Impact of Post-Thaw Incubation Time of Frozen Embryos on Clinical Pregnancy Rate : Journal of Human Reproductive Sciences

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Impact of Post-Thaw Incubation Time of Frozen Embryos on Clinical Pregnancy Rate

Ranji, G. Geovin; Shankar, Kundavi; Asokan, Yamini; Veerasigamani, Geetha; Vittal, Rashmi Gingade; Naaram, Nithya M.; Hema Niveda, KR

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Journal of Human Reproductive Sciences 16(1):p 64-69, Jan–Mar 2023. | DOI: 10.4103/jhrs.jhrs_180_22
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Vitrification of embryos following a single-controlled ovarian stimulation has been the strategy practised now in many in vitro fertilisation clinics to minimise the risk of early ovarian hyper stimulation syndrome, to reduce multiple pregnancy rates and to improve cumulative pregnancy rates. In recent years, advances in vitrification techniques and improved culture conditions have led to good post-thaw embryo survival rates, thereby increasing pregnancy rates of frozen embryo transfer (FET) cycles.


The aim of this study was to analyse the effect of post-thaw incubation time of frozen embryos on the clinical pregnancy rates (CPRs) of frozen embryo transfer (FET) cycles.

Settings and Design: 

This was a retrospective, comparative study done at a teaching hospital in assisted reproductive treatment.

Materials and Methods: 

Three hundred and ten FET cycles were analysed, of which 125 had day 2 freezing and 185 had day 3 freezing. Depending upon the day of thawing and day of transfer, FET cycles were divided into six groups: Group 1 (day 2 thawing and day 3 transfer), Group 2 (day 2 thawing and day 4 transfer), Group 3 (day 2 thawing and day 5 transfer), Group 4 (day 3 thawing and day 3 transfer), Group 5 (day 3 thawing and day 4 transfer) and Group 6 (day 3 thawing and day 5 transfer).

Statistical Analysis Used: 

Statistical analysis was performed using version 14 R software version 4.0.1 (2020-06-06) (R foundation for Statistical Computing, Vienna, Austria). A P < 0.05 is taken as significant.


The CPR of Group 4 was 42.4% which was more than that of the other groups but it did not reach statistical significance.


Short incubation time of 2–4 h is as effective as an extended incubation time in terms of CPRs of FET cycles.


In the initial days of in vitro fertilisation (IVF), a high number of embryos were transferred due to a low success rate; however, with advances in the understanding of physiology, novel culture systems and availability of better hormone preparations, there was an improvement in pregnancy rate with the added disadvantage of increased higher order pregnancies. Multiple pregnancies are associated with increased maternal and perinatal morbidity and mortality. Hence, the current norm is to transfer one or two embryos and the excess embryos are being cryopreserved. The first pregnancy following slow freezing and then thawed blastocyst replacement was reported in 1983.[1] Since then worldwide freezing of embryos became popular.

The shift from fresh transfers towards frozen cycles is to make use of a single-controlled ovarian stimulation, to reduce the risk of ovarian hyperstimulation syndrome, to reduce the number of embryos transferred, to have more number of cycles possible and thereby improve the cumulative pregnancy rates.[2] Advances in vitrification and improvement in culture systems have improved the results favouring cryopreservation of embryos.[3]

As fewer embryos are being transferred, to increase the live birth rate and clinical pregnancy rate (CPR), the best embryo has to be chosen. During frozen embryo transfer (FET), there are two common ways to select the viability of the thawed embryos: (1) In the short culture with 2–4 h incubation time-blastomere survival and (2) in long culture, when the incubation time is more than 24 h, depends upon the ability of embryo to undergo further division of cleavage.[4]

There are two schools of thought-one considering the short culture with 2–4 h incubation time as superior whereas a few studies have shown the selection of embryos after they undergo further cell division gives better pregnancy rates. There is no final consensus on the optimum thaw culture time required.[5] This study was done to find the best day of thawing, culture and day of transfer of embryos by comparing their clinical pregnancies to improve our clinical outcome.


