Clinical pregnancy rates after blastocyst culture at a stable temperature of 36.6°C versus 37.1°C: a prospective randomized controlled trial.
Is there a difference in clinical pregnancy rates (CPRs) in good prognosis patients after single embryo transfer (SET) on Day 5, in case of stable culture at 36.6°C or 37.1°C?
CPR (with heartbeat at 7 weeks) after blastocyst transfer do not differ after culturing at 36.6°C or 37.1°C.
Since the beginning of IVF, embryo culture has been performed at 37.0°C; however, the optimal culture temperature remains unknown. Changes in incubator types have led to significant improvements in temperature control. Stable temperature control, i.e. with temperature differences of max. 0.1°C between chambers, is possible in some incubators. A previous prospective pilot study showed that embryo development on Day 5/6 was not affected when embryos were cultured at a stable temperature of 36.6°C or 37.1°C, but culture at 37.1°C resulted in an increased CPR when compared to culture at 36.6°C (74.2% vs 46.4%).
A prospective randomized controlled trial was performed in a tertiary fertility centre between February 2017 and November 26, 2022. A sample size of 89/89 patients with fresh single embryo transfer (SET) was required to achieve 80% power to detect a difference of 0.22 between group proportions (0.43-0.65) at a significance level of 0.05 using a two-sided z-test with continuity correction.
Patients were recruited on the day of oocyte retrieval based on inclusion criteria with final randomization after denudation once six mature oocytes were present. The primary endpoint was CPR (heartbeat at 7 weeks); secondary endpoints were fertilization rate, blastocyst development, biochemical pregnancy rate, live birth rate (LBR), and cumulative live birth rate (CLBR).
A total of 304 patients were eligible for the study; of these 268 signed the consent, 234 (intention-to-treat) were randomized and 181 (per-protocol) received a SET on Day 5: 90 received culture at 36.6°C and 91 at 37.1°C. Patients were on average 32.4 ± 3.5 versus 32.5 ± 4.2 years old, respectively. No differences were observed in embryological outcomes per cycle between culture at 36.6°C versus 37.1°C: 12.0 ± 3.8 vs 12.1 ± 3.8 COCs retrieved (P = 0.88), 10.0 ± 3.1 versus 9.9 ± 2.9 mature oocytes inseminated (P = 0.68), with a maturation rate of 84.2% (901/1083) versus 83.5% (898/1104) (P = 0.87); and 8.0 ± 3.1 versus 7.9 ± 2.7 normally fertilized oocytes with a fertilization rate of 79.7% (720/901) vs 80.5% (718/898) (P = 0.96), respectively. On average 1.5 ± 1.7 versus 1.4 ± 1.9 (P = 0.25) and 1.1 ± 1.1 versus 0.9 ± 1.0 (P = 0.45) supernumerary blastocysts were vitrified on Day 5 and Day 6, respectively. The utilization rate per fertilized oocyte was 46.1% vs 41.5% (P = 0.14). A SET was performed for 181 patients, leading to a biochemical pregnancy rate of 72.2% (65/90) versus 62.7% (57/91) (P = 0.17), respectively. The CPR per fresh transfer cycle was 51.1% (46/90) versus 48.4% (44/91) [OR (95% CI) 1.11 (0.59-2.08), P = 0.710]. To date, a CLBR of 73.3% (66/90) versus 67.0% (61/91) (P = 0.354) has been observed, respectively. In each group, seven patients without live birth have remaining blastocysts frozen. The CPR for the intention-to-treat groups were 38.3% vs 38.6% [OR (95% CI) 0.98 (0.56-1.73), P = 0.967], respectively, for culture at 36.6°C versus 37.1°C.
Only selected patients with expected good prognosis were eligible for the study.
Embryos tend to tolerate small changes in temperature deviations during culture to the blastocyst stage, as demonstrated by their similar implantation potential at two slightly different temperatures.
There is no funding or conflicts of interest to declare.
NCT03548532.
