Effect of vitrification on clinical outcomes of cleavage-stage embryos with poor quality in human embryo cryopreservation : Reproductive and Developmental Medicine

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Original Article

Effect of vitrification on clinical outcomes of cleavage-stage embryos with poor quality in human embryo cryopreservation

Liu, Tao1, 2, 3, 4; Lian, Ying1, 2, 3, 4; Liu, Ping1, 2, 3, 4; Li, Rong1, 2, 3, 4; Yan, Jie1, 2, 3, 4,*; Qiao, Jie1, 2, 3, 4, 5, 6

Author Information
Reproductive and Developmental Medicine: March 2022 - Volume 6 - Issue 1 - p 20-25
doi: 10.1097/RD9.0000000000000004
  • Open

Abstract

Introduction

Embryo cryopreservation is an important and essential therapeutic step and standard procedure during the in vitro fertilization-embryo transfer (IVF-ET) cycle[1,2]. Embryo cryopreservation improves clinical outcomes by increasing the cumulative pregnancy rate (PR) in a controlled ovarian hyperstimulation cycle[3] while facilitating the choice of an adequate cycle to thaw and transfer the embryo to prevent ovarian hyperstimulation syndrome and/or delay embryo transfer in patients suffering from infection[4]. Importantly, it also increases the success rate of singleton live births via singleembryo transfer[5].

Following the development of assisted reproductive technology, different embryo cryopreservation technologies have been established. To date, embryo cryopreservation has included 2 different methods: slow freezing and vitrification. The first successful pregnancy was via a slow-cooled embryo in 1983[6]. Recently, vitrification has been increasingly used in the IVF-ET cycle as it can reduce the cryoprotectant concentration and prevent intra- or extra-cellular ice formation[7] as well as improve the post-thaw survival, clinical pregnancy, and live delivery rate[8-10]. However, conflicting studies have described the limitations of vitrification in animal models and human oocyte or embryo cryopreservation[11,12].

Many factors may affect the clinical outcomes of embryo vitrification, including the protocol of vitrification and the different developmental stages of the embryos[13]. A direct relationship between developmental potential and embryo quality has already been reported[14,15]. However, few studies have systematically analyzed the influence of embryonic factors in early cleavage-stage embryos on the success of embryo cryopreservation[16,17]. Therefore, this study aimed to analyze the relationship between the quality of early cleavage-stage embryos and clinical outcomes after vitrification. Here, we compared the survival of embryos after vitrification and warming, as well as the implantation and PRs.

Materials and methods

This was a retrospective study of the relationship between the parameters of cleavage-stage embryos before vitrification and the clinical outcomes of vitrification for cleavage-stage embryos on day 3. The study was approved by the Institutional Review Board of Peking University Third Hospital (2013S2017), and all patients provided written informed consent before participating in the study. Frozen embryo transfer (FET) cycles were performed between 2012 and 2013 at the Infertility Unit of the Peking University Third Hospital. Analysis was performed on 921 FET cycles performed on day 3 post-ovulation. Single-embryo transfer cycles and cycles in which there were 2 or 3 embryos with the same morphology were transferred and selected for this study.

Embryo culture and assessment

Controlled ovarian stimulation protocols and laboratory procedures were performed as described previously[18]. Metaphase II oocytes were subjected to routine fertilization and/or intracytoplasmic sperm injection between 36 and 38 hours after human chorionic gonadotropin (hCG) administration. On day 3, the 2 best embryos were transferred. When the number of remaining embryos was ≤4 or if the quality was poor, the remaining embryos were allocated for vitrification for cryopreservation[19-21]. Embryo quality was assessed according to morphology before vitrification, and embryos were grouped on day 3 according to the number of blastomeres (4 blastomeres, 5 to 6 blastomeres, 7 to 8 blastomeres, and 9 blastomeres); symmetry (equal size and unequal size); and cytoplasmic fragmentation (≤10%, 10%-20%, 20%-50%). A good quality embryo was defined as an embryo with >6 blastomeres of equal size, and the cytoplasmic fragmentation was <20%. Each embryo grading was reviewed in real-time by 2 senior embryologists for verification and consistency.

