GnRH agonist trigger fresh embryo transfers have similar outcomes but a lower dropout rate than freeze-all strategy: a retrospective analysis

doi:10.21203/rs.3.rs-4949899/v1

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Research Article

GnRH agonist trigger fresh embryo transfers have similar outcomes but a lower dropout rate than freeze-all strategy: a retrospective analysis

https://doi.org/10.21203/rs.3.rs-4949899/v1

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Background: To compare pregnancy and delivery rate of the first fresh versus the first freeze all warming embryo transfer following a GnRH antagonist stimulation protocol with a GnRH agonist trigger. The secondary objective was to evaluate the dropout rate and the safety of the two approaches.

Methods: A cohort retrospective study, set in a single university tertiary care fertility center. This study included all the first embryo transfers derived from oocyte retrievals performed between 01/01/2012 and 31/12/2020, following 1,396 GnRH antagonist induction cycles with GnRH agonist trigger. A total of 429 fresh (group A) and 967 warming embryo transfers (group B) were included.

Results: The pregnancy and delivery rate resulted comparable in the two groups (Group A: 41.72%; Group B: 42.92%, p 0.678 and Group A: 35.43%; Group B: 33.20%, p 0.415, respectively). The only significant differences recorded were an increased miscarriage rate in Group B (p=0.008) and an increased multiple birth rate (p<0.001) and a lower dropout rate (p 0.010) in Group A.

Conclusion: GnRH antagonist stimulation protocol with GnRH analogue trigger has been demonstrated to be an effective and safe technique. In addition, a subsequent fresh embryo transfer still represents a valid therapeutic option for physicians providing the desired outcomes in the first embryo transfer, with a lower dropout rate than the freeze-all strategy.

ClinicalTrials.gov registration number: NCT05362734, retrospectively registered on 11^rd May 2022

GnRH antagonist stimulation

fresh embryo transfer

freeze all

GnRH agonist trigger

dropout rate

A severe iatrogenic complication associated with assisted reproduction technology (ART) is the ovarian hyperstimulation syndrome (OHSS) that consists in an exaggerated response to ovarian stimulation (OS) [1–4]. The first action physicians can take in reducing the occurrence of OHSS is prevention, and there are several options to achieve this result. Firstly, the correct identification of “high risk” patients [5–9] whose characteristics are usually young age (< 35 years), high ovarian reserve, polycystic ovary (PCO) appearance at ultrasound (US) with or without the biochemical and clinical characteristics of polycystic ovary syndrome (PCOS), history of high response in a previous ART cycle and low body mass index (BMI) [2, 4–5, 9–10]. In order not to develop OHSS in these “high risk” patients, the strategy may be the use of gonadotropin hormone-releasing hormone (GnRH) antagonist protocol that was introduced for OS in clinical practice with the aim of inhibiting a premature rise in luteinizing hormone (LH), which could lead to premature luteinization and arrest in the maturation of follicles [2, 8, 11–13]. If the GnRH antagonist protocol is used, final oocyte maturation can be achieved using GnRH agonist (GnRHa) as trigger. The combination of antagonist protocol with agonist trigger carries potential advantages including reduced vascular endothelial growth factor (VEGF) production and consequently decreased risk of OHSS [2, 8, 11, 12–13]. Therefore, the use of GnRHa as trigger has become extremely common as it increases the safety of ART by consistently decreasing the risk of OHSS [5, 9, 11, 13–14]. Notwithstanding the potential advantages brought by GnRHa trigger, the first experiences led to increased early pregnancy loss and reduced ongoing pregnancy rate (OPR) [15], probably due to an inferior number of retrieved oocytes, poorer oocyte competence or early luteolysis and consequent insufficient luteal phase [11, 15–16]. To reduce the occurrence of such drawbacks several studies have focused on the importance of the luteal phase support (LPS) to sustain implantation after GnRHa trigger [15]. Despite initial unsatisfactory results, it is now believed that triggering final oocyte maturation with GnRHa is non inferior in terms of reproductive outcomes to trigger with human chorionic gonadotropin (hCG) when support to the luteal phase with estradiol plus progesterone is applied by adding also low dose of hCG either at the time of GnRH agonist trigger (dual trigger) or at the time of oocyte retrieval (rescue protocol) [11, 13, 17–18]. A further option for preventing OHSS is the cryopreservation of all obtained embryos and oocytes, with the so called “freeze-all” technique [2]. Such an approach is gaining favor over time as it also helps in selecting the highest quality embryos for transfer in subsequent cycles, due to the prolonged embryo culture until the blastocyst stage [13]. The debate regarding which is the best ART protocol in terms of oocyte quality, pregnancy outcomes and reduced occurrence of complications such as OHSS is still open also because data on these outcomes specifically evaluated at the first embryo transfer (ET) are still scarce. With the aim of contributing to answer some of those questions, the objective of this retrospective observational study was to evaluate in clinical practice primarily the effectiveness, in terms of pregnancy and delivery rate, and secondarily other pregnancy outcomes and safety of GnRH agonist trigger in GnRH antagonist stimulation protocols comparing the first ETs in two different approaches, i.e. with a fresh ET following the OS, after dual triggering or rescue protocol, or a frozen-thawed ET, after freeze-all strategy.

