Abstract
Purpose
To compare reproductive outcomes following a euploid embryo transfer, between those embryos vitrified-warmed twice to those vitrified-warmed once.
Methods
We retrospectively analysed 694 single euploid frozen embryo transfer cycles following preimplantation genetic testing for aneuploidy (PGT-A). For cycles in group 1 (N = 451), embryos were biopsied for PGT-A at blastocyst stage and vitrified. For cycles in group 2 (N = 146), embryos were vitrified at blastocyst stage, before being warmed and biopsied for PGT-A and vitrified again. For cycles in group 3 (N = 97), embryos were vitrified on day-3, before being warmed, cultured to day-5 and biopsied for PGT-A and re-vitrified.
Results
The pregnancy, clinical pregnancy and livebirth rate in group 2 were not statistically different to group 1 (pregnancy rate, adjusted OR 1.09, 95% CI 0.62–1.91; clinical pregnancy, aOR 0.89, 95% CI 0.58–1.37; live birth rate, aOR 0.85, 95% CI 0.56–1.28). There was also no significant difference between group 3 and group 1, with similar pregnancy rate (aOR 1.22, 95% CI 0.74–1.99), clinical pregnancy rate (aOR 1.21, 95% CI 0.75–1.96) and live birth rate (aOR 1.15, 95% CI, 0.73–1.80). There was no significant difference in miscarriage rates between all three groups. The age at the oocyte collection, embryo quality and day of biopsy were associated with pregnancy, clinical pregnancy and live birth rate.
Conclusion
This study suggests that vitrifying and warming embryos twice at blastocyst or at cleavage and then blastocyst stage, can lead to similar reproductive outcomes to embryos vitrified-warmed once, after a single euploid embryo transfer.
Keywords: Blastocyst, Euploid, Vitrified-warmed embryo transfer, Live birth, Miscarriage, Double vitrification
Introduction
The number of frozen embryo transfers (FET) has been steadily increasing [1], following advances in laboratory techniques which have led to higher success rates. Evolution in embryo cryopreservation technology and advances in DNA sequencing techniques have also resulted in preimplantation genetic testing for aneuploidy (PGT-A) being increasingly offered to women of advanced maternal age and history of recurrent miscarriage or repetitive in-vitro fertilisation (IVF) failure [2]. While the debate surrounding the use of PGT-A continues [3–6], many couples consider it depending on their individual circumstances. In order to utilise PGT-A, most women undergo ovarian stimulation, oocyte collection, IVF and embryos culture to blastocyst stage, before being biopsied and cryopreserved for future use. Other couples may, however, already have embryos cryopreserved and subsequently decide to utilise PGT-A before a subsequent transfer. In this approach, the embryos will be vitrified-warmed twice. In a third group of patients, women with a low ovarian reserve or poor responders may have multiple cycles with embryos cryopreserved on day-3 and subsequently warmed, cultured to day-5 or day-6, prior to undergoing biopsy for PGT-A and vitrification. All these scenarios involve increased embryo micro-manipulation with a potentially damaging effect, thus highlighting the importance of reviewing outcomes to enable appropriate counselling for prospective patients prior to embarking upon PGT-A and embryo cryopreservation.
Most of the early studies reporting twice freezing–thawing of embryos, included embryos cultured from one embryo developmental stage to the next (2PN to cleavage, cleavage to blastocyst) with most reporting similar pregnancy outcomes [7, 8] and some suggesting a higher miscarriage rate [9, 10]. A recent larger study suggested that vitrifying and warming an embryo twice at blastocyst stage decreases its implantation potential [11]. When euploid embryos following one biopsy for PGT-A were vitrified-warmed twice, initial studies were reassuring [12, 13] but the latest study [14] demonstrated a significantly lower live birth rate compared to embryos vitrified-warmed and biopsied once.
The objective of this study was to compare livebirth and clinical miscarriage rates in women using PGT-A who underwent embryo transfer in the following groups: (1) ovarian stimulation, embryo culture to blastocyst stage, biopsy and vitrification; (2) ovarian stimulation, embryo vitrification at blastocyst stage, prior to being warmed, biopsied and re-vitrified; (3) ovarian stimulation for embryo batching, who underwent embryo vitrification at day-3, prior to warming, further culture to blastocyst stage, prior to biopsy and re-vitrification.