Embryo vitrification and warming

Embryos were vitrified in a Cryotop (Kitazato, Japan) after a 3-step procedure at 37 °C. First, embryos were transferred into a basal solution (Quinns Advantage medium with HEPES (SAGE, Trumbull, CT) containing 20% (v/v) human serum albumin (HSA, Vitrolife, Sweden) for 1-minute pre-equilibration. Blastocysts were then transferred into an equilibration solution consisting of 7.5% (v/v) ethylene glycol (Sigma Chemical Co., MO) and 7.5% (v/v) dimethyl sulfoxide (Sigma) in basal solution. After 2 minutes, embryos were transferred into a vitrification solution consisting of 15% (v/v) ethylene glycol, 15% (v/v) dimethyl sulfoxide, and 0.65 mol/L sucrose in basal solution. While the embryos were in the vitrification solution, 2 to 4 were loaded onto the surface of the Cryotop. The Cryotop was immediately plunged into liquid nitrogen. The Cryotops were transferred to standard canes and stored in liquid nitrogen.

For embryo warming, a 3-step procedure was performed at 37 °C. The carrier was removed from the liquid nitrogen tank. After removing the protective sleeve, the Cryotop was quickly immersed into warming solution 1 (basal solution containing 0.33 mol/L sucrose). After 2 minutes, embryos were transferred into warming solution 2 (basal solution containing 0.2 mol/L sucrose) for 3 minutes; next, embryos were transferred into the basal solution for 5 minutes, and finally, embryos were transferred into culturing medium (G-2, Vitrolife, Sweden) and incubated in a Galaxy incubator (Germany Eppendorf Company) with 6% CO2, 5% O2, and 89% N2 for 2 hours. Embryo survival was evaluated as the percentage and the number of intact surviving cells before transfer. An embryo was considered to have survived if ≥50% of the initial number of blastomeres were intact. Embryo morphology was evaluated after warming by 2 senior embryologists.

Endometrial preparation therapy and embryo transfer

In 109 FET cycles, endometrial preparation therapy included the natural cycle, programmed ovarian stimulation cycle, and microstimulation protocols[18]. One to 2 surviving embryos with 4 or more blastomeres underwent embryo transfer. Luteal support was provided with intra-muscular injections of progesterone (20-40 mg) from the night of the transfer. Serum hCG levels were measured 14 days after transfer. An ultrasound examination was performed between 4 and 5 weeks after a positive pregnancy test. Clinical pregnancy was defined as the presence of high levels of ß-hCG. Implantation was defined as the detection of the gestational sac by ultrasonography 4 to 5 weeks after embryo transfer.

Statistics

Fisher's exact and Chi-squared tests were used to assess differences between categorical variables using SPSS version 19.0 (IBM Corp., Armonk, NY). Statistical significance was set at P < 0.05. Bonferroni's method was used to determine the correlation of P values in pairwise comparisons.

Results

The background characteristics of the study cycles are presented in Table 1. Patient age varied from 23 to 56 years, with a mean of 33.05 ± 4.14 years. The body mass index was 21.90 ± 3.09 kg/m2, with the duration of infertility being 4.86 ± 3.24 years. The main factors associated with infertility were tubal (41.28%) and male factors (33.94%). Ovarian dysfunction accounted for 23.85% of cases, while uterine and unknown factors accounted for 8.26% and 0.92% of cases, respectively. The number of embryos available for transfer was approximately 5.26 ± 1.85 in each patient.

Table 1 - The background characteristics of the study cycles

Parameters

Data (mean ± SD)

Cycles (n)

921

Maternal age (year)

33.05 ± 4.14

BMI (kg/m2)

21.90 ± 3.09

Duration of infertility (year)

4.86 ± 3.24

Main diagnosis % (n)

Tubal factor

41.28%

Male factor

33.94%

Premature ovarian failure

16.51%

Uterine factor

8.26%

Anovulation

7.34%

Unknown

0.92%

Basal endocrine

FSH (mIU/mL)

6.21 ± 2.19

E2 (pmol/L)

169.80 ± 106.91

P (nmol/mL)

1.20 ± 0.71

PRL (ng/mL)

13.37 ± 6.71

LH (mIU/mL)

4.06 ± 2.75

T (nmol/mL)

3.48 ± 8.68

A (nmol/mL)

