Kontrollü ovarian hiperstimülasyonda yeni kavramlar: Luteal fazda stimülasyon, dual trigger, oosit pooling Dr. L. Cem Demirel Ataşehir Memorial Hastanesi, IVF Departmanı İstanbul

Luteal faz ovarian stimülasyon........ standard regimens of ovarian stimulation are started during the early follicular phase of the menstrual cycle. One of the disadvantages of follicular-phase ovarian stimulation is that with the development of multiple follicles stimulated by exogenous gonadotropin, a premature LH surge is sometimes elicited. This is due to the positive feedback of high E2 on the pituitary and results in premature luteinization and suboptimal oocyte quality. Therefore, GnRH analog cotreatment is believed to be necessary to prevent the premature LH surge that occurs in the current practices, but this regimen makes the stimulation complex (7). Another detriment of ovarian stimulation is the serious complications caused by ovarian hyperstimulation syndrome (OHSS), a rare but potentially life-threatening condition. Few studies consider the possibility of performing ovarian stimulation during the luteal phase of the menstrual cycle except for a small number of case reports on cancer patients (8–10). In the context of fertility preservation, luteal-phase administration of FSH and a GnRH antagonist was reported to produce mature oocytes/embryos for cryopreservation (8–10), but no data on pregnancy outcomes were available in the limited samples. So we extended the concept to a routine IVF setting that can be used independently of menstruation. Given the asynchrony of the endometrium and embryo in such settings, all fertilized oocytes had to be cryopreserved for a later transfer.

The traditional process of folliculogenesis involves the recruitment of various antral follicles in each ovary during the late luteal phase of the preceding menstrual cycle. Then, during the initial or middle stage of the follicular phase, a single follicle is selected, while the others undergo atresia (1, 2). However, some studies have demonstrated that small antral follicles observed during the luteal phase may not necessarily be in atresia but may rather be in the early stages of follicular development (3, 4). This indicates waves of follicular development within a single interovulatory period, with the presence of healthy follicles in the luteal phase as determined by oocyte and granulosa cell viability

Luteal faz stimülasyon Background Luteal faz IVM: yetkin ve döllenebilen oositler Fertilite prezervasyon için emergency stimülasyon uygulamarından elde edilen deneyim Luteal-phase in vitro maturation provides sound evidence that the oocytes retrieved during the luteal phase can be competent to mature and be fertilized (5, 6). These observations suggest the possibility that follicles are continuously available for stimulation by gonadotropins during the menstrual cycle.

Emergency ovarian stimülasyon hCG GnRH antagonist 0.25 mg / d rFSH 150-225 IU /d 18 mm +3 Patients received GnRH antagonists to induce immediate luteolyis. At the same time, ovarian stimulation was initiated by daily injection of recombinant FSH. The combination of both medications was continued until follicular maturity was assessed and ovulation induction was initiated (as described above). The combination of GnRH antagonists and recombinant FSH resulted in luteolysis within 2 (midluteal phase) to 4 (early luteal phase) days, followed by follicle growth. They received GnRH antagonists, resulting in a rapid fall of progesterone followed by menses 2–4 days later. Ovarian stimulation was followed 4 days after application of GnRH antagonists, using hMG 150 IU/day. Th We did not await luteolysis before starting ovarian stimulation, but we initiated both GnRH antagonist and recombinant FSH at the same time, irrespective of the day of menstrual cycle. This protocol was designed to reduce the time frame needed until oocyte collection without significantly reducing the number of the collected oocytes.

