a routine IVF setting that could be used independently of menstruation.
The study demonstrated that luteal phase stimulation (LPS) is appropriate in producing competent oocytes, and consequently, embryos with good pregnancy outcomes, with the advantage of eradicating the ovarian hyperstimulation syndrome (OHSS) or premature luteinization. Other authors corroborated the LPS protocol feasibility (Lin et al., 2016; Wang et al., 2016; Wei et al., 2016), and the same group, later, described the safety for the offspring originated from that protocol (Chen et al., 2015).
The theory that folliculogenesis occurs in a wave-like fashion and that there are multiple follicular recruitment waves in the same menstrual cycle (Baerwald et al., 2012), coupled to the LPS protocol success, was an inspiration for another stimulation, proposed to benefit patients with poor ovarian response (POR). Also in 2014, Kuang proposed a new protocol for ovarian stimulation, called Shangai Protocol, because it was presented during the BCGIP-COGI in Xangai.
The strategy is to use luteal phase ovarian stimulation following oocyte retrieval, in the same cycle when follicular phase ovarian stimulation had already been carried out. With the main purpose of retrieving more oocytes in a short period of time, they used letrozole or clomiphene citrate plus hMG, ovarian LH surge suppression with GnRH-antagonist and its triggering with GnRH-agonist, associated with total embryo vitrification. The one thing they did different with this protocol was the sequential stimulation including the luteal phase. As the established protocols are unable to make the poor responder to have a normal response, this approach, called double ovarian stimulation (Kuang et al., 2014), aims to obtain the highest number of oocytes in the shortest time, thus avoiding the waste of time, crucial in this type of patient, in repeated attempts (Zhang, 2015).
Ubaldi et al. (2016) proposed the double stimulation protocol, which they called DuoStim, for patients with reduced ovarian reserve, taking into account the “time as an important factor for all patients, but it is crucial for those with have a foreseeable rapid loss/decrease of fertility”. Different from the Xangai protocol, which used letrozole or clomiphene citrate plus hMG, they used recombinant gonadotrophins (FSH and LH), and after 5 days of the oocyte retrieval, a luteal phase stimulation was started like the previous stimulation. The aim of the study was to exploit the ovarian reserve to increase the offer of euploid embryos to transfer per intention to treat. They could increase the rate of euploid embryos from 41.9% (from oocytes exclusively obtained from follicular phase stimulation) to 69.8% considering cumulate oocytes from both follicular and luteal phase stimulation.
Poor ovarian response (POR) during stimulation for IVF is a challenging and frustrating condition for patients and clinicians. The incidence of patients who respond poorly is estimated to be about 5–24%. The cause of poor ovarian response may be associated with diminished ovarian reserve. Various treatment protocols with high doses of gonadotrophins, along with various adjuvant drug therapies, were used to improve ovarian response and pregnancy rates .
Although standard definitions for POR are lacking, the European Society for Human Reproduction and Embryology has proposed that at least two of the following three features must be present in poor ovarian response, Bologna Criteria :
advanced maternal age or any other risk factor for poor ovarian response;
previous poor ovarian response;
or an abnormal ovarian reserve test
Recently, mild ovarian stimulation during a gonadotrophinreleasing hormone (GnRH) agonist cotreatment cycle using a low dose of exogenous gonadotrophins, ovarian stimulation for IVF using oral compounds (e.g. anti-oestrogens or aromatase inhibitors), or both, have been proposed as costeffective, patient-friendly regimens that optimize the balance between outcomes and risks of treatment. For patients with POR, mild ovarian stimulation resulted in IVF outcomes similar to conventional ovarian stimulation, and even yielded slightly better pregnancy rates than conventional ovarian stimulation in poor responders older than 37 years.
The classic IVF procedure starts with ovarian stimulation in the early follicular phase and retrieves oocytes when follicles are mature. Luteal-phase stimulation was originally used to produce mature oocytes and embryos for cryopreservation in case reports of emergency fertility preservation like paients undergoing treatment for cancer.
Stage one of treatment Protocol :
Patients screened by transvaginal ultrasound and serum FSH testing on day 3 of their menstrual cycle. Clomiphene citrate 25 mg/day cotreatment and letrozole 2.5 mg/day given from cycle day 3 onwards. Letrozole only given for 4 days and clomiphene citrate continuously used before the trigger day. Patients given injection human menopausal gonadotrophin (HMG) 150 IU every other day beginning on cycle day 6. Follicular monitoring started on cycle day 10 and carried out every 2–4 days using a transvaginal ultrasound examination to record the number of developing follicles and serum FSH, LH, oestradiol and progesterone concentrations. When one or two dominant follicles reached 18 mm in diameter, the final stage of oocyte maturation is induced with triptorelin (Decapeptyl100 g, followed by ibuprofen 0.6 g, which is used on the triggering day and the next day, forpreventing possible follicle rupture before oocyte retrieval .
