Clinical Study Of Dual Trigger Of Oocyte Maturation With GnRH Agonist And Individualizing Dose Of HCG For Ovarian Normal Responders In GnRH Antagonist Cycles

Nowadays, assisted reproductive technology (ART) is the most effective treatment for infertility. Controlled ovarian stimulation (COS) is the key to the success of ART, which induces multi-follicle development and obtain more embryos for transfer per cycle by using ovulation stimulants. The application of COS greatly improves the clinical pregnany rate of ART.Gonadotropin-releasing hormone agonist (GnRHa) protocol is the current, most widely used in our contry, due to its high and stable pregnancy rate. While GnRH antagonist protocol has progressively replaced GnRHa protocol in Europe and America. Compares to GnRHa protocol, GnRH antagonist protocol has many advantages, such as a lower dosage of Gonadotropin (Gn), a shorter duration of therapy, a greater flexibility and so on. What’s more, it can reduce the risk of developing ovarian hyperstimulation syndrom (OHSS), a potential iatrogenic cause of death in otherwise healthy women undergoing fertility treatment with COS, and reduce their physical pain, psychological distress and financial burden. At present, there is still lack of clinical practice experience in application of GnRH antagonist in our country. The inferior clinical pregnancy rate of GnRH antagonist protocol has affect its wide application in clinical.In conventional GnRH antagonist cycles, human chorionic gonadotropin (hCG) is usually administered as a surrogate luteinizing hormone (LH) surge for final oocyte maturation. However, hCG is the predominant cause of OHSS. GnRH agonist induces an initial activation (flare-up) of gonadotrophins by activating the GnRH receptor, and has been suggested as an alternative trigger of ovulation instead of hCG. Contrasts to hCG, GnRHa is effective in the prevention of OHSS, due to shorter half life of LH elicited by GnRHa administration. However, GnRHa trigger do markedly detrimental impact on corpus luteal function and the capacity of the endometrium, resulting in disappointing reproductive outcome of fresh embryo transfer. Therefore, some scholars proposed the concept of a "dual trigger" that combines a single bolus of GnRH agonist with hCG at the time of triggering. On one hand, GnRH antagonist protocol is characterized by follicular inhomogeneity, which is different from GnRHa protocol. The administration of GnRHa can promote the follicular development and maturation by inducing follicle stimulating hormone (FSH)/LH surge. On the other hand, the administration of hCG may resuce luteal phase function and then improve the clinical outcome. Several studies focusing on high responders have demonstrated that dual trigger with GnRHa trigger and low-dose hCG can effectively minimize the risk of OHSS and improve clinical outcome[1-4]. For normal responders, can dual trigger optimize clinical outcome? Few studies were reported so far. Among them, three researches demonstrated significantly improved pregnancy rates and live birth rate for the dual trigger group compared with hCG trigger group[5-7]. In contrast, Decleer et al. found that pregnancy rate in the dual trigger group was lower than that obtained in the group triggered by hCG alone[8]. However, the difference was not statistically significant. The standard dose of hCG for normal responders is 5000IU～10000IU. The important point that needs to be emphasized is that the inherent risk of OHSS associated with hCG triggering, even though normal responders are not at high risk for OHSS.Thus, we aim to investigate whether dual trigger of final oocyte maturation with a combinantion of GnRHa and standard dosage of hCG can optimize clinical outcome in normal responders in GnRH-antagonist cycles, and explore the effects of dual trigger with GnRHa and individualizing dose of hCG, so as to reduce the risk of developing OHSS and improve clinical outcome, and to give the basis of management in clinics.Part I Comparison of clinical outcome between dual trigger of oocyte maturation with gonadotropin-releasing hormone agonist and standard dose human chorionic gonadotrop and hCG trigger alone for ovarian normal responders in GnRH-antagonist cycles[Objective]To investigate whether dual triggering of final oocyte maturation with a combination of gonadotropin-releasing hormone (GnRH) agonist and standard dosage of human chorionic gonodotropin (hCG) can optimize clinical outcome in normal responders in GnRH-antagonist in vitro fertilization/intracytoplasmic sperm injection (IVF/ICSI) cycles.[Materials and Methods]1. Study Design and Study ParticipantsA prospective non-randomized clinical study about dual trigger with GnRHa agonist and hCG was performed between January 2015 and December 2015 at a university-affiliated center (Center for Reproductive Medicine, Nanfang Hospital, Southern Medical University, Guangzhou, China). So we chose those with hCG trigger in the same period as control group for the retrospective case-control study. 