Study population

This was a retrospective observational single-center study. The dataset included all the first ETs derived from oocytes retrievals performed between 01/01/2012 and 31/12/2020 at Humanitas Fertility Center in Rozzano (Milan) after GnRH antagonist protocol stimulation and trigger with GnRH agonist. Cycles in which freeze-all strategy was performed for pre-implantation genetic testing (PGT), oncological banking or deferring motherhood preferences and cycles performed with gametes obtained in other fertility centers were excluded from the study. Patients could authorize for the cryopreservation of either oocytes, embryos obtained by fertilization or both; otherwise, they could choose not to cryopreserve any gamete or embryo. Cycles have been divided in two groups according to the modality of first embryo transfer: cycles in which fresh ET was performed first, after a dual triggering or a rescue protocol (Group A) and cycles where a frozen-thawed ET was performed first, after being chosen a freeze-all strategy (Group B). The risk of OHSS development according to a pre-defined standard assessment drove physicians’ decision to perform either fresh ET (moderate risk) or freeze-all strategy (severe risk). As a supplementary study, pregnancy and safety outcomes were evaluated also in the completed cycles, i.e. including not only the first ET, but all the subsequent cryopreserved ETs resulting from that stimulation cycle. In this case, patients might have been enrolled more than once and in both groups if they performed more than one ART cycle in the study period. ART cycles where the couple had not exhausted yet the entire pool of cryopreserved embryos or oocytes, excluding the ones that had obtained a live birth, were excluded.

Intervention description

Induction protocols were defined on the basis of international guidelines (ESHRE 2011) [19] and the dosage of gonadotropins was prescribed on the basis of patient’s antral follicular count (AFC), anti-Mullerian Hormone (AMH) and body mass index (BMI). Ultrasound monitoring was performed on day 5–6 of menstrual cycle concomitantly to the start of administration of GnRH antagonist (Cetrorelix/Ganirelix), which was given subcutaneously at a dose level of 0.25 mg/day until the day of the trigger. Trigger was performed when at least 2 or 3 follicles reached a diameter ≥ 18 mm, and the decision on the approach to be used was taken considering the total number of follicles and their diameter:

Number of follicles < 25 with diameter > 12 mm ◊ GnRHa (Triptorelin 0.2 mg, Fertipeptyl Ferring S.p.a or Decapeptyl Ipsen, 2 vials subcutaneously 34 hours before oocyte retrieval) + u-hCG (1,500 IU, Gonasi HP, Ibsa) administered the day of the trigger (dual trigger) or in the operating room the day of oocyte retrieval (rescue protocol).
Number of follicles > 25 with diameter > 12 mm à GnRHa (Triptorelin 0.2 mg, 2 vials subcutaneously 34 hours before oocyte retrieval) and elective cryopreservation of the embryos and oocytes.

Independently of the number of follicles, if the number of oocytes retrieved was > 20, elective cryopreservation of the embryos and oocytes was always performed. Furthermore, the dual trigger, in which a dose of 1,500 IU of u-hCG is administered together with Triptorelin 0.2 mg, was prescribed in patients in which a basal LH level < 1 mIU/mL predicted a scarce retrieval of oocytes. The ET procedure refers to the intrauterine transfer of one or more embryos under transabdominal ultrasound guidance from day 2 to 6 after oocyte retrieval. In case of fresh embryo transfer, if the patient had already received hCG at the time of GnRHa trigger (dual trigger), LPS was performed, by administering either Micronized Progesterone (Prometrium, Rottapharm S.p.a., or Progeffik, Effik S.p.a.) 200 mg x2/day or Progesterone Gel (Crinone 8%, Merk Serono) 80 mg x2/day, together with Estradiol Valerate (Progynova, Bayer AG) 2 mg x2/day. If the patient did not receive 1,500 IU of u-hCG at the time of trigger, it was administered on the day of oocyte retrieval (rescue protocol) together with either Micronized Progesterone 200mg x2/day or Progesterone Gel 80 mg x2/day, and Estradiol Valerate 2 mg x2/day. In patients at high risk of OHSS embryo transfer was suspended and both obtained embryos and extra oocytes were cryopreserved. In these patients cabergoline (Dostinex, Pfizer) was administered via vaginal route, with the dosage of 1 mg/day for 7 days. Also, high-protein and low-sodium diet was advised together with the intake of at least 1,500 ml of liquids every day.