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Luteal faz ovarian stimülasyon Triptorelin 100 µg Letrozole 2.5 mg / d hMG 225 IU /d 12 mm 18 mm +3 Participants were required to test their urine using an LH kit beginning on cycle day 10. When the LH surge indicator line appeared, they came to the clinic for an ultrasound and a serum hormone test. These ultrasound scans, along with serum P concentration testing, helped detect spontaneous ovulation. For the patients with follicles of <8 mm on 1–3 days remaining after ovulation, an hMG (Anhui Fengyuan Pharmaceutical Co.) 225 IU IM injection and letrozole (Jiangsu Hengrui Medicine Co.) 2.5 mg were administered every day; weekly follow-up visits were conducted. For monitoring, a transvaginal ultrasound examination was performed to record the number of developing follicles, and serum FSH, LH, E2, and P4 concentrations were measured. Letrozole administration was stopped when the dominant follicles each reached diameters of 12 mm. Daily administration of medroxyprogesterone acetate (MPA) 10 mg was added to the treatment regimen for cases in which on day 12 postovulation follicle size was smaller than 14 mm and stimulation needed to continue for several more days. This was done to postpone menstruation and avoid oocyte retrieval during menstruation, to prevent the risk of infection from the procedure. The final stage of oocyte maturation was induced with triptorelin (Decapeptyl, Ferring GmbH) 100 mg, injected when at least three follicles reached diameters of 18 Kuang, Fertil Steril ; 2014, 101:105-11

N: 242 Oosit elde edilen siklus n 242 Ortalama oosit sayısı 13.1 Ortalama MII oosit sayısı 11.2 Fertilizasyon oranı % 70 Klivaj oranı % 87 Ortalama dondurulan embryo / blast 6.2 Ortalama hMG kullanım süresi 10.2 gün Ortalama letrozole kullanım süresi 8.3 gün All highest quality embryos (including grade 1 and grade 2 8-cell blastomere embryos) were frozen by vitrification on the third day after oocyte retrieval. The embryos that were not of top quality were placed in extended culture until the blastocyst stage. At this stage, on day 5 or day 6, only good-morphology blastocysts were frozen. The procedure for freezing and thawing Kuang, Fertil Steril ; 2014, 101:105-11

Luteal faz stimülasyon Kuang, Fertil Steril ; 2014, 101:105-11

Luteal faz stimülasyon No OHSS No premature LH surge No cases incurred a premature LH surge or moderate/severe OHSS. The results provided evidence of a breakthrough in overcoming the limitations in the current ovulation induction protocols that are used during the menstrual cycle and provide a new approach to follicular recruitment. The method can also serve as an emergency procedure for newly diagnosed cancer patients who wish to preservefertility, with stimulation to be performed immediately upon diagnosis. In this study, ovarian stimulation during the luteal phase resulted in lower serum LH concentrations on the day of ovulation triggering; no cases incurred a premature LH surge. In contrast, a high occurrence of premature LH surges (20%–25%) was reported during the cycles with hMG administration during the follicular phase; the consequent premature luteinization resulted in suboptimal oocyte quality and a high cancellation rate (14). Our study provided sound evidence of luteal-phase LH suppression and the need to artificially induce an LH surge by a GnRH agonist. The phenomenon, which is related to the absence of a spontaneous LH surge in women undergoing luteal-phase ovarian stimulation, will have far-reaching significance in removing the scruples of blocking an endogenous LH surge for IVF treatment. It primarily simplifies ovarian stimulation protocols and makes it easy to handle procedure monitoring. Determining the potential mechanism of LH suppression awaits further research.

Luteal faz ovarian stimülasyon Triptorelin 100 µg Letrozole 2.5 mg / d hMG 225 IU /d 12 mm 18 mm +3 There were two reasons why OHSS did not occur, namely, the GnRH agonist used to trigger ovulation lowered the risk of OHSS compared with hCG (7) and FET reduced late-onset OHSS. We speculate that other factors related to the role of the corpus luteum may also be associated with the low incidence of OHSS; however, any benefit of this protocol in such a context needs to be further investigated.

Kuang, Fertil Steril ; 2014, 101:105-11

Luteal faz stimülasyon Tüm IVF için yapılan KOH uygulamalarında Kanser hastalarında, fertilite koruma amaçlı acil KOH uygulamalarında (KT öncesi KOH için zaman olmayan vakalarda) Elde edilen tüm embryolar endometrium – embryo asenkronizasyonu nedeniyle dondurulmalıdır. Because of the asynchrony between the endometrium and embryo in this study, all fertilized embryos had to be cryopreserved for a later transfer. Using vitrification, the quality of the frozen-thawed embryos was confirmed and the thawed embryo survival rate had the potential to reach over 96% in our study. The safety of FET was proved to be similar to that of fresh embryo transfer (17). The high implantation rate in FET cycles was associated with superior endometrial receptivity in the relatively normal hormone environment

Dual trigger.......