6.92 ± 3.52

Antral follicles count

5.69 ± 3.45

Oocyte retrieval (n)

14.70 ± 5.00

Fertilization rate

75.42%

Number of embryos available for transfer

5.26 ± 1.85

Number of embryos cryopreserved

4.20 ± 1.83

Endometrial thickness (mm)

9.87 ± 1.365

Type of FET therapy

Natural

55.0%

Stimulation

30.3%

Microstimulation

12.8%

A: Androsterone; BMI: Body mass index; E2: Estradiol; FET: Frozen embryo transfer; FSH: Follicle-stimulating hormone; LH: Luteinizing hormone; P: Gesterone; PRL: Prolactin; SD: Standard deviation; T: Testosterone.


When we analyzed the embryo survival rates (SRs) according to the number of blastomeres, the percentage of cytoplasmic fragments and blastomere symmetry differed between groups, and the overall SR of good quality embryos was 91.6% (381/416). Specifically, embryos with >9 blastomeres yielded 100% survival, and embryos with 7 to 8 blastomeres yielded SRs of 86.3%. No statistically significant difference was observed between these 2 groups. However, a lower SR was found in embryos with 5 to 6 blastomeres (57.5%), and the lowest SR was observed in embryos with only 4 blastomeres (41.4%). The differences compared to the other 2 groups were statistically significant. In terms of blastomere symmetry, the SRs of embryos with equal sized blastomeres were significantly higher than those of embryos with unequal sized blastomeres (82.5% vs. 64.6%, P < 0.05). When grouped according to the degree of fragmentation, we found that as fragmentation increased, the SR declined from 92.1% to 20.6% (P < 0.05). Embryos with >20% fragmentation showed only 20.6% survival after vitrification (Table 2).

Table 2 - Survival rates and embryo parameters

Parameters

Number of surviving embryos

Number of vitrified embryos

Survival rate (%)

Number of blastomeres

4

41

99

41.4*

5-6

181

315

57.5

7-8

546

633

86.3

≥9

33

33

100

P < 0.05

Blastomere symmetry

Equal

476

577

82.5

Unequal

325

503

64.6

P < 0.05

Cytoplasmic fragmentation

≤10%

443

481

92.1*

10%-20%

351

565

62.1

20%-50%

7

34

20.6

P < 0.05

*,†,‡ Rate followed by different letters are statistically different in pairwise comparison.

Table 3 - Pregnancy and implantation rates according to embryo parameters before vitrification

Parameters

Number of pregnancies

Number of FET cycles

Pregnancy rate (%)

Number of implanted embryos

Number of embryos transferred

Implantation rate (%)

Number of total blastomeres transferred

<8

4

21

19.0*,†

4

21

19.0*,†

8-12

15

76

19.7*

15

123

12.2*

13-16

102

258

39.5

126

523

24.1

>16

18

52

34.6*,†

20

134

14.9*,†

P < 0.05

P < 0.05

Blastomere symmetry

Equal

62

170

36.5*

75

316

23.7*

Unequal

20

92

21.7

21

170

12.4

P < 0.05

P < 0.05

Cytoplasmic fragmentation

≤10%

60

163

36.8

67

296

22.6

10%-20%

33

103

32.0

40

201

19.9

20%-50%

0

1

0NA

0

2

0NA

P > 0.05

P > 0.05

FET: Frozen embryo transfer; NA: Not analyzed statistically.

*,† Rate followed by different letters are statistically different in pairwise comparison.

When we analyzed pregnancy and implantation rates (IRs) according to the 3 embryonic parameters before vitrification, there were significant differences between the groups (Table 3). In terms of the total number of 1 or 2 embryos' blastomeres transferred in each FET cycle, embryos with 13 to 16 blastomeres before vitrification yielded the highest PR and IR (39.5% and 24.1%, respectively), although the difference was not statistically significant. In terms of blastomere symmetry, PRs and IRs of embryos with equally sized blastomeres were significantly higher than those of asymmetrical embryos (36.5% vs. 21.7% and 23.7% vs. 12.4%, respectively, P < 0.05). In terms of fragmentation, increased fragmentation was associated with a decrease in pregnancy and IRs; however, the difference did not reach significance (P>0.05). It should be noted that the number of pregnancies from embryos with 20% to 50% fragmentation before vitrification was too low to include them in the statistical analysis of the IR.