Material and methods
This study included all women who embarked on a FET with a euploid embryo between 1st January 2015 and 30th June 2020 at the Centre for Reproductive and Genetic Health (CRGH) in London. Indications for PGT-A included advanced maternal age, or a history of implantation failure or miscarriage, or patient request. Cycles involving oocyte cryopreservation, donated oocytes or embryos and surrogates were excluded. Cycles including preimplantation genetic testing for single gene disorders or chromosomal rearrangements, the transfers of mosaic embryos, embryos transported from another clinic or embryos with inconclusive results requiring a second biopsy were also excluded.
Study groups
A total of 694 single euploid FET cycles were included. Each cycle was divided between three groups, depending on the number of vitrification-warming episodes. Embryos that underwent PGT-A biopsy at blastocyst stage and cryopreserved (biopsied day-5—vitrified) were included in Group 1 (N = 451). Cycles where embryos were cryopreserved at blastocyst stage, subsequently warmed and biopsied for PGT-A, then cryopreserved for a second time (vitrified day 5- biopsied—re-vitrified) were included in Group 2 (N = 146). Group 3 (n = 97) included embryos that were cryopreserved on day 3, subsequently warmed and cultured to blastocyst stage, biopsied for PGT-A and re-cryopreserved (vitrified Day 3- biopsied day 5—re-vitrified). The approach used for Group 3 was employed for women who had diminished ovarian reserve and/or poor responders. They underwent embryo batching by undergoing a minimum of two ovarian stimulation cycles with the purpose to warm, culture and biopsy all embryos together for PGT-A.
Ovarian stimulation, fertilisation and embryo culture
All women underwent controlled ovarian stimulation with long agonist, short antagonist or mild stimulation cycles depending on their ovarian reserve or previous response. Oocyte maturation was triggered from day 10 of the cycle onwards when three or more follicles ≥ 17 mm in diameter were identified on ultrasound with either 10,000 units of HCG (Gonasi®) or 1-ml Buserelin acetate (Suprecur®) if deemed at risk of ovarian hyperstimulation syndrome. Transvaginal oocyte retrieval was undertaken 36–37 h later under ultrasound guidance.
The collected oocytes were fertilized with IVF, intracytoplasmic sperm injection (ICSI), or split IVF/ICSI. A small number of cycles used intracytoplasmic morphologically selected sperm injection or physiologic intracytoplasmic sperm injection. The sperm used for fertilization was either fresh or cryopreserved partner sperm or donor sperm. The embryos were cultured in either a benchtop incubator or a time-lapse incubator (EmbryoScope, Vitrolife, Denmark) as previously described [15], and underwent assisted hatching with non-contact diode laser for zona drilling on day-3 post insemination in Group 1, and after the first warming episode on day-5 in Group 2 and on day-3 in Group 3.
Blastocyst morphologic assessment
All blastocysts were graded at first cryopreservation using modified Gardner and Cornell’s criteria [16]. We have further sub-categorized the B grade for trophectoderm and inner cell mass into a B + and a B − based on the number of cells present and how tightly packed or fragmented the cells are respectively. For this study, excellent and good quality blastocysts (AA, AB + , B + A, AB, BA, B + B + , AB − , BB) were considered as high quality (HQB), and any grade lower than this was categorised as low quality (LQB).
Trophectoderm biopsy and embryo vitrification
Embryos were biopsied on day 5 or 6 of their development when the embryos became blastocysts and several trophectoderm cells were herniating from the zona pellucida. From the hatching blastocyst, 5–10 trophectoderm cells were aspirated into the biopsy pipette. Laser pulses were aimed at the cell junctions just outside of the biopsy pipette while tension was created by further aspirating and pulling the engulfed cells from the blastocyst. Occasionally, manual excision was required by engulfing 5–10 cells within the biopsy pipette and rubbing the two pipette tips past each other with some pressure. Ploidy status was assessed by next generation sequencing (NGS). Prior to 2017, array comparative genomic hybridisation (aCGH) was implemented to determine the ploidy status of the biopsied embryos. Blastocysts that were not biopsied had artificial collapsing of the blastocoelic cavity by a laser pulse. All blastocysts were vitrified using Sydney IVF Blastocyst Vitrification Kit (Cook Medical), as soon as possible to avoid full re-expansion of the cavity.
