Impact of Previous-day versus Same-day Warming of Cleavage-stage Embryos on Reproductive Outcomes: A Retrospective Cohort Study.

Globally, there has been an increase in the uptake of assisted reproductive technology (ART) treatment for treating couples with infertility.[ 1 ] According to the International Committee for Monitoring ART (ICMART) World Report, approximately 3,411,005 ART cycles were performed in 2019.[ 1 ] There has been a change in ART practice over the years, with an increasing number of frozen embryo transfers (FETs) being performed across the world. This increase in the number of FET cycles is primarily due to the cautious approach adopted by clinicians to mitigate the risk of ovarian hyperstimulation syndrome and an improvement in cryopreservation techniques. The other reasons for higher FET cycles include a rising number of pre-implantation genetic testing and oocyte donation cycles.[ 2 3 4 ] In contemporary practice, the blastocyst stage is preferred for cryopreservation as it allows self-selection of embryos, has a higher implantation potential and facilitates single embryo transfer (SET) which helps reduce multiple pregnancy rates.[ 2 5 6 ] While blastocyst transfer results in higher live birth per embryo transfer, in clinical practice, embryo cryopreservation is also often performed at the cleavage stage due to various factors such as low oocyte yield and poor embryo development.[ 7 8 ] The cryopreserved embryos can be warmed on the same day and transferred or warmed and cultured for a day or more before embryo transfer. It is of interest to know if the warming of cryopreserved embryos on the same day of embryo transfer or the previous day improves the FET cycle outcomes. The decision to culture for an additional day can have a clinical impact due to the potential developmental arrest of the embryo and cycle cancellation. Furthermore, the information is also important due to logistical reasons for a busy ART unit. In 2021, a retrospective cohort study compared culturing of warmed cleavage-stage embryos for 2 days until blastocysts before transfer to transfer of warmed blastocysts.[ 9 ] They concluded that culturing post-warmed cleavage embryos for 2 days increased the chances of ongoing pregnancy and live birth as compared to warmed blastocysts. Another prospective study also reported that blastocyst development following warming of cleavage-stage embryos was a good indicator for embryo viability and treatment outcomes.[ 10 11 ] In a study done by Zhao et al . in 2018, it was observed that extended culture of day 3 embryos for 7–8 h improved the clinical outcomes as embryos went on to cleave or develop into a morula with good developmental potential.[ 12 ] While many studies have assessed the impact of culturing warmed cleavage-stage embryos to blastocyst in FET cycles, there is a paucity of studies that have evaluated the impact of warming of day 3 embryos and culturing for a day before embryo transfer (day 4) versus same-day warming and transfer of embryos (day 3). In ART cycles with less numbers and poor quality of embryos, culturing warmed cleavage-stage embryos (day 3) for 2 days might result in cycle cancellation due to the unavailability of embryos. Due to the paucity of data, we planned the current study to compare the FET treatment outcomes between cryopreserved day 3 embryos, warmed and transferred the next day, i.e. day 4, versus transfer of warmed day 3 embryos on the same day, i.e. day 3.

During the study period, 2144 FET cycles were performed at our centre. Around 1560 FET cycles were excluded. Those FET cycles in which the embryos were vitrified other than day 3 were excluded. Furthermore, only one FET cycle of each patient was included in the study. Finally, 584 FET cycles fulfilled the inclusion criteria and were included in the study. After case–control matching according to age and body mass index, 209 FET cycles were analysed in either group [ Figure 1 ]. CONSORT diagram The demographic characteristics of the participants are summarised in Table 1 . The mean age of the patients was similar between the groups (31 ± 4.35 years in the day 3 group vs. 31 ± 4.45 years in the day 4 group, P = 0.93). The groups had comparable BMI (25.80 ± 4.12 vs. 25.79 ± 4.05, P = 0.87) for day 3 and day 4 groups, respectively. The aetiologies of infertility and infertility status (primary vs. secondary) were comparable, as well. Comparison of baseline characteristics between day 3 versus day 4 embryo transfer a Values are represented as mean±SD. n (%) Data expressed as number of women (percentage) for categorical variables. BMI=Body mass index, SD=Standard deviation The FET treatment cycle characteristics are represented in Table 2 . The most common reason for performing ART was male factor infertility in both the groups, and there was no significant difference between the groups for the number of ART and FET cycles as well as the number of embryos transferred during each FET cycle. For endometrial preparation, there was a significant difference between the groups with the use of HRT protocol being higher