23 October 2017.
10 November 2017.
Wouters K
，Mateizel I
，Segers I
，Van de Velde H
，Van Landuyt L
，De Vos A
，Schoemans C
，Jankovic D
，Blockeel C
，Drakopoulos P
，Tournaye H
，De Munck N
... -
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Impact of letrozole co-treatment during ovarian stimulation on oocyte yield, embryo development, and live birth rate in women with normal ovarian reserve: secondary outcomes from the RIOT trial.
Does letrozole (LZ) co-treatment during ovarian stimulation with gonadotropins for in IVF impact follicle recruitment, oocyte number and quality, embryo quality, or live birth rate (LBR)?
No impact of LZ was found in follicle recruitment, number of oocytes, quality of embryos, or LBR.
Multi-follicle stimulation for IVF produces supra-physiological oestradiol levels. LZ is an aromatase inhibitor that lowers serum oestradiol thus reducing negative feedback and increasing the endogenous gonadotropins in both the follicular and the luteal phases, effectively normalizing the endocrine milieu during IVF treatment.
Secondary outcomes from a randomized, double-blind placebo-controlled trial (RCT) investigating once-daily 5 mg LZ or placebo during stimulation for IVF with FSH. The RCT was conducted at four fertility clinics at University Hospitals in Denmark from August 2016 to November 2018 and pregnancy outcomes of frozen-thawed embryo transfers (FET) registered until May 2023.
One hundred fifty-nine women with expected normal ovarian reserve (anti-Müllerian hormone 8-32 nmol/l) were randomized to either co-treatment with LZ (n = 80) or placebo (n = 79). In total 1268 oocytes were aspirated developing into 386 embryos, and morphology and morphokinetics were assessed. One hundred twenty-nine embryos were transferred in the fresh cycle and 158 embryos in a subsequent FET cycle. The effect of LZ on cumulative clinical pregnancy rate (CPR), LBR, endometrial thickness in the fresh cycle, and total FSH consumption was reported.
The proportion of usable embryos of retrieved oocytes was similar in the LZ group and the placebo group with 0.31 vs 0.36 (mean difference (MD) -0.05, 95% CI (-0.12; 0.03), P = 0.65). The size and number of aspirated follicles at oocyte retrieval were similar with 11.8 vs 10.3 follicles per patient (MD 1.5, 95% CI (-0.5; 3.1), P = 0.50), as well as the number of retrieved oocytes with 8.0 vs 7.9 oocytes (MD 0.1, 95% CI (-1.4; 1.6), P = 0.39) in the LZ and placebo groups, respectively. The chance of retrieving an oocyte from the 13 to 16 mm follicles at trigger day was 66% higher (95% CI (24%; 108%), P = 0.002) in the placebo group than in the LZ group, whilst the chance of retrieving an oocyte from the ≥17 mm follicles at trigger day was 50% higher (95% CI (2%; 98%), P = 0.04) in the LZ group than in the placebo group. The proportion of fertilized oocytes with two-pronuclei per retrieved oocytes or per metaphase II oocytes (MII) (the 2PN rates) were similar regardless of fertilization with IVF or ICSI with 0.48 vs 0.57 (MD -0.09, 95% CI (-0.24; 0.04), P = 0.51), and 0.62 vs 0.64 (MD -0.02, 95% CI (-0.13; 0.07), P = 0.78) in the LZ and placebo groups, respectively. However, the MII rate in the ICSI group was significantly lower with 0.75 vs 0.88 in the LZ vs the placebo group (MD -0.14, 95% CI (-0.22; -0.06), P = 0.03). Blastocysts on Day 5 per patient were similar with 1.5 vs 2.0, P = 0.52, as well as vitrified blastocysts per patient Day 5 with 0.8 vs 1.2 in (MD -0.4, 95% CI (-1.0; 0.2), P = 0.52) and vitrified blastocysts per patient Day 6 with 0.6 vs 0.6 (MD 0, 95% CI (-0.3; 0.3), P = 1.00) in the LZ vs placebo group, respectively. Morphologic evaluation of all usable embryos showed a similar distribution in 'Good', 'Fair', and 'Poor', in the LZ vs placebo group, with an odds ratio (OR) of 0.8 95% CI (0.5; 1.3), P = 0.68 of developing a better