Dual trigger Antagonist sikluslarda : Tek GnRH agonist bolus + normal / azaltılmış hCG dozu Nevertheless, when compared with the conventional hCG triggered cycles, significantly reduced implantation rate and higher abortion rate were observed for GnRH-agonist triggered cycles (3, 4). The inferior pregnancy outcomes were attributed to defective luteal phase function and decreased endometrial receptivity induced by the GnRH-agonist trigger (14). To completely eliminate OHSS, the optimal dose of hCG administered at periovulation or, alternatively, recombinant LH administered during the luteal phase still needs to be determined.

Endometrial reseptivitede artış ? Oliveira JB, Baruffi R, Petersen CG, Mauri AL, Cavagna M, Franco JG Jr. Administration of single-dose GnRH agonist in the luteal phase in ICSI cycles: a meta-analysis. Reprod Biol Endocrinol 2010;8:107. With all the embryo quality parameters (proportions of top quality embryos and blastocyst progression) equivalent between the control and dualtrigger groups, the difference in cycle outcome was most likely related to the disparity in endometrial receptivity that resulted from the different triggering agent used. With the identification of GnRH receptors in the extrapituitary sites such as the endometrium (21), myometrium (22), fallopian tube (23), ovaries (24, 25), placenta (26, 27), and preimplanting embryo (28), GnRH has been suggested to play multiple roles in the regulation of endometrial receptivity. Gonadotropin-releasing hormone has been shown to modulate matrix metalloproteinases (MMPs) in the placental trophoblasts that mediate trophoblast cell invasion and extracellular matrix degradation (31, 3 In a recent meta-analysis by Oliveira et al. (42), the administration of a single-dose GnRH-agonist in the luteal phase significantly increased implantation rates in cycles treated with either a GnRH-agonist or GnRH-antagonist protocol, while significant improvement in clinical pregnancy and ongoing pregnancy rates were only noted in cycles treated with a GnRH antagonist protocol. These findings along with the results from our present study further confirm the beneficial effects of a GnRH agonist on embryo implantation and pregnancy outcomes in GnRH-antagonist cycles.

Normal cevaplı hastalarda dual tetikleme 250 mg of recombinant hCG plus 0.2 mg of triptorelin (Decapeptyl; Ferring GmbH). The mean number of oocytes retrieved and mature metaphase II (MII) oocytes were both statistically significantly greater in the dual-trigger group. The mean number of embryos obtained and top quality embryos were similar between the two groups, as was the rate of blastocyst progression. the dual-trigger group demonstrated a statistically significantly higher implantation rate (29.6% vs. 18.4%; P<.001), clinical pregnancy rate (50.7% vs. 40.1%; P¼.042), and live-birth rate (41.3% vs. 30.4%; P¼.047) in comparison with the hCG trigger group. The difference in abortion rate between the two groups was not statistically significant. There was one case of moderate OHSS in the control group, but it did not require hospitalization; none occurred in the dualtrigger group. 187 hasta dual trigger, 191 hasta kontrol grup

GnRHa triggered LH + FSH surge Another advantage of triggering with a GnRH agonist for oocyte maturation is the simultaneous induction of a midcycle FSH surge that is similar to the hormone surge in a natural cycle. Animal studies have confirmed the role of FSH in promoting the formation of luteinizing hormone (LH) receptor sites in rat granulosa cells (43, 44). The increase in LH receptors is crucial for preparing the maturing follicle for an LH surge that triggers the events of ovulation and subsequent luteinization of the granulosa cells. Furthermore, FSH has been shown to promote the resumption of oocyte meiosis (45, 46) and cumulus expansion (47, 48) in animal models. The importance of the presence of FSH during human oocyte maturation process is also supported by studies in which the groups triggered with a GnRH agonist consistently resulted in retrieval of a significantly greater number of mature oocytes (4,