Type of study

This was a retrospective, comparative study.

Period of study

The period of study was January 2017–October 2021.

Inclusion criteria

Subfertile couples who underwent egg collection, intracytoplasmic sperm injection (ICSI) and had their embryos frozen in our unit and had FET done in our institution during the period of study were included in our study.

Exclusion criteria

Fresh embryo transfers where embryos were not vitrified were excluded from the study.

Subfertile couples who attended our outpatient department were evaluated and who needed IVF were counselled and explained about the process. Antagonist protocol was used for ovarian stimulation. The women were monitored with periodic hormonal analysis and transvaginal ultrasound (TVS). When two or more follicles reached 18 mm, trigger was given with human chorionic gonadotropin (hCG) and/or agonist. Egg collection was done after 35 h after the trigger. Fresh or frozen semen samples obtained from the husband/donor as per their elective decision with informed written consent were prepared and ICSI was performed uniformly for all patients.

After ICSI, embryos were cultured; pronuclei check was done 17 ± 1 h post-ICSI. Fertilised embryos were cultured in G1 media (Vitrolife, Sweden) and were frozen using Kitazato vitrification kit[6] which contains two different media with different time exposure needed for the water content from inside the embryos to be replaced with cryoprotectant thus making it suitable for vitrification-12 min in the equilibration media, followed by 1 min in the vitrification media on day 2 or day 3. The embryos were loaded into CryolockR (Irvine Scientific) and plunged into liquid nitrogen (−196°C) and stored in canisters.

During the frozen embryo transfer cycle, endometrium was prepared in the natural cycle, hormone replacement therapy cycle or stimulated cycle. Endometrial thickness was monitored with sequential TVS. When endometrial thickness reached 8 mm and above, progesterone was started with 100 mg injection daily, the duration of days needed was dependent upon the day of transfer decided on an individual basis.

When the woman was ready for the frozen embryo transfer, we used Kitazato media[6] for thawing the vitrified embryos. In the sequential thawing method, the embryos were exposed to three different solutions of decreasing the concentration of cryoprotectants in a specified time as mentioned by the manufacturer. First, the embryos were placed in the Thawing media for 1 min, followed by the dilution media for 3 min and then in the Washing media for 6 min. Depending on the day of freezing-day 2 and day 3, they were further cultured in G1 and G2 media (Vitrolife, Sweden), respectively, in Cook MINC Benchtop Incubator. We assessed the quality of the embryos and accordingly we transferred them.

The day of transfer was dependent upon the patient's characteristics, availability of staff and patient preference. During the frozen embryo transfer cycle, depending upon the day of thawing and transfer, we have six groups:

Group 1: day 2 thawing and day 3 transfer, Group 2: day 2 thawing and day 4 transfer, Group 3: day 2 thawing and day 5 transfer, Group 4: day 3 thawing and day 3 transfer, Group 5: day 3 thawing and day 4 transfer and Group 6: day 3 thawing and day 5 transfer [Figure 1].

Figure 1:
Groups according to the day of thawing and day of transfer

Group 4 with 2–4 h incubation time is described as 'short incubation period' and the rest of the groups are described together as 'long incubation period'.

The women were given luteal support with oral and injectable progesterone. Serum beta hCG, which is the biochemical marker of pregnancy, was tested 16 days after embryo transfer. Early pregnancy scan was done 1 week after a positive pregnancy test.

Clinical pregnancy was diagnosed when 1 or more pregnancy sacs were observed on ultrasound. The following formulae are used.[7]

This study was approved by the institutional ethics committee (ECR/140/Inst/TN/2013/RR-20). The patients have given informed written consent for using their anonymised data for research purposes and this study adheres to the principles of the Declaration of Helsinki with its amendments (2013).

Statistical analysis

Statistical analysis was performed using version 14 R software version 4.0.1 (2020-06-06) (Manufacturer: R foundation for Statistical Computing, Vienna, Austria). Power was not calculated. Parametric and non-parametric tests were used as needed. Continuous variables were expressed as mean ± standard deviation (SD) and analysed using Student's t-test. Categorical variables were expressed as percentages and analysed using Chi-squared or Fisher's exact test depending on the sample size. Statistical significance was defined as P < 0.05.