Primary Outcomes

The primary outcome of the present study was to compare the ongoing pregnancy and delivery rate [20] of the first ET in GnRH antagonist stimulation protocol triggered with a GnRH agonist, in case of a subsequent fresh embryo transfer or a freeze-all strategy. For ongoing pregnancy rate the number of viable pregnancies that had completed at least 12 weeks of gestation on the total number of ETs performed was considered [21]. For delivery rate the number of deliveries of one or more live births over the total performed ET was considered. A live birth was defined as the complete expulsion or extraction from a woman of a product of fertilization, after 22 completed weeks of gestational age which, after such separation, breaths or shows any evidence of life, such as heartbeat, umbilical cord pulsation or definite movement of voluntary muscles, irrespective of whether the umbilical cord has been cut or the placenta is attached. It includes deliveries that resulted in the birth of one or more live births and/or stillbirths. The delivery of a singleton, twin or other multiple pregnancy is registered as one delivery. If more than one newborn is delivered, it is often recognized as a delivery with multiple births [20].

Secondary Outcomes

The secondary outcomes were the other pregnancy outcomes, the dropout rate and the safety, in terms of severe OHSS occurrence, of the two approaches. As for ectopic pregnancy rate, it was considered as the number of pregnancies outside the uterine cavity, diagnosed by ultrasound, surgical visualization or histopathology [20, 22–23] expressed per 100 pregnancies. As for miscarriage rate, it was considered as the number of spontaneous losses of an intra-uterine pregnancy prior to 12 completed weeks of gestational age expressed per 100 pregnancies. As for dropout rate, it was considered as the number of cycles in which any live birth (from either ART and/or spontaneous conceive) was not achieved and still some embryos or oocytes had to be thawed. At last, an OHSS was considered severe if it led to hospitalization. Hospitalization took place if a systemic response characterized by severe abdominal discomfort and/or other symptoms of ascites, hemoconcentration (hematocrit > 45%) and/or other serious biochemical abnormalities had occurred, as a result of ovarian stimulation interventions [2, 20]. The temporal criterion allows further identification of two OHSS classes [2, 5]: early onset OHSS when symptoms appear < 10 days after hCG administration and late onset OHSS when symptoms appear in a timeframe ≥ 10 days after oocyte retrieval, as a consequence of the hCG produced by the embryonic trophoblast at the time of implantation. As a supplementary analysis, the following parameters were evaluated, on the total cycles started during the study period in which the exhaustion of the entire pool of cryopreserved oocytes occurred: the time to first live birth, defined as months from the start of the stimulation protocol until the first live birth; the cumulative pregnancy rate defined as the number of cycles resulting in at least one clinical pregnancy i.e. a pregnancy diagnosed by ultrasonographic visualization of one or more gestational sacs or definitive clinical signs of pregnancy, expressed per 100 cycles [20] and the cumulative delivery rate, defined as the number of deliveries with at least one live birth resulting from one cycle expressed per 100 cycles, including all cycles in which fresh and/or frozen embryos were transferred, until one delivery with a live birth occurred or until all embryos were used, whichever occurred first [24].

Statistical Analysis

Data were described as number and percentage, if categorical, or mean and standard deviation, if numerical. Median and range have been used whenever continuous variables distribution was significantly different form normal. Differences between groups were explored with chi square test if the variable was categorical, or with Mann Whitney test, if the variable was numerical. In the supplementary analysis that included all started cycles, time to first live birth, described by Kaplan Meier curve, and survival median, defined as the time where the survival curve reached 50%, were also explored. Association of variables with cumulative delivery rate were explored with logistic regression analysis, and results were expressed as odds ratio (OR) with 95% confidence interval (95% CI). The potential contributing factors for the logistic regression included: female and male partner’s age, duration of infertility, female BMI, female active smoking, the presence of primary infertility, previous deliveries, previous abortion, ovarian reserve (expressed as Follicle Stimulating Hormone (FSH) and AMH) and the main indication for treatment. The program used for data-analysis was STATA 17.0 (StataCorp. 2021. Stata Statistical Software: Release 17. College Station, TX. StataCorp LLC). Statistical significance was set at p < 0.05.

Ethical Approval

and Data Protection

The Humanitas Ethical Committee approved the study protocol (n. 21/18) on 18th September 2018, and the protocol was registered on ClinicalTrials.gov (ID NCT05362734) on May 11th, 2022. Data were collected by a specific internal web-based database, which allowed to organize, store, and readily retrieve information about patients and ART cycles. Patients’ data were protected by an advanced threat prevention system, requiring a periodical need of password renewal. Patients included in the study signed a written consent for the usage of their medical records for research purpose and publication of data, being assured that their privacy and anonymity were protected.