OHSS riski olan high responder hastalarda ‘Dual triggerring’ Although there have been several strategies proposed, GnRH agonist (GnRHa) to induce oocyte maturation has been shown to be the most effective method to reduce the risk of OHSS (2–4). Studies have shown lower conception and higher miscarriage rates in patients triggered with GnRHa compared with hCG (5, 6). These poor outcomes are most likely due to defective corpora lutea (CL) function after GnRHa trigger leading to impaired endometrial receptivity. Therefore, intensive luteal-phase steroid supplementation has been shown to result in improved luteal-phase steroid profiles and excellent conception rates The authors hypothesized that the higher LH levels on the day of trigger observed in patients with peak E2 >4,000 pg/mL may induce the rescue of some CL function accounting for the higher conception rates. It is therefore possible that some form of adjuvant low-dose LH supplementation would result in the rescue of a few CL, resulting in optimal steroidal secretion and improvement in pregnancy rates in patients with peak E2 levels<4,000 pg/mL. The dose of hCG used in that study was based on a patient's weight and risk of OHSS and ranged from 1,000 to 2,500 IU (8). Owing to a concern for the development of OHSS with an hCG dose of 2,500 IU combined with GnRHa trigger, we chose a fixed dose of hCG 1,000 IU to minimize the risk of significant OHSS. A fixed low dose of 1,000 IU hCG in addition to GnRHa for trigger of oocyte maturation may rescue a few CL to function and improve the chance of conception without increasing the risk of significant OHSS. We therefore explored the use of dual trigger of oocyte maturation with GnRHa and low-dose hCG (1,000 IU) in high responders with peak E2 <4,000 pg/mL.

In comparison to HCG, GnRH agonist administration is associated with a significantly reduced likelihood of achieving a clinical pregnancy (0.21, 0.05–0.84;P=0.03). The odds of first trimester pregnancy loss is increased after GnRH agonist triggering; however, the confidence interval crosses unity (11.51,0.95–138.98; P=0.05). In conclusion, the use of GnRH agonist to trigger final oocyte maturation in IVF, where inhibition of premature LH surge is achieved with GnRH antagonists, yields a number of oocytes capable to undergo fertilization and subsequent embryonic cleavage, which is comparable to that achieved with HCG. However, the likelihood of an ongoing clinical pregnancy after GnRH agonist triggering is significantly lower as compared to standard HCG treatment.

Neden dual trigger? Tek başına GnRHa ile tetikleme canlı doğum oranlarında önemli bir düşüş yaratmaktadır Cochrane Database Syst Rev 2011;1:CD008046. Ama dual tetiklemede; GnRH agonist OHSS riskini azaltırken, Azaltılmış doz hCG de luteal fonksiyonu idame ettirmektedir. In a recent Cochrane review, the routine use of a GnRH agonist as the lone trigger for final oocyte maturation in fresh IVF autologous cycles was discouraged because of the significant decrease in live-birth rates (OR 0.44; 95% CI, 0.29–0.68; 4 RCTs) and ongoing pregnancy rates (OR 0.45; 95% CI, 0.31– 0.65; 8 RCTs) compared with a conventional hCG trigger (15). Therefore, modifications in luteal phase support have been suggested in IVF cycles triggered with a GnRH agonist. One method is intensive luteal progesterone and an estrogen supplement, as proposed by Engmann et al. (9). By maintaining the serum estradiol level above 200 pg/mL and progesterone level above 20 ng/mL, investigators have achieved a remarkable ongoing pregnancy rate of 53%. Another method that adds a reduced dosage of hCG either at oocyte retrieval (5, 6) or intermittently during luteal phase (16) has also showed promising results. Although the risk of OHSS was effectively minimized by triggering with a GnRH agonist, proper luteal function was also salvaged by the added hCG. Several studies focusing on high responders have demonstrated significant improvements in both ongoing pregnancy rates (17, 18) and live-birth rates (19) when a dual trigger was used instead of a GnRH-agonist trigger, all without conferring a significant increase in the OHSS rate. GnRHa trigger causes an abnormal endogenous LH surge which may lead to abnormal CL function (15–17) resulting in an abnormal luteal-phase steroid profile (4, 18, 19) and lower conception rates (5, 6). To completely eliminate OHSS, the optimal dose of hCG administered at periovulation or, alternatively, recombinant LH administered during the luteal phase still needs to be determined.

