{"paper_id":"fbec42f7-637e-46e4-b289-ce462a3baca8","body_text":"The increasing number of women who delay pregnancy and must undergo assisted\nreproductive technology (ART), poses the challenge of finding increasingly efficient\novarian stimulation protocols, since oocyte donation is not always accepted. It is\nknown that 76% of the blastocysts produced from women older than 40 years are\naneuploid ( Harton  et al. ,\n2013 ). In these women, the ovarian response is below ideal and worsens\nwith the interval between treatments.\nThe number of oocytes used in  in vitro  fertilization (IVF) is\ndirectly related to the reproductive outcome. Patients with few oocytes are less\nlikely to get pregnant and pose a great challenge for specialists ( Polyzos & Devroey, 2011 ). Therefore, the\nshorter the time, the greater the number of eggs obtained and the higher the\nlikelihood of reaching an embryo with potential for implantation and development of\na full-term pregnancy. However, an aggressive stimulation, in addition to the risk\nof hyperstimulation, may recover lower quality oocytes due to the risk of premature\nluteinization.\nBaerwald  et al . (2003) \ndemonstrated that, during the luteal phase, remaining small antral follicles could\nbe in the early stages of follicular development, suggesting that the ovary could\nhave been continuously stimulated during the menstrual cycle. That possibility has\nproved to be especially useful in fertility preservation for patients in a hurry to\ninitiate cancer treatment. Aware of that, and excited about a patient's outcome who\naccidentally had a luteal-phase ovarian stimulation (LPS),  Kuang  et al.  (2014)  studied the efficacy of\ninitiating ovarian stimulation in the luteal phase, so they could extend the concept\nto a routine IVF setting that could be used independently of menstruation. The study\ndemonstrated that luteal phase stimulation (LPS) is appropriate in producing\ncompetent oocytes, and consequently, embryos with good pregnancy outcomes, with the\nadvantage of eradicating the ovarian hyperstimulation syndrome (OHSS) or premature\nluteinization. Other authors corroborated the LPS protocol feasibility ( Lin  et al ., 2016 ;  Wang  et al ., 2016 ;  Wei  et al ., 2016 ), and the\nsame group, later, described the safety for the offspring originated from that\nprotocol ( Chen  et al .,\n2015 ).\nThe theory that folliculogenesis occurs in a wave-like fashion and that there are\nmultiple follicular recruitment waves in the same menstrual cycle ( Baerwald  et al ., 2012 ), coupled\nto the LPS protocol success, was an inspiration for another stimulation, proposed to\nbenefit patients with poor ovarian response (POR). Also in 2014, Kuang proposed a\nnew protocol for ovarian stimulation, called Shangai Protocol, because it was\npresented during the BCGIP-COGI in Xangai. The strategy is to use luteal phase\novarian stimulation following oocyte retrieval, in the same cycle when follicular\nphase ovarian stimulation had already been carried out. With the main purpose of\nretrieving more oocytes in a short period of time, they used letrozole or clomiphene\ncitrate plus hMG, ovarian LH surge suppression with GnRH-antagonist and its\ntriggering with GnRH-agonist, associated with total embryo vitrification. The one\nthing they did different with this protocol was the sequential stimulation including\nthe luteal phase. As the established protocols are unable to make the poor responder\nto have a normal response, this approach, called double ovarian stimulation ( Kuang  et al ., 2014 ), aims to\nobtain the highest number of oocytes in the shortest time, thus avoiding the waste\nof time, crucial in this type of patient, in repeated attempts ( Zhang, 2015 ).\nUbaldi  et al . (2016) \nproposed the double stimulation protocol, which they called DuoStim, for patients\nwith reduced ovarian reserve, taking into account the \"time as an important factor\nfor all patients, but it is crucial for those with have a foreseeable rapid\nloss/decrease of fertility\". Different from the Xangai protocol, which used\nletrozole or clomiphene citrate plus hMG, they used recombinant gonadotrophins (FSH\nand LH), and after 5 days of the oocyte retrieval, a luteal phase stimulation was\nstarted like the previous stimulation. The aim of the study was to exploit the\novarian reserve to increase the offer of euploid embryos to transfer per intention\nto treat. They could increase the rate of euploid embryos from 41.9% (from oocytes\nexclusively obtained from follicular phase stimulation) to 69.8% considering\ncumulate oocytes from both follicular and luteal phase stimulation.\nWe started to offer the double stimulation protocol, in May 2016 for fertility\npreservation (oocyte cryopreservation) and IVF to patients with POR, fertilization\nfailure in previous IVF cycles, embryonic development failure, in the cases of IVF\nwith genetic tests where the patient had no blastocyst development for biopsy, as\nwell as total aneuploidy. The DuoStim protocol was offered with the aim of\nincreasing the number of oocytes and consequently of embryos for transfer or genetic\nevaluation. Most of these patients had already performed IVF cycles at other clinics\nbefore initiating treatment with us, with unfavorable outcomes such as low ovarian\nresponse or embryo development failure. Some patients were submitted to IVF in our\nservice, with an unfavorable outcome. The objective of our study was to evaluate the\nefficacy of the double stimulation protocol over conventional ovarian stimulation in\nthose patients.