Endometrial preparation
All FETs were performed with either hormone replacement treatment (HRT), during a natural cycle or with superovulation and were categorized as either HRT or ovulatory cycles (natural and superovulation). All HRT cycles had luteal support with intramuscular, oral and/or vaginal progesterone as described previously [17]. Ovulatory cycles had either no luteal support or were supplemented with Crinone® 8% (Merck Serono, UK), which was applied intravaginally twice daily.
Outcomes
The primary outcome was live birth and secondary outcomes were pregnancy, clinical pregnancy, and clinical miscarriage. Live birth rate (LBR) was defined as the number of live births after 24 completed weeks of pregnancy divided by the number of embryo transfer cycles. A twin birth for this data analysis was considered as one live birth. Pregnancy was defined as having a positive urine pregnancy test or serum β-hCG > 5 IU/L two weeks after an embryo transfer. Clinical pregnancy was defined as a pregnancy with one or more gestational sacs confirmed on ultrasound [18]. Clinical miscarriage was defined as the spontaneous loss of a clinical pregnancy before 24 weeks [18].
Statistical analysis
Continuous variables are expressed as median and interquartile range and categorical values are presented as percentages. Calculation of odds ratios (OR) and 95% confidence intervals (CI) was performed with generalised estimating equations (GEE) with an independent correlation matrix, as repeat cycles from each patient are not independent. Linear GEE was used for the analysis of continuous values in demographics and binomial GEE for the categorical values and outcomes. The outcomes are presented as unadjusted and adjusted values for the age at the oocyte collection, body mass index (BMI), endometrial thickness, semen source, endometrial preparation method, biopsy day, embryo quality category and any significant 2-way interactions. P values < 0.05 were considered statistically significant. Where there were no associations observed, to estimate the degree of certainty, instead of a retrospective power analysis we interpreted the confidence intervals of odds ratios according to rules previously proposed for the strength of observed associations. We calculated the Cohen’s d for the 95% CI of the ORs based on the outcome proportion of the control group, and with Cohen’s d of 0.2, 0.5 and 0.8 we reported a small, moderate or a significant likelihood that the two groups could differ if the group sizes were bigger [19, 20]. All statistical analyses were performed with IBM Corp. IBM SPSS statistics version 25, Armonk, NY, USA.
Ethics approval
All women received an explanation of the IVF process, the evidence regarding PGT-A including the uncertainties and risks, and the vitrification-warming process. Every woman signed institutional and Human Fertilisation and Embryology Authority (HFEA) consent forms prior to embarking upon their treatment. No woman or embryo was subjected to research and as such, ethical approval was not required for this retrospective cohort.
Results
The baseline characteristics of the 694 FET cycles are summarised in Table 1. Women in Group 1 underwent oocyte collection at a median age of 38 years (36–40). Women in Group 2 underwent oocyte retrieval at a significantly younger age (36 years, 34–38) compared to the other two groups (Group 1: 38 years; Group 3: 39 years P < 0.001). The mean time from oocyte retrieval to FET (1.8 years) was also significantly higher in group 2 (1.8 years) compared to Group 1 (0.6 years) and Group 3 (0.4 years), and the proportion of high quality embryos transferred was lower in Group 2 (50%) than in Group 1 (67%). There was no significant difference in BMI, endometrial thickness, semen source, endometrial preparation protocol or biopsy day between any of the groups.
Table 1.