Between the 1st of January 2012 and the 31st of December 2020, a total of 1,543 ART cycles performed with GnRH antagonist stimulation and GnRH agonist as trigger were eligible. As shown in Fig. 1, a total of 1,396 ART cycles were eventually enrolled in the present study, 429 in Group A and 967 in Group B. Table 1 reports the descriptive statistics for the baseline characteristics of cycles belonging to the total population, to Group A and B. The variables reported in Table 1 have been introduced in the model used for the primary analysis of this study with the aim of correcting for potential confounders. The analysis showed that the two groups were extremely homogenous, being only level of AMH and basal AFC count significantly different between the two groups. Table 1 also reports the retrieval results, including the characteristics of retrieved oocytes and obtained embryos in the two groups and describes oocyte quality expressed as MII rate in the two groups.

Table 1

Sample population characteristics and characteristics of retrieved oocytes and obtained embryos in the two groups.
Variable	Total	Group A	Group B	p
Total N. of Started Cycles	1,396	429	967
Female Age at Oocyte Retrieval (years)	34.1 ± 4.1	34.0 ± 4.3	34.1 ± 4.1	0.870
Male Age (years)	38.1 ± 5.7	38.0 ± 5.4	38.1 ± 5.8	0.903
Duration of Infertility (years)	4.0 ± 2.3	4.0 ± 2.4	4.1 ± 2.3	0.363
Female BMI (kg/m²)	21.9 ± 3.1	22.0 ± 3.2	21.8 ± 3.1	0.654
Female Active Smoking	264 (18.91%)	85 (19.81%)	179 (18.51%)	0.566
Primary Infertility	945 (67.69%)	283 (65.97%)	662 (68.46%)	0.358
Previous Deliveries	138 (9.89%)	41 (9.56%)	97 (10.03%)	0.784
Previous Abortion	374 (26.79%)	109 (25.41%)	265 (27.40%)	0.437
FSH (mUI/ml)	6.3 ± 1.8	6.4 ± 1.9	6.3 ± 1.8	0.286
AMH (ng/ml)	5.7 ± 3.8	4.7 ± 2.7	6.2 ± 4.2	< 0.001
Basal AFC	22.4 ± 11.6	18.2 ± 9.0	25.7 ± 12.3	< 0.001
Retrieval results
Retrieved Oocytes	18.4 ± 6.5	13.6 ± 3.8	20.6 ± 6.3	< 0.001
MII Rate	0.75 (0–1)	0.78 (0–1)	0.75 (0–1)	0.002
Inseminated / Injected Oocytes	10.3 ± 3.5	9.1 ± 2.7	10.8 ± 3.6	< 0.001
Fertilized Oocytes	7.7 ± 3.1	6.7 ± 2.6	8.1 ± 3.2	< 0.001
Cryopreserved Oocytes	4.1 ± 5.3	1.4 ± 2.9	5.2 ± 5.7	< 0.001
Cryopreserved Embryos	2.7 ± 1.9	1.5 ± 1.6	3.3 ± 1.7	< 0.001
Embryos Transferred per Procedure	1.5 ± 0.6	1.8 ± 0.5	1.3 ± 0.4	< 0.001
Blastocyst Stage Transferred (%)	61.1 ± 43.1	37.1 ± 40.0	71.7 ± 40.1	< 0.001

BMI = body mass index, FSH = follicle stimulating hormone, AMH = anti-Müllerian hormone, AFC = antral follicle count. Data is mean ± Standard Deviation for continuous variables and number (percentage) for categorical variables; the variable “MII rate” is shown as median (range).

In Table 2 group A and B were compared for the pregnancy outcomes and delivery distribution. The difference between the two groups in ongoing pregnancy rate (179 (41.72%) in Group A vs 415 (42.92%) in Group B) and delivery rate (152 (35.43%) in Group A vs 321 (33.20%) in Group B) appeared to be not significant, being the p values respectively 0.678 and 0.415. As for the other secondary pregnancy outcomes, ectopic pregnancy rate in Group A was 1.11%; while in Group B it was 0.24% out of a total of 3 events (p = 0.217). Miscarriage rate in Group A was 11.17% while 20.24% in Group B out of a total of 104 events (p = 0.008). The dropout rate resulted significantly higher in Group B (7.03% vs 3.50%, p = 0.010).