325 patients that fulfilled following criteria were retrospectively enrolled in a consecutive manner:(1)GnRH-antagonist cycles; (2) dual triggering of final oocyte maturation with a combination of GnRHa and 5000-10000IU hCG, or triggering with 5000～10000IU hCG alone; (3)normal response to controlled ovarian stimulation (COS):a serum estradiol (E2) level greater than 500pg/ml and less than 4000 pg/ml, and the number of retrieved oocytes 4-20; (4)age≤ 40years; (5)body mass index (BMI)>18kg/m2 and<30 kg/m2. Exclusion criteria:(1) poor ovarian response:a serum E2 level less than 500pg/ml on the day of triggering, or the number of retrieved oocytes≤3; (2)high ovarian response:a serum E2 level greater than 4000pg/ml on the day of triggering, or the number of retrieved oocytes>21; (3)day-3 follicle stimulating hormone (FSH) concentration of>10IU/L; (4)more than three IVF/ICSI attempts; (5)more than six embryos transferred; (6)unnormal uterus and endometrium; (7)presence of endocrine disorders.2. Ovarian Stimulation ProtocolsOvarian stimulation was initiated on Day 2 of withdrawal bleeding with recombinant FSH or highly purified FSH. The gonadotrophin (Gn) started at 75-375IU per day for 4 to 5 days according to age, antral follicle count (AFC), BMI, day-3 FSH concentration and previous ovarian response. Thereafter, the dose was adjusted on the basis of serum E2 and follicular growth, monitored by serial transvaginal ultrasound. GnRH-antagonist of 0.25 mg SC was commenced once at least one of the following criteria was fulfilled:(1) presence of at least one follicle measuring 14 mm;(2)serum E2 levels> 600 pg/ml; (3)serum LH levels> 10 IU/L. GnRH antagonist was continued until the day for triggering final oocyte maturation. When at least two follicles reached 18 mm in diameter or three follicles reached 17 mm in diameter, individualized trigger agent was administered for final oocyte maturation according to the number of growing follicles and serum E2 level on trigger day. Trans-vaginal ultrasound-guided oocyte retrieval was performed 35-36 hours after trigger3. Outcome Variables and Statistical AnalysisAccording to trigger agent selection, patients were classified as dual trigger group (received GnRHa in addition to standard dose hCG, n=224) and hCG trigger group (received standard dose hCG alone, n=101). Number of oocytes retrieved, number of mature metaphase Ⅱ (Mil) oocytes, number of embryos obtained, number of top quality embryos obtained, implantation rate, clinical pregnancy rate and the OHSS incidence were calculated. SPSS (version 16.0) was used for analysis. Continuous variables were presented as mean with standard deviation (SD). Proportions were compared with χ2 test and continuous variables were compared with Student’s t-test or Mann-Whitney for independent samples depending on the normality of their distribution. P<0.05 was considered statistically significant.[Results]1. As for baseline characteristics, statistically significant difference in age and AFC was observed between dual trigger group and hCG trigger group (32,06±4.26 vs 33.50±3.89; 13.30±5.85 vs 9.33±4.29). No significant difference in BMI and basic sexual hormone concentration were observed between two groups2. Compared with hCG trigger group, patients in dual trigger group presented a significant lower initial dose of Gn, total Gn dosage and serum E2 level on the day of triggering (232.23±65.11 vs 294.10±78.88,2367.95±623.06 vs 2818.56±901.43,1763.99±646.49 vs 1994.69±737.60). The mean number of retrieved oocytes was similar between two groups, while the mean number of mature oocytes,2PN, embryos obtained and top quality embryos were both statistically significantly greater in the dual trigger group (6.65±3.61 vs 5.90±3.29,5.86±2.97 vs 4.95±2.35,4.16±2.54 vs 3.38±1.76,2.81±2.21 vs 2.29±1.55).3. In terms of clinical outcome, compared with hCG trigger group, dual trigger group demonstrated higher implantation and clinical pregnancy rates (43.4% vs 36.3%,65.1% vs 52.6%, respectively). However, after adjustment for age, there were no significant differences. In addition, there was one case of severe OHSS in hCG trigger group, none occurred in the dual trigger group.[Conclusion]1. Compared to hCG trigger of oocyte maturation, dual trigger with GnRHa and standard-dose hCG in normal responders improves number of mature oocytes retrieved, embryos obtained, and top quality embryos.2. Dual trigger shows a increasing trend in implantation rate and clinical pregnancy rates in GnRH-antagonist cycles.3. The antagonism between GnRHa and hCG may play an important pole in reducing risk of OHSS. However, the inherent risk of OHSS consist in those patients triggered with a standard dose hCG for oocyte maturation, even though the patient in our study did not profile of a high-risk population for incurring OHSS. Therefore, further research focusing on the minimal effective dose of hCG for normal responders is needed.Part Ⅱ Effects of triggering oocytes maturation by GnRHa and individualizing dose of hCG on the outcome of IVF-ET treatment for ovarian normal responders in GnRH-antagonist cycles[Objective]To explore the effects of dual trigger of oocyte maturation by GnRHa and individualizing dose of hCG on the outcome of IVF-ET treatment in ovarian normal responders in GnRH-antagonist cycles, so as to reduce the risk of developing OHSS and improve clinical outcome, and to give the basis of management in clinics.