in day 4 (84.2%) versus day 3 (67.9%) ( P = 0.001). Comparison of frozen embryo transfer cycle treatment characteristics between day 3 versus day 4 embryo transfer group n (%) Data expressed as number of women (percentage) for categorical variables. HRT=Hormone replacement therapy The FET outcomes are presented in Table 3 . The clinical pregnancy rate per transfer (41.1% vs. 38.2%, P = 0.548) and LBR per transfer (29.1% vs. 27.7%, P = 0.745) were comparable between the day 3 versus day 4 transfer groups, respectively. Furthermore, no significant difference was observed in miscarriage rate per clinical pregnancy, implantation rate and multiple pregnancy rate per clinical pregnancy between day 3 versus day 4 group (20.9% vs. 21.2%, P = 0.957; 26.2% vs. 26.9%, P = 0.900; and 27.9% vs. 32.5%, P = 0.519). Comparison of clinical outcomes between day 3 and day 4 embryo transfers a Values are represented as mean±SD. n (%) Data expressed as number of transfer (percentage) for categorical variables. SD=Standard deviation Although the cycle cancellation rate was comparable in both the groups (0.8% in day 3 vs. 1.4% in day 4, P = 0.673), cryosurvival of embryos was more in the day 3 group versus day 4 (86.3% vs. 81.7%), and the difference was significant ( P < 0.001). Univariate logistic regression analysis for LBR did not show any significant association for number of embryos transferred, number of FET cycles and endometrial preparation protocol [ Table 4 ]. Multivariate logistic regression analysis demonstrated that the likelihood of achieving live birth did not depend on day of embryo transfer (odds ratio: 1.059, 95% confidence interval: 0.688–1.628, P = 0.793) [ Table 5 ]. Univariate logistic regression analysis for live birth rate a Values are represented as mean±SD. HRT=Hormone replacement therapy cycle, SD=Standard deviation Multiple logistic regression analysis for live birth rate *Adjusted for number of embryos transferred, FET treatment protocol and total number of FET cycles. CI=Confidence interval, OR=Odds ratio, aOR=Adjusted OR

The study findings suggest no evidence of the benefit of an extended culture of vitrified-warmed day 3 cleavage-stage embryos by an additional day, instead of transferring them on the day of warming itself, on the FET outcomes. From a clinical perspective, there was no significant difference in the treatment outcomes. However, for a busy ART unit, these findings enable flexibility in planning their workflow, which will help optimise the treatment outcome. Our study findings need to be further validated in multicentre prospective studies. Concept and design: MSK; analysis and interpretation of data: JS, SS and MSK; drafting the manuscript: SS, MSK, ATK and AM; critical revision of the manuscript: all authors. All authors approved the final version submitted. MSK is EIC of the journal. However, he was not involved in the editorial process for the manuscript. The data supporting the findings of the study are available from the corresponding author on reasonable request and subject to approval of regulatory authority.

The current study did not find any significant difference in the LBR amongst women who underwent FET after culturing of warmed day 3 embryo for an additional day (day 4) versus those who underwent transfer of vitrified-warmed day 3 embryo on the same day of warming (day 3). While the study did find significantly lower cryosurvival rates in the day 4 group compared to the day 3 group, the overall cancellation rate was comparable between the two cohorts. The study suggests no major clinical benefit or harm of extending the culture of vitrified warmed day 3 cleavage-stage embryo by an additional day, but this finding provides flexibility to the ART unit to plan their workflow without compromising the treatment outcomes. In a retrospective study by Joshi et al ., in which supernumerary embryos were cryopreserved on day 2, the pregnancy rate was compared between cohorts: the first cohort ( n = 415) in whom embryos were transferred after an overnight culture and 2 nd cohort ( n = 89) in whom embryos were transferred within 2 h of warming. No statistically significant difference was found in the pregnancy rate between the two cohorts which was in agreement with the current study, even though the day of cryopreservation was different (24.3% versus 20.3%).[ 14 ] A randomised clinical trial by Eftekhar et al . in 2012, in which cryopreserved day 2 embryos, after warming, were cultured until the blastocyst stage in the first cohort ( n = 67), while they were cultured for a day until the day 3 cleavage stage in the second cohort ( n = 67). The mean number of embryos transferred in the blastocyst group was significantly lower than the cleavage-stage group (1.82 ± 0.58 versus 2.73 ± 0.44, P < 0.001). The authors reported a significantly higher implantation rate (30% versus 17.4%, P = 0.00) and ongoing pregnancy rate (42.9% versus 24.6%, P = 0.023) in the blastocyst group as compared to the cleavage group.[ 10 ] In both studies mentioned earlier, Joshi et al . and Efthekhar et al .