Demographics
| Group 1 Biopsied Day-5—Vitrified |
Group 2 Vitrified day 5-Biopsied- Re-Vitrified |
Group 3 Vitrified Day 3- Biopsied day 5- Re-Vitrified |
Differences among groups | |
|---|---|---|---|---|
| N cycles | 451 | 146 | 97 | |
| Age at transfer (years, range) | 38 (36–40) | 38 (36–40) | 40 (38–42) |
1 vs 2 NS* 1 vs 3 < 0.001 2 vs 3 = 0.001 |
|
Age at Oocyte collection (years, range) |
38 (36–40) | 36 (34–38) | 39 (37–41) |
1 vs 2, 3 < 0.001 2 vs 3 < 0.001 |
| VEC to FET (years) |
0 (0–1) MM** 0.6 |
1 (1–3) MM 1.8 |
0 (0–1) MM 0.4 |
1 vs 2 < 0.001 1 vs 3 < 0.05 2 vs 3 < 0.001 |
| BMI (kg/m2) | 22.6 (20.7–25.3) | 21.7 (20.2–25.3) | 22.6 (20.7–26.0) | NS |
| Partner vs. Donor sperm | 94.9% | 91.8% | 92.8% | NS |
| HRT vs Ovulatory | 93.6% | 91.1% | 90.7% | NS |
| Endometrial thickness (mm, range) | 8.9 (8.1–10.0) | 8.8 (8.1–9.8) | 8.9 (8.1–10.3) | NS |
|
Biopsy day 5 vs 6 |
80.3% | 76.7% | 73.2% | NS |
| Embryo quality, % of transfers with HQB vs LQB | 67.8% | 50.0% | 57.7% |
1 vs 2 < 0.001 1 vs 3 NS 2 vs 3 NS |
Continuous variables are expressed as median and interquartile range and categorical values are presented as percentages
* NS: not significant
** MM: estimated Marginal Mean (Mean adjusted for the multiple cycles some patients had) is presented to highlight differences not obvious by the median.
HQB: High quality blastocyst, LQB: Low quality blastocyst
The three groups had similar odds of pregnancy, clinical pregnancy and live birth, even after adjusting for baseline characteristics (Table 2). Using Group 1 as the reference group, Group 2 had similar odds of pregnancy (aOR 1.09, 95% CI 0.62–1.91), clinical pregnancy (aOR 0.89, 95% CI 0.58–1.37) and live birth (aOR 0.85, 95% CI 0.56–1.28) after adjusting for baseline characteristics. Likewise, Group 3 had similar odds of pregnancy (aOR 1.22, 95% CI 0.74–1.99), clinical pregnancy (aOR 1.21, 95% CI 0.75–1.96) and live birth (aOR 1.15, 95% CI, 0.73–1.80). As a significant difference was not observed between the groups, we examined the adjusted CIs for pregnancy, clinical pregnancy and live birth and based on Cohen’s d values, the effect size is small suggesting that with a larger sample we could observe only a small difference in the groups compared. If we were to estimate the lower limit of that difference for the live birth rate, we could not exclude the possibility of the live birth rate for Group 2 and Group 3, being up to 14% and 8% lower than Group 1, respectively.
Table 2.
Pregnancy, live birth and miscarriage rate among the 3 groups
| Group 1 Biopsied Day-5—Vitrified |
Group 2 Vitrified day 5-Biopsied- Re-Vitrified |
Group 3 Vitrified Day 3- Biopsied day 5- Re-Vitrified |
(GEE) p, OR, CI 95% |
|
|---|---|---|---|---|
| Pregnancy rate | 65.2% (294/451) * | 67.8% (99/146) | 66.0% (64/97) | |
| Ref. group | 1.09 (0.62–1.91), p = 0.77 | 0.97 (0.61–1.53), p = 0.88 | Unadjusted | |
| 1.07 (0.68–1.64), p = 0.78 | 1.22 (0.74–1.99), p = 0.44 | Adjusted | ||
| Clinical pregnancy rate | 62.3% (281/451) | 62.3% (91/146) | 62.9% (61/97) | |
| Ref. Group | 1.00, (0.66–1.50), p = 0.99 | 1.02, (0.65–1.61), p = 0.91 | Unadjusted | |
| 0.89, (0.58–1.37), p = 0.60 | 1.21, (0.75–1.96), p = 0.44 | Adjusted | ||
| Live birth rate | 56.8% (256/451) | 54.8% (80/146) | 56.7% (55/97) | |
| Ref. Group | 0.93 (0.55–1.56), p = 0.77 | 1.00 (0.65–1.55), p = 0.99 | Unadjusted | |
| 0.85 (0.56–1.28), p = 0.43 | 1.15 (0.73–1.80), p = 0.54 | Adjusted | ||
| Clinical miscarriage | 7.8% (22/281) | 11.0% (10/91) | 9.8% (6/61) | |
| Ref. Group | 1.45, (0.67–3.17), p = 0.35 | 1.28, (0.50–3.30), p = 0.60 | Unadjusted | |
| 1.35, (0.59–3.06), p = 0.47 | 1.24, (0.50–3.10), p = 0.64 | Adjusted |
The outcomes are presented as unadjusted and adjusted values for age at the oocyte collection, BMI, endometrial thickness, semen source, endometrial preparation method, biopsy day, embryo quality category and any of their 2-way interactions that were statistically significant.