Table 2

Pregnancy outcomes and delivery distribution per pregnancies in each group: total number, (percentages) and p-values.
	Total	Group A	Group B	p
Total N. of Cycles	1,396	429	967
Ongoing pregnancies (% of total N. of started cycles)	594 (42.55%)	179 (41.72%)	415 (42.92%)	0.678
Delivery (% of total N. of started cycles)	473 (33.88%)	152 (35.43%)	321 (33.20%)	0.415
Ectopic Pregnancy Rate	3 (0.51%)	2 (1.11%)	1 (0.24%)	0.217
Miscarriage rate < 12w	104 (17.51%)	20 (11.17%)	84 (20.24%)	0.008
Lost to follow up	1 (0.17%)	0	1 (0.24%)
Delivery Distribution				< 0.001
Singleton	434 (89.64%)	119 (78.29%)	305 (95.02%)
Twins	47 (9.94%)	32 (21.05%)	15 (4.67%)
Triplets	2 (0.42%)	1 (0.66%)	1 (0.31%)
Dropout	83 (5.95%)	15 (3.50%)	68 (7.03%)	0.010

Lastly, the analysis aimed at investigating if episodes of severe OHSS had a different distribution in the two groups over the total number of stimulation cycles started, as reported in Table 3. Table 3 also shows the distribution of the latter cases concerning the temporal criterion of onset.

Table 3

OHSS incidence per stimulation cycle in each group: total number, (percentages) and p-values.
	Total	Group A	Group B	p
Total N. of Started Cycles	1,396	429	967
Severe OHSS	19 (1.36%)	5 (1.17%)	14 (1.45%)	0.805
Early Onset OHSS	15 (78.95%)	2 (40.00%)	13 (92.86%)
Late Onset OHSS	4 (21.05%)	3 (60.00%)	1 (7.14%)
OHSS = Ovarian Hyperstimulation Syndrome

Supplementary Fig. 1 shows, on the total started cycles, the time to first live birth, defined as months from the start of the stimulation protocol, until the first live birth, as a Kaplan-Meier curve. Supplementary Table 1 reports the results regarding cumulative pregnancy and cumulative delivery rates over the total of 1,185 cycles started during the study period, in which the exhaustion of the entire pool of cryopreserved oocytes occurred. Moreover, a subsequent evaluation was focused on identifying possible characteristics that could have influenced the outcome of the above supplementary analysis. Supplementary Table 2 represents the results of the logistic regression analysis, performed with this purpose.