Table 4 - Pregnancy and implantation rates according to embryo parameters after warming

Parameters

Number of pregnancies

Number of FET cycles

Pregnancy rate (%)

Number of Implanted embryos

Number of embryos transferred

Implantation rate (%)

Number of total blastomeres transferred

<8

5

30

16.7*,†

5

30

16.7*,†

8-12

26

110

23.6*

28

199

14.1*

13-16

95

232

40.9

116

480

24.2

>16

14

35

40.0*,†

16

88

18.2*,†

P < 0.05

P < 0.05

The percentage of intact blastomeres

100%

102

267

38.2

121

521

23.2*

80%-100%

25

84

29.8

29

174

16.7*

50%-80%

13

56

23.2

15

106

14.2*

P > 0.05

P < 0.05

FET: Frozen embryo transfer.

*,† Rate followed by different letters are statistically different in pairwise comparison.

In the case of cryopreserved embryos, the percentage of blastomeres that survived the freeze-thawing process and the total number of intact blastomeres from all embryos in each FET cycle were also determined and included in the analysis. Embryos with 13 to 16 blastomeres after warming yielded the highest pregnancy (40.9%, P < 0.05) and IRs (24.2%, P < 0.05). Higher percentages of intact blastomeres were associated with higher pregnancy and IRs, increasing from 23.2% to 38.2% and 14.2% to 23.2%, respectively (Table 4).

Discussion

For some time, vitrification has been recognized as a more effective freezing protocol for embryo cryopreservation than slow freezing methods[8,22]. The pregnancy outcomes achieved using different vitrification protocols were excellent[7,23,24]. However, our experience suggests that vitrification does not improve clinical outcomes for certain types of embryos of relatively poor quality. The number of blastomeres, their symmetry, and the level of cytoplasmic fragmentation are the main morphological parameters associated with the survival and implantation capacity of an embryo in FET cycles using vitrification.

We have extensive experience using vitrification for human embryo cryopreservation, and our vitrification system is highly consistent[25]. The present study, involving >1,000 warmed embryos and a larger sample size compared to that from any previous study, confirms the inferior outcomes of the vitrification system for embryos of poor quality.

We evaluated the open vitrification of embryos of different qualities according to their morphology and observed SRs varying from 20.6% to 100.0%, clinical PRs of 12.5% to 39.7%, and IRs of 0% to 23.5%.

With embryos of good quality, vitrification yielded better pregnancy outcomes, which were similar to the rates of survival (94.8%-96.9%), clinical pregnancy (40.5%-59.0%), and implantation (16.6%-49.0%) reported in previous studies[7,26]. In contrast, with embryos of poor quality, pregnancy outcomes were slightly poorer than those observed with good quality embryos in terms of survival, pregnancy, and IRs.

When analyzing the correlation between embryo quality and survival, we found that the SR was positively correlated with the number of blastomeres on day 3; thus, development of embryos from 4 cells to >9 cells increased their survival from 41.4% to 100.0%. It is not surprising to find that a higher number of blastomeres was associated with a greater ability to maintain intact blastomeres when exposed to freezing damage, consistent with the findings of previous studies[16,27].

Furthermore, the relationship between the implantation capacity of an embryo in FET cycles and the number of blastomeres before freezing and after warming was analyzed. Cryopreservation of embryos with a total number of 13 to 16 blastomeres yielded the best pregnancy and IRs, while embryos vitrified with <13 or >16 blastomeres produced poorer outcomes. Similarly, after warming, the surviving embryos with a total number of 13 to 16 blastomeres yielded the best pregnancy and IRs, while for embryos with <13 or >16 cells, the rates were reduced. This finding is consistent with those of previous studies on embryo cryopreservation by slow freezing and vitrification[27,28]. In conclusion, embryos dividing at a slower or faster rate are not conducive to successful implantation[16].