OHSS riski olan hastalarda dual trigger uygulamasında hCG için güvenli en düşük doz nedir? 1000 ? 1500 ? 2000 ? 2500 ? Acceptable rates of fertilization, implantation, clinical pregnancy, ongoing pregnancy, and early pregnancy loss were achieved in high responders after triggering final oocyte maturation with a combination of leuprolide acetate and hCG (1,000 to 2,500 IU). These findings, along with the absence of ovarian hyperstimulation syndrome, suggest that this dual trigger is safe and effective for oocyte maturation in patients with significant risk factors for ovarian hyperstimulation syndrome.

1 mg leuprolide + 1000 IU hCG Patients were triggered once three follicles reached R17 mm in diameter with either 1 mg leuprolide acetate (Lupron; TAP Pharmaceuticals) subcutaneously or 1 mg leuprolide acetate and 1,000 IU hCG subcutaneously. The serum P and E2 levels 3 days after oocyte retrieval were significantly higher in the dual-trigger group than in the GnRHa group. Midluteal serum E2 levels were significantly higher in the dual-trigger group compared with the GnRHa group, whereas the midluteal serum P levels did not differ significantly between the two groups

1 mg leuprolide + 1000 IU hCG The implantation (41.9% vs. 22.1%; P<.01), clinical pregnancy (58.8% vs. 36.8%; P¼.03) and live birth (52.9% vs. 30.9%; P¼.03) rates were significantly higher in the dual-trigger group compared with the GnRHa group (Table 3). There was one case of mild OHSS in the dual-trigger group but no cases of OHSS in the GnRHa-alone group. Dual trigger of final oocyte maturation with GnRHa and low-dose hCG resulted in improved implantation, clinical pregnancy, and live birth rates compared with GnRHa alone without increasing the risk of clinically significant OHSS. Higher luteal-phase serum P and E2 levels after dual trigger may be suggestive of a rescue of partial CL function. In view of the low luteal-phase serum LH levels and the absence of continuous hCG stimulation of the CL after GnRHa trigger, it has been suggested that LH supplementation in the form of adjuvant low-dose hCG at the time of trigger (8) or at oocyte retrieval (20), intermittent low-dose hCG supplementation (21), and luteal-phase recombinant LH supplementation (22) may improve implantation rates. Unfortunately, all of these approaches may potentially increase the risk of OHSS development.

Oocyte Pooling..... The setbacks of LR are the number of oocytes retrieved and suboptimal oocyte maturation, embryo quality and cycle/transfer cancellation rates with respect to age-matched normoresponders (Karande and Gleicher, 1999). LR management is a challenge, as the limited follicular reserve of these patients results in a small cohort of oocytes (Pellicer et al., 1998). A variety of regimens have been employed to optimize the ovarian response of these women, with disappointing results (Garcia-Velasco et al., 2005; Kyrou et al., 2009; Schoolcraft et al., 2008). Moreover, the drop-out rate due to the low probability of becoming pregnant is high, which naturally leads to disappointment and frustration (Verberg et al., 2008). An ideal treatment would provide a similar number of embryos for transfer as that produced by women responding adequately to ovarian stimulation. Therefore, a strategy for obtaining a similar number of oocytes as in normal responders is needed. After ovarian stimulation and oocyte retrieval, mature oocytes were vitrified and stored for use in a subsequent cycle. After an interval of at least one menstrual cycle, a new ovarian stimulation was performed. Oocytes were either vitrified or inseminated together with those from previous cycles that had been vitrified and later warmed. The procedure was repeated in two or more stimulation cycles. The decision about whether to vitrify the oocytes obtained in a given ovarian stimulation cycle or to inseminate those harvested in previous cycles was based on two factors: (i) the total number of oocytes likely to be available after vitrification (estimated survival rate) (Cobo et al., 2008) and the need for a total number of five embryos to be transferred in consecutive cycles (the number needed to reach a 52% cumulative live-birth rate (LBR), a standard situation for normoresponders (Garrido et al., 2010)); and (ii) the patient’s own decision.