Our data confirmed that GnRH antagonist protocol with analogue trigger represents a valid and safe therapeutic option for women considered more at risk of developing severe OHSS. The present study improves clinicians’ awareness when choosing between a fresh ET or a freeze-all strategy. Indeed, it showed that for women in which a fresh ET is performed firstly following the OS, after a dual triggering or a rescue protocol (Group A), such an approach can be easily adopted as it guarantees the same efficacy, in terms of pregnancy and delivery rate, and safety as a first frozen ET, in case of freeze-all strategy, but with a lower dropout rate. The modality of first embryo transfer (fresh versus frozen in case of freeze-all strategy) determined in this study the allocation of performed cycles in two groups, which resulted to be very homogeneous in terms of baseline characteristics of the population. As seen in Table 1, in fact, the only variables that resulted to be significantly different were the AMH value and the basal AFC (p < 0.001). In particular, they were found to be significantly higher in Group B. AMH and AFC are both related to the ovarian reserve and both directly correlate with the number of oocytes retrieved after ART procedures [25], which was one of the parameters taken into account for addressing patients to a freeze-all strategy due to the high risk of OHSS. On the other hand, oocytes and embryos characteristics resulted to be significantly different between the two groups (p < 0.001). In particular, the number of oocytes retrieved, inseminated or injected, fertilized and cryopreserved, and the number of embryos cryopreserved resulted to be higher in Group B versus Group A. This difference could be explained by the inclusion criteria of the groups: Group B comprises patients that, for Humanitas Fertility Center’s protocol, are candidates to elective embryo cryopreservation (freeze-all strategy) as an additional preventive tool to avoid the occurrence of OHSS. As said before, one of the characteristics for adopting such strategy is the number of oocytes retrieved (more than 20), and therefore the higher values observed in Group B for oocytes and derived embryos can be justified. The difference in the number of cryopreserved oocytes and embryos observed in the study can be also explained considering the Italian Legislation. Although less restrictive as compared to the original formulation, the current law is still limiting the total number of oocytes that can be injected or inseminated for each ART cycle. In May 2009, indeed, the Italian Constitutional Court modified the law regulating ART that had been approved in 2004 by the Italian Parliament. Some of the initial restrictions were removed, in particular allowing an individualized definition of the optimal number of embryos to be obtained to give the best chance of achieving a pregnancy while limiting the number of cryopreserved embryos to the minimum feasible [26]. As a consequence, it is not surprising that the number of cryopreserved embryos is higher in Group B considering that, in fresh transfer, some of the obtained embryos are immediately used. A variable that was found to be higher in Group A is the number of embryos transferred per procedure, being 1.8 ± 0.5 in Group A and 1.3 ± 0.4 in Group B, with a p value of < 0.001. This fact is due to the internal Fertility Center guidelines which suggest transferring more than one embryo in case of cleavage stage, while one embryo only in case of blastocyst stage. Indeed, the average percentage of embryos transferred at blastocyst stage was significantly higher in Group B (71.7 ± 40.1) as compared to Group A (37.1 ± 40.0; p < 0.001). Previous studies highlighted the fact that compared to hCG, GnRHa trigger results in similar proportion of mature oocytes (MII), ongoing pregnancy and live birth rates [18]. Oocyte maturity rate is generally related to nuclear maturity and represents the potential value of being a marker of the efficiency of ovarian stimulation and triggering. In the current study, an overall median MII rate of 0.75 was found, which is in line with what has been reported by the Vienna Consensus of 2017 [27]. However, a significant difference in MII rate was observed between the two groups. This could be potentially explained by the larger number of yielded oocytes in Group B, which could have impacted the full development of all the oocytes. Such an observation is in line with the findings of an Israelian study [28].where oocyte maturation rate was found to be significantly lower in the high ovarian response patients’ group. However, this study showed comparable implantation and clinical pregnancy rates to the normal ovarian response patients’ group. These findings confirm that, as already reported in the literature [29], an impaired maturity score has no impact on live birth rate, so also the difference documented in the current study can be considered negligible for the purposes of our analysis. Despite the observed differences in terms of oocytes and embryos characteristics, both groups in the present study share a favorable prognosis in terms of pregnancy and delivery outcomes even at the first ET, as documented in Table 2, and in terms of safety, considering the severe OHSS occurrence rate, as documented in Table 3. In particular, the positive results for Group A confirm the appropriateness of the luteal phase support routinely adopted at Humanitas Fertility Center, in the absence of which the rate of success for ART is significantly reduced. Many studies have documented the influence of luteal phase support to sustain implantation after GnRHa trigger, which is essential to prevent implantation failure and increased miscarriage rate due to luteal phase deficiency [15]. Benadiva and Engmann in 2018 reported that the administration, on top of estradiol and progesterone supplementation, of a low-dose hCG bolus (1,500 IU) on the day of trigger (dual trigger) or on the day of retrieval (rescue protocol) is in both cases effective in maintaining successful pregnancy outcomes. The same authors suggested that the number of follicles the day of trigger could be used as a potential indicator to proceed with the administration of low dose hCG or to perform freeze-all strategy [30]. Overall, the secondary analysis of this study showed that GnRH antagonist stimulation protocol with GnRH agonist trigger is effective also in terms of cumulative pregnancy and delivery rates (69.79% and 63.04%, respectively), as shown in Supplementary Table 1. Moreover, significant differences between Group A and B were observed. Since GnRHa trigger has proven to be effective in achieving a successful cumulative delivery rate, the study subsequently aimed at investigating if there were any variables that could have decreased or increased the probability of reaching such outcome. The fact that the adoption of freeze-all strategy per se positively influenced the achievement of an effective delivery (as shown in Supplementary Table 2) is fully supported by literature data especially in patients at risk for OHSS [31–33], indeed it has been reported that freeze-all strategy could be more advantageous than fresh embryo transfer in terms of pregnancy rate and live birth rate if high numbers of oocytes are collected. However, when pregnancy is achieved, the median time to first live birth is very similar between the two groups, namely 9.24 months in Group A and 9.44 months in Group B as shown in Supplementary Fig. 1. The survival analysis was performed after removing patients who underwent a single ET which did not result in live birth from both groups and considering as starting time for Group B the date of thawing process. This was done to reduce the potential bias of the difference in time between the two groups linked to the time interval needed to perform ET after freeze-all which, according to Humanitas Fertility Center’s protocol, is performed after the second menstrual cycle following the oocyte retrieval procedure. Moreover, the curves also suggest that the majority of live births occurred after the first (most of the cases) or second ET performed, since most (50th centile) events were located around 9 months. After having assessed that freeze-all strategy had an impact on the probability of delivering, the study further progressed to investigate if other characteristics could have determined a higher or lower probability on its achievement. The logistic regression analysis (shown in Supplementary Table 2) highlighted the fact that the presence of primary infertility and higher AMH level were associated to increased probability of achieving an effective delivery; while female and male partner’s age, duration of infertility and previous miscarriages were associated with a decreased probability. Rather than suggesting that primary infertility could lead to an increased probability of delivery, it is the strong association with a significant lower age of mothers with this diagnosis in the study sample (p = 0.001) which could explain