*The percentages for reproductive outcomes are used to demonstrate the magnitude of the outcomes and are not directly comparable. Since patients had more than one treatment cycle included in the study and as the treatment cycles of the same patient are not independent of each other, the outcomes can be compared with the Odd Ratios from GEE.
In the regression model, increasing age at the oocyte collection was associated with lower odds of pregnancy (aOR 0.93, 95% CI 0.89–0.98, p = 0.005), clinical pregnancy (aOR 0.92, 95% CI 0.88–0.97, p = 0.001) and live birth (aOR 0.94, 95% CI 0.90–0.98, p = 0.009). While embryo quality or the day of the biopsy was not predictive of these outcomes, their interaction was a significant predictor suggesting a potential synergistic effect. Transferring a LQB that underwent the biopsy on day 6 was associated with significantly lower odds of pregnancy (aOR 0.19, 95% CI 0.08–0.43, p < 0.001), clinical pregnancy (aOR 0.20, 95% CI 0.09–0.45, p < 0.0001), and live birth (aOR 0.31, 95% CI 0.14–0.68, p = 0.003), compared to transferring a HQB that had the biopsy on day 5.
There was also no significant difference in the odds of clinical miscarriage between the groups even after adjusting for baseline characteristics (Table 2). As there was a small number of clinical miscarriages in our cohort, the confidence interval of the adjusted odds ratio for Group 2 and Group 3 is wide and examining the Cohen’s d value, we cannot exclude the possibility of identifying up to a moderate difference if the study population was bigger. If we were to estimate the upper limit of that difference, it could be up to 13% higher for Group 2 and Group 3 compared to Group 1, which while statistically possible, has not been observed to date in any patient group having PGT-A in the literature. In the regression model neither age, embryo quality, day of biopsy nor their interaction appeared to be significant predictors for clinical miscarriage rate, suggesting that these factors are likely to be more important for implantation than for maintaining a pregnancy.
Discussion
Our findings are in agreement with other studies which have shown that pregnancy, clinical pregnancy, live birth and miscarriage rates are similar for euploid embryos cryopreserved once or twice. This has important clinical significance, providing essential and reassuring information for women wishing to undergo the process of PGT-A on embryos already in storage. We demonstrate herein that embryos that have been vitrified-warmed twice at blastocyst stage (Group 2) led to similar pregnancy, live birth and miscarriage rate to euploid embryos vitrified once (Group 1). While this is in agreement with an early small study (N = 17) [21], a subsequent study (N = 37) suggested that additional cryopreservation procedures are associated with a lower live birth per transfer, although the findings were not statistically significant likely due to the small study numbers (Group 1 vs Group 2, LBR 50.0% vs 38.5%) [13]. The largest study investigating euploid embryos cryopreserved twice (N = 155) [12] demonstrated similar ongoing pregnancy and clinical miscarriage rates to embryos vitrified once (63.2% vs 66.8%, 15.5% vs 9.8%, respectively), although live birth rate was not reported.
A recent study reporting live birth rate suggested that biopsied blastocysts vitrified-warmed twice (Group 2, N = 95) vs vitrified-warmed once (Group 1, N = 2603) had a significantly lower live birth rate (adj. RR 0.57, 95% CI 0.41 to 0.79), but a similar miscarriage rate [14]. The live birth rate reported for those vitrified-warmed twice (28.4%) was much lower than that reported in our study (54.5%), and other literature for the same group of patients 38.5% in Bradley et al. [13] and 50.0% in Taylor et al. [21]. This difference may be attributed to many factors including laboratory protocols and culture media, vitrification and embryo biopsy techniques. As it has been previously demonstrated that the removal of more DNA from an embryo is associated with lower pregnancy rates [22], the significant variation between studies may also be explained by the amount of DNA removed using different embryo biopsy techniques.