Low responderlarda drop out (tedaviyi bırakma) of 482 women underwent 587 stimulation cycles, resulting in a mean per woman of 1.21 cycles (95% CI 1.14–1.28). Drop-out rates were >75% after each attempt. The mean age of patients included in this group was 36.7 years (95% CI 36.4–37.0). First, this study confirmed the high drop-out rate in women with a poor prognosis in a regular assisted reproduction cycle LR management is a challenge, as the limited follicular reserve of these patients results in a small cohort of oocytes (Pellicer et al., 1998). A variety of regimens have been employed to optimize the ovarian response of these women, with disappointing results (Garcia-Velasco et al., 2005; Kyrou et al., 2009; Schoolcraft et al., 2008). Moreover, the drop-out rate due to the low probability of becoming pregnant is high, which naturally leads to disappointment and frustration (Verberg et al., 2008). An ideal treatment would provide a similar number of embryos for transfer as that produced by women responding adequately to ovarian stimulation. Therefore, a strategy for obtaining a similar number of oocytes as in normal responders is needed. A potential alternative to the management of LR is to create a large stock of oocytes by accumulating vitrified metaphase-II oocytes over several stimulation cycles and inseminating them all at the same time. Theoretically, this could help to increase the chances of success by endowing patients with a ‘normoresponder-like’ status. To confirm this hypothesis, it is necessary to evaluate both the incidence of treatment drop-out among LR patients and the outcome of vitrified–warmed supernumerary embryos

The embryo-transfer cancellation rate was approximately 4-fold higher in the LR-fresh group than in the LR-Accu-Vit group. In addition, the mean number of embryos transferred was statistically higher in the LR-Accu-Vit group (P < 0.05). Although the LBR per patient was statistically similar between groups, the cumulative LBR was statistically higher among patients belonging to the LR-Accu-Vit group (36.4% versus 23.7%, P < 0.05). Additionally, no statistical differences were found between the groups with regards to implantation rate or LBR when calculated on a per embryo-transfer basis LBR/patient was higher in the LR-Accu-Vit group, which confirms the efficiency of this method for managing LR patients The positive effects of this strategy are even more evident when cumulative outcome is considered. Larger oocyte/embryo cohorts in LR patients allow the cumulated success rate after cryotransfer of surplus cryopreserved embryos to be increased.

The protocol is based on initial stimulation by letrozole 2 The protocol is based on initial stimulation by letrozole 2.5 mg/d and clomid 25 mg/d from the second day of the cycle, for the first 4 days. On day 6, the letrozole is being replaced with hMG, 150 IU/d every second day while the clomid continue. Monitoring is done with ultrasound and estrogen serum concentration to determine the day of GnRH-agonist administration, which induces final stage of oocyte maturation to be followed by egg collection. All embryos developed are being cryopreserved. The regimen for the second stimulation, which is started 2-3 days after the first egg collection, is started with letrozole 2.5mg and HMG 225IU every day. After 7-8 days of stimulation an ultrasound examination is performed to follow the treatment. When the leading follicle reach the diameter of 14 mm letrozole is being stopped while hMG still continue in a dose of 225 IU/d. Final oocyte maturation is again being stimulated by GnRH agonist (triptoreline 0.1mg), to follow by egg collection 32-36 h following the drug administration. Again, all available embryos are being cryopreserved.

Faydalar DOR hastalarında yüksek drop out oranı Tekrarlayan başarısızlıkların psikolojik stresi Daha az transfer iptali Daha yüksek kümülatif gebelik oranı Although at first glance it would appear that this strategy does not involve any clinical advantage, it does bring two remarkable benefits related to the previously discussed drop-out rate in the LR population and is sure to palliate the psychological distress caused by repeated failures. This should bring these patients closer to achieving their goal of a healthy new-born without having to resort to using donated gametes