Transvaginal ultrasound-guided oocyte retrieval is conducted 32–36 h after GnRH agonist administration. All follicles of less than 10 mm are not retrieved and left for the second-stage stimulation in the luteal phase. Fertilization of the aspirated oocytes is carried out in vitro, by either conventional insemination or ICSI, depending on semen parameters. Embryos are examined for the number and regularity of blastomeres and the degree of embryonic fragmentation, and graded according to Cummins’s criteria. All highest-quality embryos (including grade 1 and grade 2, eight-cell blastomere embryos) are cryopreserved on the third day after oocyte retrieval. The non-top-quality embryos are placed in extended culture until the blastocyst stage. During this stage, on day 5 or day 6, only good morphology blastocysts are cryopreserved. Both cleavage-stage embryos and blastocysts cryopreserved by vitrification. In brief, the cryotop carrier system are used for vitrification and 15% (v/v) ethylene glycol, 15% (v/v) Dimethylsulphoxide and 0.5 M sucrose as the cryoprotectant . For warming, 1 M, 0.5 M and 0 M sucrose solutions are used for cryoprotectants dilution step by step. All vitrification and warming steps are carried out at room temperature except the first warming step at 37°C.
Stage two of treatment protocol: ovarian stimulation and oocyte retrieval
Transvaginal ultrasound examination is carried out after oocyte retrieval to determine whether to continue the second ovarian stimulation. The criterion for continued stimulation is the presence of at least two antral follicles 2–8 mm in diameter. A total of 225 IU HMG and letrozole 2.5 mg are administered daily from the day of, or the day after, oocyte retrieval. The initial second stage follicular monitoring is conducted 5–7 days later, and then every 2–4 days, using a transvaginal ultrasound examination to record the number of developing follicles, and serum FSH, LH, oestradil and progesterone concentrations. Letrozole administration is stopped when the dominant follicles reached diameters of 12 mm, given that large follicles have redundant LH and FSH receptors, and good response to exogenous hormone stimulations. Daily administration of medroxyprogesterone acetate 10 mg is added beginning on stimulation day 12 for cases in which post-ovulation follicle size was smaller than 14 mmin diameter and stimulation needed to continue for several more days. This is done to postpone menstruation and avoid oocyte retrieval during menstruation, to prevent the risk of infection from the procedure. When three dominant follicles reached diameters of 18 mm or one mature dominant follicle exceeded 20 mm, the final stage of oocyte maturation is induced again with triptorelin 100 g by injection. Again, ibuprofen 0.6 g is used on the day of oocytematuration triggering and the day after. Transvaginal ultrasound-guided oocyte retrieval is conducted 36–38 h after GnRH agonist administration. All oocytes collected are treated as in study stage one.
It is also beneficial for cancer patients needing emergency fertility preservation. As the number of women who survive cancer increases, the demand for effective and individualized fertility preservation options grows. Among the strategies for the preservation of fertility, embryo cryopreservation or oocyte vitrification seem to be the most effective approach. This protocol would minimize delays and allow more oocytes and embryos to preserve in a limited period of time for the cancer patients before starting chemotherapy, radiotherapy, or both. Another important finding in reproductive endocrinology is the relative ovarian insensitivity to HMG stimulation during the luteal phase compared with the follicular phase. Use of relatively larger dose of HMG to perform ovarian stimulation after first oocyte retrieval.
A study reported that the mean dose of HMG per oocyte retrieved in the luteal phase stimulation was nearly two times more than those of classical protocol (Buendgen et al., 2013). In the pituitary gland, the FSH surge and LH surge induced by the same dose of GnRH agonist were much higher in the first trigger than the second trigger. This suggested that ovarian sensitivity to the HMG stimulation was significantly reduced during the luteal-phase ovarian stimulation. One of the impacting factors was pituitary suppression of co-existing high progesterone during the luteal phase. The inherent mechanism of the corpus luteum on follicle growth waits for further study. There was a wide range of oestradiol and progesterone levels on the day of and the day after the second trigger. Further analysis showed the fluctuation of progesterone was negatively associated with HMG duration during the second stimulation.
For the cases with longer lutealphase HMG stimulation (>9 days), the progesterone level had a decreasing trend which approximately coincided with the formation and recession of the corpus luteum. In addition, this pilot study (Shanghai Protocol) opened the door for the possibility of performing double stimulation in patients with POR. As mentioned above, double stimulation during the follicular and luteal phases in the same menstrual cycle provided more opportunities to retrieve oocytes in poor responders, with the resulting embryos having similar development potential. Double stimulation and subsequent cryopreserved embryo transfer is a promising approach both for patients with POR, especially for the cases that repeatedly did not have oocytes retrieved or viable embryos using conventional IVF regimens, and for cancer patients needing emergency fertility preservation.