The age of the women who underwent embryo transfers ranged between 23 and 50 years. Their mean ± SD was 32.2 ± 5.2 years.

Three hundred and ten FET cycles were analysed, of which 125 had day 2 freezing and 185 had day 3 freezing. The CPR of day 3 thawing at 38.4% was greater than day 2 thawing at 35.2%, but it was not found to be statistically significant; P = 0.57 and Chi-square test were used. Fifty four percentage (167) of our embryo transfers were Day 3, followed by 33% (102) on day 4 and 13 % (41) on day 5 [Figure 2].

Figure 2:
Showing the groups according to day of thawing and day of transfer

The CPR of the FET cycles with short incubation time between 2 and 4 h was 42.4% and long incubation time between 24 and 96 h was 34.9%, P = 0.21.

When comparing CPR between groups, Group 4 had the highest CPR of 42.4%.

In the group with short incubation time, i.e. day 3 thawing and day 3 transfer and when the incubation time was between 72 and 96 h, all the transfers where the beta hCG was positive, there was a gestational sac seen in the ultrasound whereas when the culture time was between 24 and 72 h, out of the 75 women, 5 had biochemical pregnancy or early implantation failure only [Table 1].

Table 1:
Relation between hours of incubation, positive pregnancy test and positive scan

In our centre, we have a policy of limiting the number of blastocysts transferred to two, and in the other stages of embryos to three. We rarely transferred four embryos when there were no risk factors for carrying multiple pregnancies and when there were previous ICSI failures. We did a subgroup analysis based on the number of embryos transferred-<3 and 3 or more embryos.

The CPR was significantly higher (P = 0.008, significant and Chi-square) when 3 or more embryos were transferred when compared to <3 embryos but the multiple pregnancy rate, all were twin pregnancies, though was higher when the number of embryos ≥3, it was not of statistical significance (P = 0.46) [Table 2].

Table 2:
Comparison of clinical pregnancy rate and multiple pregnancy rates with number of embryos

The CPR of the FER cycles with short incubation time between 2-4 hours was 42.4% and long incubation time between 24-96 hours was 34.9%, P = 0.21 [Table 3].

Table 3:
Relation between incubation time and clinical pregnancy rate

When comparing CPR among the six groups, the Group 4 had the highest CPR of 42.4% [Table 4].

Table 4:
Numbers and clinical pregnancy rate in each group


The culture system plays a crucial role in embryo viability, implantation and pregnancy. Twenty-four-hour culture could be a natural selection process for embryos.

Results from our study show that CPR is higher (42.4%) when the post-thaw incubation time is 2–4 h than the long culture (34.9%) but the results did not reach statistical significance [Table 3]. These results are similar to the study by Wang et al.,[8] in which the implantation rate was 41.1% when the incubation time was 2 h.

Even though there is one study which advocates the longer culture and emphasising upon the resumption of mitosis,[9] but this was an older study done in 1998, where the overall implantation rate was only 9%. This study recommends keeping the embryos for overnight culture and checking for further rounds of cleavage as a method of selection of viable embryos to improve the implantation rate and to reduce multiple pregnancies. With recent updated culture systems, even the shorter duration of incubation and same-day transfer gives comparable results [Tables 1 and 4].

It is interesting to note that there were no biochemical pregnancies in Groups 3 and 4 [Table 1].

In a retrospective cohort study of 392 frozen embryo cycles[10]-'the transfer of morula stage embryos following the overnight culture of post-thawed cleavage embryos led to an improvement in the clinical outcomes of FET cycles'; highlighting the formation of morula after an overnight culture and their higher implantation rates.

In another retrospective study published in 2013 by Guo et al. comparing the different incubation times post-thawing, there was no difference in CPR, even after doing subdividing analysis according to age group.[11] These results are similar to our observations, mostly because a well-kept culture system can mimic the tubal environment in growing the embryos.