class embryo. Two hundred and ninety-five of the 386 embryos were cultured in an embryoscope. Morphokinetic annotations showed that the odds of having a high KIDscore™ D3 Day 3 were 1.2 times higher (CI (0.8; 1.9), P = 0.68) in the LZ group vs the placebo group. The CPR per transfer was comparable with 31% vs 39% (risk-difference of 8%, 95% CI (-25%; 11%), P = 0.65) in the LZ and placebo group, respectively, as well as CPR per transfer adjusted for day of transfer, oestradiol and progesterone levels at trigger, progesterone levels mid-luteal, and number of oocytes retrieved (adjusted OR) of 0.8 (95% CI (0.4; 1.6), P = 0.72). Comparable LBR were found per transfer 28% vs 37% (MD -9%, 95% CI (-26%; 9%), P = 0.60) and per randomized women 24% vs 30% (MD of -6%, CI (-22%; 8%), P = 0.60) in the LZ group and placebo group, respectively. Furthermore, 4.8 years since the last oocyte aspiration, a total of 287 of 386 embryos have been transferred in the fresh or a subsequently FET cycle, disclosing the cumulative CPR, which is similar with 38% vs 34% (MD 95% CI (8%; 16%), P = 0.70) in the LZ vs placebo group.
Both cleavage stage and blastocyst transfer and vitrification were permitted in the protocol, making it necessary to categorize their quality and pool the results. The study was powered to detect hormonal variation but not embryo or pregnancy outcomes.
The similar utilization rate and quality of the embryos support the use of LZ co-treatment for IVF with specific indication as fertility preservation, patients with previous cancer, or poor responders. The effect of LZ on mature oocytes from different follicle sizes and LBRs should be evaluated in a meta-analysis or a larger RCT.
Funding was received from EU Interreg for ReproUnion, Sjaelland University Hospital, Denmark, Ferring Pharmaceuticals, and Gedeon Ricther. Roche Diagnostics contributed with assays. A.P. has received grants from Ferring, Merck Serono, and Gedeon Richter, consulting fees from Preglem, Novo Nordisk, Ferring, Gedeon Richter, Cryos, & Merck A/S, speakers fees from Gedeon Richter, Ferring, Merck A/S, Theramex, & Organon, and travel support from Gedeon Richter. The remaining authors declare that they have no competing interests in the research or publication.
NCT02939898 and NCT02946684.
Bülow NS
，Warzecha AK
，Nielsen MV
，Andersen CY
，Holt MD
，Petersen MR
，Sopa N
，Zedeler A
，Englund AL
，Pinborg A
，Grøndahl ML
，Skouby SO
，Macklon NS
... -
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Cumulative live birth rates after one ART cycle including all subsequent frozen-thaw cycles in 1050 women: secondary outcome of an RCT comparing GnRH-antagonist and GnRH-agonist protocols.
Are cumulative live birth rates (CLBRs) similar in GnRH-antagonist and GnRH-agonist protocols for the first ART cycle including all subsequent frozen-thaw cycles from the same oocyte retrieval?
The chances of at least one live birth following utilization of all fresh and frozen embryos after the first ART cycle are similar in GnRH-antagonist and GnRH-agonist protocols.
Reproductive outcomes of ART treatment are traditionally reported as pregnancies per cycle or per embryo transfer. However, the primary concern is the overall chance of a live birth. After the first ART cycle with fresh embryo transfer, we found live birth rates (LBRs) of 22.8% and 23.8% (P = 0.70) for the GnRH-antagonist and GnRH-agonist protocols, respectively. But with CLBRs including both fresh and frozen embryos from the first oocyte retrieval, chances of at least one live birth increases. There are no previous randomized controlled trials (RCTs) comparing CLBRs in GnRH-antagonist versus GnRH-agonist protocols. Previous studies on CLBR are either retrospective cohort studies including multiple fresh cycles or RCTs comparing single embryo transfer (SET) with double embryo transfer (DET).