In our study, embryos cryopreserved on day 3, warmed and then cultured to blastocyst stage, prior to biopsy and re-vitrification (Group 3) had similar pregnancy rate, clinical pregnancy, live birth rate and miscarriage rate with cycles with euploid embryos cryopreserved once (Group 1). Early case series observing embryos slow-frozen on day 3 and refrozen on day 5, not biopsied for PGT-A, reported higher miscarriage rate [9, 10] and lower clinical pregnancy [23] and live birth rates [10]. The improved outcomes demonstrated herein are highly likely to be related to the utilisation of vitrification as a cryopreservation technique. The only recent study including a similar cohort to our Group 3 (euploid embryos vitrified day 3- biopsied day 5 and re-vitrified) reported outcomes together with those cryopreserved twice at blastocyst stage [24]. While it reported similar clinical pregnancy rates to cycles with those cryopreserved once, we cannot directly compare it to our findings.
Our study also suggested that the age at the oocyte collection was a significant predicting factor for pregnancy, clinical pregnancy and live birth rates. This finding is in agreement with a recent study with > 8000 euploid blastocyst transfers, which confirmed that increasing age is associated with decreasing live birth rate [25]. A smaller study with 750 euploid transfers did not confirm a lower implantation potential of euploid embryos with maternal age but suggested a lower number of euploid embryos available, which aligns with the biological concept of increasing aneuploidy rate with advancing age [26].
Our regression analysis did not find the day of embryo biopsy or the embryo quality to be independent factors for any of the outcomes, but their interaction was predicting pregnancy, clinical pregnancy and live birth rate. This is consistent with other studies suggesting that embryos biopsied on day 5 have better live birth rates compared to embryos biopsied on day 6 [25, 27–29] and that blastocyst morphology [27–32] and blastocyst expansion [29] are significant factors affecting live birth.
Our study is limited by including outcomes from a single embryology laboratory, which creates the possibility for bias and therefore limits applicability to other laboratories at different IVF centres. However, this may also highlight the importance of optimising and standardising laboratory techniques and protocols in order to maximise PGT-A outcomes. This can then in return be applied across multiple centres to assess whether such outcomes are sustained. As our study did not observe a significant difference in the outcomes, the strength of the observations is related to the effect size and examining the CIs, we cannot exclude a small true difference among the groups for the OR of pregnancy, clinical pregnancy and live birth. As the number of miscarriages observed in our cohort was low, we cannot exclude a moderate true difference between the groups, which would require a study with significantly higher numbers of participants. As our study is not aiming to change the standard practice of performing a biopsy at the blastocyst stage and freezing and thawing once before transfer (Group 1), the above uncertainty may be acceptable for patients that have to take a different to the standard approach. While we could not adjust for the number of previous miscarriages which appears to be a significant predicting factor [28], our study reports similar live birth rates following euploid transfers of embryos cryopreserved at two different developmental stages. While previous studies have suggested normal neonatal outcomes for twice cryopreserved-warmed embryos [10, 11, 13], further studies are required to assess the long-term health of resultant offspring.
Conclusion
Our study suggests that vitrifying and warming a euploid embryo twice at blastocyst stage or at both cleavage and blastocyst stage can lead to the same reproductive outcomes of euploid embryos vitrified-warmed once. Whereas this does not condone or support unnecessary embryo manipulation, our findings can be used to help counsel women considering PGT-A who have already cryopreserved embryos of uncertain genetic status.
Author contribution
Efstathios Theodorou conceived and designed the study, performed the statistical analysis, and wrote the first draft manuscript. Efstathios Theodorou, Daniella Cardenas and Carleen Heath collected and verified the data. Benjamin Jones and Jara Ben Nagi helped write and revise the manuscript. All authors critically reviewed, revised the manuscript and approved the final version.
Data availability
Not applicable.
Code availability
Not applicable.
Declarations
Ethics approval and consent to participate
As this is an observational study the Internal Ethics Committee has confirmed that no ethical approval is required.
Consent for publication
Not applicable.
Conflict of interest
The authors declare no competing interests.
Footnotes
Publisher's note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
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