In another study published by Joshi et al. in 2010,[12] which emphasises the selection of embryos following thawing, they compared pregnancy rates between two groups-2 h incubation after thawing and overnight culture, and found comparable results. Furthermore, they did a subgroup analysis on further cleaved and uncleaved embryos-'revealed that highly significant pregnancy rates were observed when ≥2 cleaved embryos were transferred (42.2%) than when 1 cleaved embryo (16.9%, P = 0.0002) or embryos without cleavage were transferred (3.2%, P ≤ 0.0001)'. However, we could not analyse in similar lines as we do not transfer uncleaved embryos. In the same study also, when the number of embryos was 3, their pregnancy rate was 29% whereas it was 10.7% with <3 embryos. In our study, this difference reached a statistical significance (P = 0.008), 45.1% versus. 30.4%, respectively [Table 2].

It is to be noted that when blastocysts were transferred, no more than two embryos were transferred. Hence, in the group with more than three embryos which consisted of the cleavage stage and morula. Although the implantation rate of blasts is higher, the CPR of more than three embryos was higher, possibly the lesser implantation rate expected was combated by the number of embryos transferred.

The increase in CPR was not at the cost of increasing multiple pregnancies. The multiple pregnancy rate was 31.3% as compared to 25.5% when the number of embryos was more than or equal to three and <3, respectively [Table 2]. However, it is important to note that the multiple pregnancy rates in both these groups are high and extensive informed counselling is necessary if the transfer of more than two embryos is considered.

In a recent study in 2015–2016,[13] where similar vitrification and transfer protocols were followed, analysing 366 frozen embryo transfer cycles, they got CPRs of extended culture and same day culture as 30.8% versus 24.1%, respectively, but it did not reach statistical significance (P = 0.15). It concluded that 'overnight culture and confirmation of mitosis resumption were not essential for FET cycles in vitrification method'. There are three randomised clinical trials[14,15,16] comparing two culture times and all studies concurred similar pregnancy rates. This could be because of avoiding prolonged culture and building up of toxic metabolites as well by maintaining the embryonic potential.

There are a small number of studies emphasising short incubation time – A retrospective study done for 8-year period by Rato et al.[4] concluded 'that a short post-thaw culture period is associated with higher implantation and live birth rates per embryo' and 'supports selection of frozen-thawed embryos strictly based on blastomere cryo survival and raises the hypothesis that environmental factors may have an important role on embryo implantation and developmental potential during post-thaw culture'. In future, more research is needed on culture systems and metabolomics of the spent media.

Another retrospective cohort study by Zhou et al.,[17] when the comparison was done between different ages, 'cleavage-stage FET was found to be more suitable for younger women, while FET of blastocysts cultured from cleavage-stage embryos was better suited for women of advanced age', implying the need for blastocyst transfer when there is expected poor implantation as in advanced age >34 years, sufficient embryos to freeze to avoid cycle cancellation.

We compared the CPR between day 2 and day 3 thawing of embryos, and found 35.2% and 38.4%, respectively, P = 0.57; A study analysing the clinical data of FET cycles by Yao et al.[18] and a study by Salumets et al.[19] comparing stages of freezing showed the same result and highlights good cryopreservation techniques.


This being a retrospective study shares the limitations of any retrospective study in study population selection.

  1. We were not able to analyse the cause of infertility and other population characteristics, quality of oocytes and sperms
  2. We did not have control over allocating into each group resulting in an inequal distribution of numbers in different groups
  3. In many of our reference studies, larger samples were analysed. Adding prospective samples will give us definite conclusions.


Post-thaw incubation time of 2–4 h and checking blastomere survival gives comparable CPR when compared to longer incubation time. Larger sample size studies are required to improve our understanding.

Financial support and sponsorship


Conflicts of interest

There are no conflicts of interest.

Data availability

The data is available with the corresponding author upon reasonable request.


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Clinical pregnancy rate; embryo culture; frozen embryo transfer; vitrification

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