[ 10 14 ] embryos were cryopreserved in day 2 which is not in agreement with the current study in which cryopreserved day 3 embryos were vitrified and warmed for transfer. In another study[ 9 ] in which the investigators compared warmed cleavage-stage embryos and culturing them for transfer as blastocysts (day 3–5, n = 232) versus transfer of vitrified-warmed blastocysts (day 5, n = 231) on the same day, reported improvement in pregnancy and perinatal outcomes in day 3–5 group versus day 5 group, which is not consistent with current study. Although stage at which embryo (day 3) was cryopreserved was same as the current study, the duration for which it was cultured was different, i.e. 2 days instead of overnight culture as seen in the current study. Multivariable regression analysis, although demonstrated a clear benefit for culturing warmed cleavage embryos for 2 additional days and transferring as blastocysts in a study done by Raghav-Koren et al . but in the current study, we found no impact of the stage of embryo transfer following multivariable regression analysis. The results of studies by Eftekhar et al . and Raghav-Koren et al .[ 9 10 ] are not in agreement with the current study, possibly due to the differences in the embryo stage on the day of transfer. To our knowledge, this is the first study to compare the day 4 transfer of a vitrified-warmed day 3 cleavage-stage embryo versus the same day transfer of a vitrified-warmed day 3 cleavage-stage embryo. To reduce bias due to confounders, we matched the two cohorts for age and BMI as well as included only one treatment cycle per couple. We further adjusted the results with potentially known confounders to minimise selection bias. We also reported on the clinically important outcome, LBR. However, the retrospective nature and lack of morphokinetic scoring and more number of FET cycles with HRT protocol in the day 4 group serve as a limitation of the present study. Although one FET cycle per couple was included in the study, the FET cycle number would differ amongst the participants, affecting the quality of embryos being transferred. Furthermore, data from a single centre limit the generalisability of the results.

We conducted a retrospective cohort study in the Department of Reproductive Medicine and Surgery at Christian Medical College, Vellore, India. All couples who underwent FET at our centre between January 2016 and December 2023 were screened for eligibility. The departmental ART database was utilised to obtain information regarding relevant clinical and treatment outcomes. Since it is a retrospective study, sample size calculation was not done. We obtained the Institutional Review Board (IRB) approval (IRB No.: 2410152), dated 16/10/2024, and waiver of consent for the study. This study was conducted in strict accordance with the ethical principles outlined in the Declaration of Helsinki (2013). Women aged <41 years with infertility who had at least one embryo that underwent cryopreservation on day 3 were included in the study. The patients were divided into two groups according to the stage of the embryo that was transferred: warmed and transferred the same day as a day 3 embryo. The other group in which embryos were warmed the previous day of embryo transfer and cultured for 1 day was transferred as a day 4 embryo. The cryopreservation stage was determined randomly according to the number of embryos, grade of embryos and indication for ART. We excluded women with hydrosalpinx visible on transvaginal ultrasound during ovarian stimulation or gross adenomyosis (uterine size ≥12 weeks) and uterine anomalies. The study groups were matched according to age (23–28, >28–33 and >33–37 years) and body mass index (BMI) (below 18.5 kg/m 2 , 18.5–24.9 kg/m 2 , 25–29.9 kg/m 2 , 30–34.9 kg/m 2 and >34.9 kg/m 2 ) by case–control matching to adjust for potential confounders for primary outcome, i.e. live birth. The ART treatment was carried out according to standard institutional protocol. Downregulation was achieved using either a long gonadotropin-releasing hormone (GnRH) agonist protocol, short agonist protocol, GnRH agonist depot ultralong protocol or an antagonist protocol. Recombinant follicle-stimulating hormone (GONAL-f, follitropin alpha, Merck Serono, Inc., Rockland, USA, or Recagon, follitropin beta, Schering-Plough, USA) alone or in combination with human menopausal gonadotropin (Menopur, Ferring Pharmaceuticals, USA) was used for ovarian stimulation at doses ranging between a total of 100 and 400 IU/day. The dose of gonadotropins was individualised according to the body mass index (BMI), female partner’s age, serum anti-Müllerian hormone and antral follicle count. Follicular monitoring was done using serial transvaginal ultrasound scans. Ovulation was triggered by urinary human chorionic gonadotropin 5000 IU (Pregnyl, Baxter Pharmaceutical Solutions LLC, Bloomington) or recombinant human chorionic gonadotropin (rhCG) 250 mcg (Ovitrelle, Merck Serono, Inc., Rockland, USA) or GnRH agonist (2 mg) (leuprolide acetate) (Lupride, Sun Pharmaceuticals Industries Ltd., India) when more than