CLBR was a secondary outcome in a Phase IV, dual-center, open-label, RCT including 1050 women allocated to a short GnRH-antagonist or a long GnRH-agonist protocol in a 1:1 ratio over a 5-year period using a web-based concealed randomization code. The minimum follow-up time from the first IVF cycle was 2 years. The aim was to compare CLBR between the two groups following utilization of all fresh and frozen embryos from the first ART cycle.
All women referred for their first ART cycle at two public fertility clinics, <40 years of age were approached. A total of 1050 subjects were allocated to treatment and 1023 women started standardized ART protocols with recombinant human follitropin-β (rFSH) stimulation. Day-2 SET was planned and additional embryos were frozen and used in subsequent frozen-thawed cycles. All pregnancies generated from oocyte retrieval during the first IVF cycle including fresh and frozen-thaw cycles were registered. Ongoing pregnancy was determined by ultrasonography at gestational week 7-9 and live birth was irrespective of the duration of gestation. CLBR was defined as at least one live birth per allocated woman after fresh and frozen cycles. Subjects were censored out after the first live birth. Cox proportional hazard model was used to evaluate the relative prognostic significance of female age, BMI, the number of retrieved oocytes and the diagnosis of infertility in relation to the CLBR.
Baseline characteristics were similar and equal proportions of patients continued with frozen-thaw (frozen embryo transfer, FET) cycles after their fresh ART cycle in the GnRH-antagonist and GnRH-agonist arms. When combining all fresh and frozen-thaw embryo transfers from first oocyte retrieval with a minimum of 2-year follow-up, the CLBR was 34.1% (182/534) in the GnRH-antagonist group versus 31.2% (161/516) in the GnRH-agonist group (odds ratio (OR):1.14; 95% CI: 0.88-1.48, P = 0.32). Mean time to the first live birth was 11.0 months in the GnRH-antagonist group compared to 11.5 months in the GnRH-agonist group (P < 0.01). The total number of deliveries from all FET cycles where embryos were thawed were higher in the antagonist group 64/330 (19.4%) compared to the agonist group 43/355 (12.1%) ((OR): 1.74; 95% CI: 1.14-2.66, P = 0.01). The evaluation of prognostic factors showed that more retrieved oocytes were associated with a significantly higher CLBR in both treatment groups. For the subgroup of obese women (BMI >30 kg/m2), the CLBR was significantly higher in the GnRH-antagonist group (P = 0.02).
The duration of the trial is a possible limitation with introduction of new methods as 'Freeze all' and 'GnRH-agonist triggering', but as these treatments were used in only few women, a systematic bias is not likely. Blastocyst culture of surplus embryos for freezing was introduced to both groups simultaneously, thereby minimizing the risk of bias. Furthermore, with a minimum of 2-year follow-up, a minority (<1%) still had cryopreserved embryos and no live birth at the end of the trial. The post hoc prognostic covariate analyses with multiple strata should be interpreted with caution. Finally, the physicians were not blinded to GnRH treatment group after randomization.
With the improvement of embryo culture, freezing and thawing methods as well as a strategy of elective SET, CLBR until first live birth provides an all-inclusive success rate for ART. When comparing GnRH-antagonist and GnRH-agonist protocols, we find similar CLBRs, despite more oocytes being retrieved in the GnRH-agonist protocol.
An unrestricted research grant is funded by Merck Sharp & Dohme Corp., a subsidiary of Merck & Co., Inc., Kenilworth, NJ, USA (MSD). The funders had no influence on the data collection, analyses or conclusions of the study. No conflict of interests to declare.
EudraCT #: 2008-005452-24. ClinicalTrial.gov: NCT00756028.
18 September 2008.
14 January 2009.
Toftager M
，Bogstad J
，Løssl K
，Prætorius L
，Zedeler A
，Bryndorf T
，Nilas L
，Pinborg A
... -
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ResearchGate
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