the observed positive effect. AMH level resulted to be correlated with a higher probability of delivery, and this, as already reported, is correlated with the known association between AMH values and number of oocytes retrieved after an ART procedure, which is associated with increased cumulative live birth rate, as reported in the literature [25].The inverse relationship between female and male partner’s age, with the probability of an effective delivery is well known and largely reported in the literature [34–37]. The findings of this study are therefore fully aligned with this evidence. Also the inverse correlation of duration of infertility and previous miscarriages with the probability of conception could be explained by the known negative effect of these characteristics on the likelihood of an effective delivery [35, 38–39]. In summary, the GnRHa trigger in combination with GnRH antagonist stimulation protocol resulted to be effective in achieving a successful delivery and none of the factors that have been assessed seemed to have had a novel or unexplained impact on the delivery outcome. As already described, the novelty of the present study is the primary analysis of the study focused on the assessment of potential differences in pregnancy and delivery outcomes based on the first embryo transfer procedure adopted in the two groups. The clinical equivalent effectiveness of both procedures has been proven by the analysis of the pregnancy outcomes, as defined by ongoing pregnancy rate and delivery rate. In fact, 41.37% of cycles in Group A and 42.92% in Group B led to an ongoing pregnancy, and 35.43% versus 33.20% respectively led to delivery (Table 2). These results are aligned with previous studies reported in the literature [13, 40–41]. The subsequent analysis of pregnancy outcomes and delivery distribution reported in Table 2 further corroborated the clinical equivalence of the concerned strategies. No differences have been observed in terms of ectopic pregnancy rate between the two groups, although miscarriage rate resulted to be significantly higher in Group B as compared to Group A (20.24% vs 11.17% respectively; p = 0.008). This could be explained by the fact that the selection protocol adopted in our center allocated to Group B most of the patients with polycystic ovary. By definition, polycystic ovary is characterized by an increased AFC at ultrasound investigation [42]. The higher prevalence of polycystic ovary in Group B is supported by the significantly higher AFC values (p < 0.001) reported in Group B, as shown in Table 1. As reported in the literature [43], the higher prevalence of polycystic ovary syndrome (PCOS) in Group B could justify the significantly increased miscarriage rate that was observed in our study. Indeed, whether or not a freeze-all strategy could lead to a different miscarriage rate compared to fresh ET is still controversial and also a recent meta-analysis could not reach a final conclusion [44]. On the other hand, the delivery distribution (singleton, twins and triplets) was significantly different between the two groups (p < 0.001). In Group A, the multiple birth rate was higher than in Group B, regarding both twins (21.05% Group A vs 4.67% Group B) and triplets (0.66% Group A vs 0.31% Group B). The fact that women undergoing fresh embryo transfer tended to deliver a larger proportion of twins or triplets could be explained by the fact that such transfers were mostly performed at an earlier stage of embryo development than for thawed transfers, as confirmed by the lower average percentage of transfers at blastocyst stage in Group A (Table 1). To compensate for the lower probability of implantation associated to transfers at cleavage stage [45], the routine protocol adopted at Humanitas Fertility Center suggests the transfer of 2 embryos instead of one, which is the number recommended for the blastocyst stage. This has been clearly demonstrated by the fact that the number of embryos transferred in Group A is significantly higher than the one in Group B (1.8 ± 0.5 vs 1.3 ± 0.4 respectively; p < 0.001). Transferring more than one embryo, indeed, increases the probability of having multiple pregnancies as described in the literature [46]. This should be kept in mind since multiple pregnancy is the most frequent iatrogenic complication in ART procedures [47]. Multiple pregnancy indeed increases the number of maternal and fetal complications [48–49]. Since the clinical effectiveness of the two approaches could be considered almost equivalent, the analysis progressed in assessing if also their relative safety could be considered comparable. Overall, the two approaches resulted to be associated with a very low incidence of severe OHSS, as it was observed in 19 cases only over 1,396 cycles of stimulation (1.36%), as shown in Table 3. Such a positive outcome was related to the adoption of GnRH agonist as trigger in all the patients. The lack of a direct stimulation of intraovarian release of vasoactive factors such as vascular endothelial growth factor (VEGF) associated with the GnRHa trigger as compared to hCG trigger is the explanatory cause of the low incidence of OHSS that was observed. The adoption of GnRHa as trigger also causes a FSH mid-cycle surge associated with a LH surge. This mid-cycle hormonal modulation is more consistent with the physiological mechanism since it mimics the natural cycle which is relevant for oocyte maturation. Additionally, LH has a much shorter (6 to 8 times) half-life compared to hCG, resulting in an earlier luteolysis which is essential for preventing OHSS [2–3. 5, 11, 50–51]. Overall, the incidence of OHSS in the moderate risk population clustered in Group A turned out to be very low (1.17%), thus confirming the suitability of the adopted fresh transfer strategy for these patients. The adoption of a freeze-all strategy in Group B, which clustered patients at severe risk of OHSS only, further protected them from the occurrence of OHSS, as demonstrated by the not significant difference recorded in the current study. In conclusion our study confirmed the clinical efficacy and safety of the GnRH antagonist protocol with agonist trigger for women at risk of developing OHSS, in both groups, though the dropout rate resulted significantly higher in cycles where freeze-all approach was set. Such a result, explained probably by the badly tolerated interval period between the ovarian stimulation and the first embryo transfer that may discourage couples, should be considered when the clinician is in doubt whether or not to freeze-all in borderline OHSS-risk patients. This observational study has one of its key strengths in its representativeness of real-world, single-center experience. The Humanitas Fertility Center treats a large number of patients every year, and this is reflected by the large population recruited for the study and their relatively long follow-up period. Another strength of the study is the novelty of the focus on the very first ET outcomes between the two approaches since it has not been investigated thoroughly in literature as far. On the other hand, concerning possible limitations, at the time of oocyte retrieval, on the basis of number of retrieved oocytes, age of the patient, patient’s clinical history, risk factors assessment and couple’s desires, the physician could have decided to proceed with fresh ET or to cryopreserve embryos overruling the indications of the standard protocol. The long period of observation (2012–2020) is also a potential bias for the study. Some factors, indeed, have changed in key aspects of the applied procedures over time, such as the reduced number of transferred embryos per procedure and the increased rate of embryos transferred at the blastocyst stage, as well as advances in technologies and knowledge. Also the inter-operator variability should be taken into account considering the high number of physicians and biologists performing the concerned procedures at our center. Finally, retrospective data from a single center cannot be easily generalized to other populations or countries. In addition, it should be considered that the Italian legislation, which limits the number of embryos that can be obtained by fertilization, could have impacted some of the outcomes of the study. This could be relevant when the results have to be extrapolated to countries where the existing legislation is less restrictive. New prospective studies on broader populations are probably needed to clarify and validate some findings of the current research. The actual reasons for the observed risk of miscarriage rate with freeze-all strategy, possibly related to issues of the population characteristics, or the final understanding if the adoption of a standard elective freeze-all strategy in all patients at risk of OHSS would increase the dropout rate, are questions which still lack a final answer. The current study found that fresh ET approach was associated with an increased multiple birth rate, for which women should be clearly informed upfront.