two follicles reached a diameter >17 mm on ultrasound. Transvaginal oocyte retrieval was performed 35–36 h after triggering ovulation under conscious sedation. Fertilisation was achieved using in vitro fertilisation or intracytoplasmic sperm injection. The resulting embryos were assessed by morphological grading and between one and three embryos were transferred on day 2, 3 or 5 following oocyte retrieval. The number of embryos transferred was determined by the woman’s age, quality of the embryo(s), couple’s preference and availability of transferable embryos. The remaining supernumerary embryos were cryopreserved by vitrification. In case there was no fresh embryo transfer, elective cryopreservation at the cleavage or blastocyst stage was done, and a subsequent FET was planned. Vitrification technique was used as an embryo cryopreservation for cleavage-stage embryos, which were cultured in sequential and single-step culture medium for both the groups. The vitrification device used was Cryotop (Kitazato, Japan). The embryo transfer was performed at least 2 h after warming for the day 3 embryo transfer group. In the group in which the previous-day warming of embryos was performed, embryo transfer was done after overnight culture. All vitrified-warmed embryos underwent laser-assisted hatching. Vitrified and warmed embryo transfers (FET) were performed in women who underwent elective cryopreservation of either all or supernumerary embryos. Endometrial preparation was achieved by hormone replacement therapy (HRT) with escalating doses of exogenous oral oestrogen (oestradiol valerate 2 mg, Progynova, Bayer plc, Germany). Ultrasound was performed on day 15 of the menstrual cycle to measure endometrial thickness and confirm ovarian suppression. This regimen was used alone or in combination with downregulation by administration of a single or two doses (28 days apart) of leuprolide acetate depot 3.75 mg deep intramuscularly (Lupride, Sun Pharmaceuticals Industries Ltd., India). Micronised progesterone 400 mg was administered twice daily vaginally when the endometrial thickness was ≥7 mm, and embryo transfer was carried out on day 3 or 4 from the day of initiating progesterone, depending on the stage at which embryos were vitrified. Alternatively, the stimulated FET protocol was used for endometrial preparation in women with polycystic ovary syndrome. Oral ovulation induction agent letrozole 2.5 mg/5 mg (Fempro, Cipla, India) was administered from the 2 nd day of the menstrual cycle for a total of 5 days, and serial transvaginal ultrasound monitoring was carried out to follow the development of a mature follicle. Serum luteinising hormone (LH) and progesterone were done once the mean follicle diameter reached 15–17 mm, and rhCG trigger was given to induce ovulation in case serum LH ≤15 mIU/l. Micronised vaginal progesterone was started 36 h after trigger or 24 h following a positive serum LH >15 mIU/l, and embryo transfer was planned at P + 1, depending on the stage of the embryo. Luteal support protocol for frozen cycles was oral and vaginal progesterone in addition to oestrogen (oestradiol valerate, 2 mg three times daily) in the HRT cycles. Pregnancy was confirmed by positive serum beta-hCG at about 18 days following progesterone initiation in vitrified-warmed embryo transfer. Following the documentation of a clinical pregnancy, all women were referred at around 8–10 weeks gestational age to the obstetric unit of their preference for further antenatal care. The follow-up of pregnancy outcomes was carried out through means of telephonic interviews and electronic communication. The primary outcome was the live birth rate (LBR). A live birth was defined as a delivery with at least one live-born infant after 22 completed weeks of gestation.[ 13 ] The secondary outcomes included the clinical pregnancy rate per woman. A clinical pregnancy was diagnosed by ultrasonographic visualisation of one or more gestational sacs or definitive clinical signs of pregnancy. In addition to intrauterine pregnancy, a clinically documented ectopic pregnancy was also included as a clinical pregnancy.[ 13 ] Miscarriage rate per clinical pregnancy (spontaneous loss of pregnancy at ≤22 weeks gestation), perinatal outcome and cycle cancellation were additional secondary clinical outcomes. The study groups were matched by case–control matching according to age (23–28, >28–33, >33–37 years and >37 years) and body mass index (below 18.5 kg/m 2 , 18.5–24.9 kg/m 2 , 25–29.9 kg/m 2 , 30–34.9 kg/m 2 and >34.9 kg/m 2 ) to adjust for potential confounders. Parametric t -test or non-parametric Mann–Whitney test was used to find the difference between the groups. The Chi-square and Fisher’s exact tests were used to find the association between categorical variables. Univariate and multivariable analysis was performed with a multiple logistic regression model to assess the potential impact of those parameters on LBR. P < 0.05 was considered statistically significant. All analyses were conducted using SPSS Statistics for Windows (2022), Version 29.0. (IBM Corp., Armonk, NY).