[Materials and Methods]1. Study Design and Study ParticipantsA prospective cohort study was performed between January 2015 and December 2015 at a university-affiliated center (Center for Reproductive Medicine, Nanfang Hospital, Southern Medical University, Guangzhou, China).273 patients that fulfilled following criteria were retrospectively enrolled in a consecutive manner: (1)GnRH-antagonist cycles; (2)dual trigger:a combination of GnRHa and hCG for oocyte maturation; (3)normal response to controlled ovarian stimulation (COS):a serum estradiol (E2) level greater than 500pg/ml and less than 4000 pg/ml, and the number of retrieved oocytes 4-20; (4)age≤ 40years; (5)body mass index>18kg/m2 and<30 kg/m2. Exclusion criteria:(1) poor ovarian response:a serum E2 level less than 500pg/ml on the day of triggering, or the number of retrieved oocytes≤3; (2)high ovarian response:a serum E2 level greater than 4000pg/ml on the day of triggering, or the number of retrieved oocytes≥21; (3)day-3 follicle stimulating hormone (FSH) concentration of>10IU/L; (4)more than three IVF/ICSI attempts; (5)more than six embryos transferred; (6)unnormal uterus and endometrium; (7)presence of endocrine disorders.2. Ovarian Stimulation ProtocolsThe same with Part Ⅰ.3. Outcome Variables and Statistical AnalysisAccording to the number of growing follicles and serum E2 level on trigger day, patients were divided into three group and administered by a combination of GnRHa and individualizing dose of hCG:(1) lower resonse group (Group A, n=49, GnRHa+low-dose hCG, including 3 cases of 1000IU,1 case of 1500IU,34 cases of 2000IU,1 case of 3000IU and 10 cases of 4000IU); (2)moderate response group (Group B, n=119, GnRHa+5000/6000IU Hcg, including 116 cases of 5000IU and 3 cases of 6000IU); (3) higher response group (Group C, n=105, GnRHa+10000IU hCG). Percent of oocytes retrieved, percent of mature oocytes, normal fertilization proportion (defined as number of nomally fertilized oocytes divided by the number of oocytes retrieved, including both IVF and ICSI cycles), implantation rate, biochemical pregnancy rate, clinical pregnancy rate and the OHSS incidence were calculated. The statistical analysis is the same with Part One.[Results]1. The mean dose of hCG in three group was 2357.14±889.761U,5025.21±157.43IU, 10000±0.00IU, respectively. Group A demonstrated a statistically significantly lower age (29.20±3.74), middle in Group B (31.17±4.28), and higher in Group C (33.05±4.02). In terms of AFC, Group A is the highest (19.82±6.53), followed by Group B (14.55±5.40), Group C is the lowest (11.91±6.01). No significant difference was observed within three groups regarding baseline characteristics, except age and AFC.2. Group A demonstrated a statistically significantly lower initial dose of Gn and total Gn dosage, middle in Group B, and higher in Group C (178.83±58.25 vs 214.92±65.45 vs 252.02±58.56,1855.31±752.04 vs 2213.31±632.43 vs 2543.21±565.88). No significant difference in days of Gn and GnRH antagonist were observed within three groups. The number of follicle≥ 10mm on trigger day, serum E2 level on trigger day and next trigger day successively decreased in three groups, while serum hCG level successively increased with the increased dose of hCG administered. All the above were significantly different. In terms of mean number of retrieved oocytes, Group A is the highest, followed by Group B, and Group C is the lowest. However, Group A demonstrated a statistically significantly lower percent of oocytes retrieved than other two groups (63.1% vs 74.5% vs 73.4%), and Group C demonstrated a statistically significantly lower percent of mature oocytes retrieved (81.7% vs 82.2% vs 68.8%). There was no significant difference in fertilization proportion (defined as number of normally fertilized oocytes (2PN) divided by the number of oocytes retrieved, both IVF and ICSI cycles included) within three groups.3. No significant difference were observed in endometrial thickness and number of embryos transferred. The number of top quality embryos transferred in Group B was higher than Group C, with a significant difference (1.89±0.65 vs 1.51±0.87). The implantation rate was similar within three groups. In respect of clinical pregnancy rate, Group B is the highest, followed by Group C, and Group A is the lowest, with no significant difference(67.4% vs 61.2% vs 51.6%). No moderate-to-severe OHSS occurred in three groups.(Conclusion]1. For normal responders in GnRH-antagonist cycles, dual trigger of oocyte maturation with GnRHa and low-dose hCG (1000-4000IU) decreased percent of oocytes retrieved, but it had no adverse effect on oocytes maturity rate and normal fertilization proportion. While percent of mature oocytes was lowest in patients with GnRHa and 10000IU hCG, which was related to the older age and poor ovarin response.2. Dual trigger with a combination of GnRHa and individualizing dose of hCG, according to the number of growing follicles and serum E2 level, effectively eliminated the incidence of OHSS and obtained favorable pregnancy outcomes. However, it showed decreasing trend in pregnancy rate in patients triggered with GnRHa and low-dose hCG.3. Dual trigger with GnRHa and 5000IU hCG may be the optimal trigger agent selection for normal responders in GnRH-antagonist cycles, and increase dose of hCG can not improve clinical pregnancy rate. However, for those patients with a relatively increasing risk of developing OHSS, a reduced hCG dose should be required according to the circumstances.