In the case of cryopreserved embryos, the percentage of blastomeres that survived the freeze-warming process was added to the analysis. Our data indicated that lesser the damage observed, the better the implantation and PRs. This is consistent with the results of a previous study[29]. This may be owing to the percentage of blastomeres that survive the freeze-warming process indicating the embryo's ability to resist freezing damage, or it could reflect the developmental potential of the embryo itself. Previously, it was found that an embryo with better developmental potential had a higher ability to survive freezing damage and yielded better implantation results[16].

Another important feature of the embryo, which influences vitrification efficiency, is the blastomere symmetry. According to our results, significantly higher rates of survival, implantation, and pregnancy were observed in freeze-thawed embryos with equal blastomere symmetry before freezing than in those with unequal blastomere symmetry. These results are consistent with those of previous reports on fresh embryo transfer cycles and FET cycles by slow freezing[16,28]. We hypothesized that uneven cleavage may result in embryos with a higher degree of aneuploidy and/or multinuclear cells, which in turn might help to elucidate their low IRs[30,31]. To the best of our knowledge, there are no published studies on the relationship between blastomere symmetry in fresh embryos and outcomes of FET cycles.

More importantly, for the first time, fragmentation has been proven to be an important factor influencing vitrification outcomes. Although embryos with a high degree (>50%) of fragmentation were not cryopreserved in our study, we explored whether embryo fragmentation affected survival and IRs (0%-50%) before vitrification. The degree of fragmentation of embryos before freezing was negatively correlated with survival after warming. When pregnancy and IRs were analyzed, no significant differences were detected between the groups (≤10% vs. 10%-20%). The cut-off point was established at 20% fragmentation in terms of implantation capacity in vitrification cycles, while 20% to 35% was the cut-off point most widely used in slow freezing cycles[16,32,33]. The number of surviving embryos with 20% to 50% fragmentation was highly limited; therefore, few surviving embryos were available for transfer. Thus, we did not perform a statistical analysis of their implantation and PRs. The relatively poor prognosis of day 3 embryos with serious fragmentation may be related to potential damage to their metabolism or epigenetic modifications caused by the vitrification process[7,11,34].

These data indicate that vitrification methods do not effectively improve the survival outcome of embryos with serious fragmentation. The data described in the present study provide solid evidence for the inadequacy of this strategy. Although it confirmed the limitation of the current vitrification protocol, in contrast, it suggested that other strategies should be employed to avoid pregnancy losses caused by embryo cryopreservation on day 3. Additionally, the lower SRs after warming and the lower IRs observed in cycles using vitrified embryos with >20% fragmentation on day 3 showed that the vitrification procedures might negatively impact this particular status of development, similar to slow freezing cycles, while embryos of good quality can be cryopreserved with equal success using slow cooling and vitrification[17]. When preserving embryos with serious fragmentation, the key question is not which cryopreservation protocol should be used, but to change the laboratory strategy altogether, such as by extending the culture time for the cleavage-stage embryo to obtain blastocyst development and cryopreservation as the benefit of this alternative over cleavage-stage vitrification and transfer could be significant[35,36].

In conclusion, this study presented solid evidence to confirm the limitations of vitrification in day 3 embryos. It will guide clinical practice to achieve a comprehensive analysis of the practical aspects of vitrification, including concerns and options regarding its limitation for cleavage-stage embryos of poor quality. However, there is a strong need to perform prospective randomized clinical trials and live birth reports, which would permit assessment of the impact of differences in cryopreservation strategies. These findings will help elucidate the practical indications for the embryo cryopreservation method to improve the cumulative success rate.

Acknowledgments

We are grateful to Ms. Ying Huo from Peking University for her contribution to the language revision in this study.

Author contributions

J.Y. and J.Q. designed the review. X.S. and R.L. provided several writing ideas in the review and gave supervision. T.L. wrote the manuscript. J.Q., M.T. and R.Y. revised the manuscript and provided edits. All authors contributed to the final manuscript and approved the submitted version.

Funding(s)

This work was supported by the China National Key R&D Program (2018YFC1004001), the Research Units of Comprehensive Diagnosis and Treatment of Oocyte Maturation Arrest (2019-I2M-5-001), and the Special Research Project of Chinese Capital Health Development (2018-2-4095).

Conflicts of interest

All authors declare no conflict of interest.

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Keywords:

Cleavage stage; Embryo vitrification; Embryological factors; Fragmentation; Poor quality

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