The use in clinical practice of GnRH agonist trigger in GnRH antagonist stimulation protocol has been demonstrated to be a safe and valid technique. Its adoption is associated with positive outcomes in terms of pregnancy and delivery rates and a low occurrence of complications such as ovarian hyperstimulation syndrome if fresh embryo transfer is performed only in moderate risk patients. This protocol resulted to be effective and safe both in case of freeze-all strategy and fresh embryo transfer, provided that an appropriate luteal phase support is promptly adopted for the latter. The current study also has found that fresh ET approach was associated with an increased multiple birth rate, for which women should be clearly informed upfront. Lastly, but not less important, our study has also highlighted that freeze-all strategy has a significant higher dropout rate, that involve abandoned embryos which surge relevant ethical issues yet to be solved.

GnRH gonadotropin hormone-releasing hormone

IRB Institutional Review Board

ART assisted reproduction technology

OHSS ovarian hyperstimulation syndrome

OS ovarian stimulation

PCO polycystic ovary

US ultrasound

PCOS polycystic ovary syndrome

BMI body mass index

LH luteinizing hormone

GnRHa gonadotropin hormone-releasing hormone agonist

VEGF vascular endothelial growth factor

OPR ongoing pregnancy rate

LPS luteal phase support

hCG human chorionic gonadotropin

ET embryo transfer

PGT pre-implantation genetic testing

AFC antral follicular count

AMH anti-Mullerian Hormone

Ethics approval and consent to participate: Approval was obtained from the ethics committee of Humanitas institutional review board. The procedures used in this study adhere to the tenets of the Declaration of Helsinki. Patients who underwent the treatment described signed an informed consent approving that their medical records could be used for research purposes, if their anonymity was protected. Hence no specific consent was needed for this study.

Consent for publication: Patients provided an informed written consent to have data from their medical records used in research, as the internal IRB and ethics committee required. All patients agreed for their data to be used anonymously for research purposes.

Availability of data and materials: The dataset underlying this article is available in Zenodo repository and can be accessed, on reasonable request to the corresponding author.

Competing interest: The authors have no relevant financial or non-financial interests to disclose.

Funding: No funds, grants, or other support was received for the present study.

Authors’ contributions: FC and PELS were involved in the study concept and design, analysed data and wrote the manuscript. CR and MC contributed to the acquisition of data, analysed data, wrote the manuscript and contributed to bibliography updating. EM analysed data and supervised the analysis. VS, AS and AB contributed to the acquisition of data. RDC critically revised the manuscript and helped for data analysis.

Acknowledgments: The authors thank all the embryologists, gynaecologist, nurses and staff working at Humanitas Fertility Centre, Rozzano, Milan, Italy.

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No competing interests reported.

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GnRH agonist trigger fresh embryo transfers have similar outcomes but a lower dropout rate than freeze-all strategy: a retrospective analysis

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