\n\nFrom May 2016 to February 2017, we performed 54 cycles of DuoStim for IVF, and 11\nfor fertility preservation. Of those 54 IVF DuoStim cases, 13 patients had been\npreviously submitted to IVF with conventional antagonist protocol stimulation in\nour clinic.\nWe analyzed the two treatment cycles from each of the 13 patients, comparing the\nnumber of oocytes collected, mature oocytes collected, fertilization rates,\nblastocyst rates, biopsied blastocyst rates and euploidy rates.\nThe study project was approved by the HUPE Research Ethics Committee.\nA baseline transvaginal ultrasound was carried out in the 2 nd  day of\nthe menstrual cycle, to check ovarian volume, number of antral follicles,\npresence of residual cysts greater than 15mm and endometrial thickness.\nAttesting the basal ovarian state, the patient receives the prescription of the\nmedications, which consists of subcutaneous human menopausal gonadotropin (hMG,\n75IU) and a subcutaneous injection of recombinant follitropin alpha (FSHr,\n225IU). Follicular development was monitored by transvaginal ultrasound starting\non day 6 of the cycle and then every two days. Daily administration of GnRH\nantagonist starts when a follicle reaches 14mm. When at least three follicles\nreach 16mm in diameter the triggering is carried out with a single subcutaneous\ninjection of recombinant hCG (hCGr, 250mcg), and oocyte retrieval is performed\nafter 35 hours.\nThe ovarian double stimulation starts exactly like the conventional protocol,\nexcept for the triggering that is carried out with GnRH agonist (triptorelin,\n0.2mg). After five days of the first oocyte pick up, the ovarian stimulation\nrestarts with the same protocol. The follow-up of this second stimulation is\ndone as in the first one, with an antagonist beginning with follicles of at\nleast 14mm, and triggering with GnRH agonist from three follicles with at least\n16mm.\nAfter oocyte pick-up and 4 hours of incubation, cumulus and corona radiata cells\nare removed by hyaluronidase treatment and pipetting, and then the MII oocytes\nare subjected to ICSI. Fertilization is checked 16 to 18 hours after ICSI and\nthen the presumptive embryos are cultured in groups, up to four embryos, in 25mL\nof Irvine continuous single culture medium (CSCM; Irvine Scientific, USA), and\ncovered with mineral oil. Culture is performed at 37 o C in 7,5% carbon\ndioxide and 5% oxygen tension within a benchtop incubator. Cleavage and embryo\nscore are evaluated on day 3 and the development to the blastocyst stage is\nevaluated while attesting the viability of the cells or up to day 7. When\nindicated, the blastocyst biopsy and the chromosome number screening are\nperformed, as follows: on day 4, at morula stage, a 10-20mm hole is opened on\nthe zona pellucida using a diode laser, the embryo goes back to the incubator\nuntil the blastocyst expands, when 3 to 7 trophectodermal cells are removed and\nsent to an outsourced genetics laboratory, in a PCR tube. All embryos, biopsied\nor not, from the DuoStim protocol are vitrified with the open method (Cryotop or\nsimilar).\nAll statistical analyses were performed in the Excel program, the student\n t -test was used for analysis for interval or reason\nvariables. All numerical variables were expressed as means and standard\ndeviations. The odds ratio (OR) was calculated, and a chi-squared test\n(χ 2 ) was performed for comparison of categorical\nvariables. Fisher's exact test was also performed when necessary.\n p  values < 0.05 were considered significant.\n\nThirteen patients were analyzed in this study for having performed DuoStim after one\ncycle of IVF using the antagonist protocol.\nThe mean age of the study population was 40.9 years, ranging from 37 to 44 years.\nFive patients were classified as poor responders according to the Bologna criteria\n( Ferraretti  et al. ,\n2011 ). Of the 13 patients analyzed, one did not perform a pre-implantation\ngenetic test in either treatment, and two had the genetic test indicated only after\nantagonist cycle failure. The main factor of infertility and the indication of the\ndouble stimulation cycle are described in  Table\n1 .\nTreatment characteristics of the study population\nPOR - Poor Ovarian Response. BMI - Body Mass Index. PGS -\nPre-implantation genetics screening.\nThe mean number of oocytes collected was 6.7 in the antagonist cycle and 11.7 in the\nDuoStim group ( p =0.007). Of the oocytes collected, the mean number\nof mature oocytes in the conventional group was 5.3, while in the DuoStim it was\n9.23 ( p =0.01). There was no statistical difference in the rates of\nfertilization and blastocyst rates, as per shown in  Table 2 , with a  p  value equal to 0.78 and 0.59,\nrespectively.\nComparison of laboratorial results of the conventional protocol with\nDuoStim\nMedian (Range).\nTen patients underwent IVF with genetic testing in both treatment cycles. From the\nantagonist cycle 20 embryos were biopsied and of these, only two were euploid. In\nthe DuoStim cycles, 32 embryos were biopsied, of which six were euploid. There was\nno significant difference between the number of embryos biopsied, the number of\neuploid embryos and euploidy rate, as per described in  Table 3 .\nComparison between two catheters for embryo transfer in relation to patient\ndata and IVF cycle outcomes\n\nThe purpose of this study was to compare the double stimulation protocol to\nconventional ovarian stimulation. The evaluated patients had already had\nunsatisfactory results with the conventional protocol at our clinic, and the\nopportunity to double stimulate in the same cycle aiming to increase the number of\noocytes and embryos was the argument for the new intent to treat.\nThe number of collected oocytes was one of the factors associated with the positive\noutcome of assisted reproduction treatment, such as IVF and oocyte cryopreservation.\nThis association between low response and poor embryo quality is not uncommon; it\noften does not reach the blastocyst stage or arrives in insufficient amounts to\nachieve an euploid embryo status, leading to cycle and transfers cancellations. This\nunfavorable situation leads to disappointment, anxiety and frustration, resulting in\na high dropout rate ( Verberg  et\nal. , 2008 ).\nAs not only the quality but also the quantity is important, some authors have\nattempted to estimate an ideal number of oocytes collected, so that patients could\nachieve the desired pregnancy. This number is even mentioned in studies that try to\nevaluate the number of oocytes that should be cryopreserved, in cases of\noncofertility preservation and for social freezing purposes ( Cil  et al. , 2013 ). Another important issue in\nassisted reproduction is the time, which is crucial for patients with rapid loss or\ndecreased fertility. Therefore, a double stimulation to enhance the number of\noocytes in the same menstrual cycle is a very attractive strategy.\nThe opportunity for egg accumulation with double stimulation was demonstrated by\n Ubaldi  et al . (2016)  and\n Kuang  et al . (2014) , but\nboth authors performed the statistical analysis between the two stages of\nstimulation: the follicular and the luteal phases. The main difference of our study\nwas that we evaluated patients who had already been submitted to conventional\nstimulation, with poor outcomes from that treatment, who had a chance for double\nstimulation. Our analysis compared the outcome of the two protocols, considering\nthat the double stimulation is the sum of the two phases.\nThe double stimulation should be considered as a single treatment cycle. Patients who\nopt for dual stimulation aim at having higher number of oocytes in a shorter period\nof time, since when performing two independent stimuli the patient must wait for a\nnew menstrual cycle and resolution of the hematic cysts after puncture.\nThe luteal phase stimulation was believed to affect the oocyte ability to mature and\nbe fertilized. However, as described in other studies, despite the particularities\nof the DuoStim protocol, there was no difference in fertilization rate and\nblastocyst rate, confirming the safety in maintaining oocyte quality ( Kuang  et al. , 2014 ).\nIn our study, the patients submitted to the DuoStim protocol had a statistically\nsignificant increase in the number of oocytes collected, increasing the mean from\n5.3 to 9.3 mature oocytes, greater than the minimum described by  McAvey  et al . (2011)  to\nachieve pregnancy. In their study, with 737 women undergoing IVF cycles with less\nthan six MII oocytes, they found a statistically significant decrease in the\nlikelihood of a live birth compared to groups with six or more oocytes. Taking into\naccount that DuoStim was offered to most patients after one year of the last cycle\nof unsuccessful IVF; it is possible that the number of oocytes obtained would have\nbeen higher if DuoStim had been the first option.\nAlthough we have seen an increase in the number of embryos biopsied in the blastocyst\nstage, we could not reach statistical significance ( p =0.899), nor\ndid we have significant fertilization and blastocyst rates between the two\nprotocols. We believe that it happened due to the small number of cases analyzed.\nSince a prospective study with these characteristics would not be ethical, for not\noffering what we believe to be the best for the patient in the group that would be\nrandomized to the traditional protocol.\n\nDouble stimulation favors patients who would need more than one stimulus to produce\nan adequate amount of oocytes. The greatest benefit of this protocol is the\naccumulation of oocytes in a single cycle of stimulation, minimizing the time in\nwhich it will be performed. In addition, it allows the production of a larger number\nof embryos, which can then be genetically evaluated, thus favoring the final\nclinical result.","source_license":"CC-BY-4.0","license_restricted":false}