There are many reasons why women choose to freeze their embryos instead of trying to transfer them immediately. The pregnancy and birth results of freezing all embryos have been shown to be just as effective as fresh embryo transfers; some studies have shown that frozen embryos produce more pregnancies than fresh transfers. For many women who, for personal choice or for medical necessity (impending cancer treatment), this is now a solid option. Also, for those who prefer to have genetic testing conducted prior to transfer, freezing is almost always required because it takes too much time to report genetic results before an embryo loses viability in culture.

Possible risks of embryo freezing include the following: bacterial contamination, ice crystal formation, premature warming, and reduced viability over time. Today’s study looks at that last risk, comparing how embryos fare over time.

Study Background

WHAT

• Does the storage time of a frozen embryo impact rates of pregnancy?

• Does the storage time of a frozen embryo impact newborns? Does it cause early delivery? Does it change birth weight? Does it cause birth defects?

WHERE

• a large, hospital-based reproductive center in Shanghai, China

WHEN

• January 2011 - December 2017

WHO

• Inclusion criteria: any type of infertility diagnosis or patient preference leading to a freeze-all embryo cycle (includes women with diminished ovarian reserve, polycystic ovary syndrome (PCOS), poor ovarian response, advanced maternal age, tubal factor, male factor, and endometriosis)

• Exclusion criteria: prior fresh or frozen embryo transfers, transferring non-biological embryos, pre-implantation genetic testing (PGD) of embryos, or transfer of mixed cleavage-stage embryos-blastocysts

HOW

Frozen Embryo Creation

• Ovulation induction (protocols not stated) then IVF +/- ICSI

• Starting Day 0: embryos in single-culture medium (Irvine Scientific, USA) in incubator (Astec, Japan) at 5% CO2/5% O2 at 98.6°F/ 37°C

• Starting Day 3:

  • blastocyst assessment, double-checked by senior embryologist—> retained blastocysts with quality higher than Grade 3CC

  • blastocyst cryopreservation by vitrification

    • equilibration solution = mHTF medium (Irvine Scientific, USA) + 7.5% (v/v) ethylene glycol + 7.5% dimethysulfoxide (DMSO) + 20% synthetic serum substitute (SSS)

    • vitrification solution = mHTF medium with 15% ethylene glycol, 15% DMSO, 0.5 mol/L sucrose and 20% SSS

    • final solution = liquid nitrogen (USA) at -320.8°F/ 196°C

Frozen Embryo Thawing

• Thawed in “basic medium” + 0.5-1.0 mol/L sucrose and 20% SSS

• Washed in “basic medium” + 20% SSS

• Cultured until transfer in mHTF medium + 10% SSS in incubator at 5% CO2/5% O2 at 98.6°F/ 37°C

Female Patient

• Completed natural, stimulated, or hormone induction pre-transfer cycles, per patient health

• Received 1-2 embryos in blastocyst or cleavage-stage, per patient age/embryo quality/embryo quantity

• Took progesterone (formulation not specified) from transfer date until eight weeks of pregnancy

• At 14 days post-transfer, blood test for hCG

• At 35 days post-transfer, vaginal ultrasound if hCG +

Statistics (for the uber curious)

• Controlled for possible confounders, including maternal age, maternal BMI, infertility type, parity, infertility causes, embryo quality, number of transferred embryos, stage of embryo development, endometrial preparation program and treatment years

• Reported as adjusted odds ratios (aOR) with 95% CI using two-sided significance of 5%

• See page 3 of study for further details

Results

There were four embryo groups. Group 1 were embryos with a storage time of 0–3 months; Group 2 was embryos with a storage time 3–6 months; Group 3 had a storage time 6–12 months and Group 4 had a storage time 12–24 months. This study excluded transfers of embryos stored longer than 24 months.

Pregnancy: This study found that the rate of clinical pregnancy dropped from 56% in Group 1 (which had the embryos frozen less than three months) to 26% pregnancy rates in Group 4 (the group that had storage times of 12-24 months). The live birth rates decreased as well, with Group 1 having 47% of women give birth vs. 26% in Group 4. Rates of miscarriage and ectopic pregnancy increased with prolonged storage time, but these numbers were not statistically significant, which means that these differences could have occurred due to random chance.

Newborns: The happiest results of the study were that, regardless of how long the embryo was frozen, it did NOT impact the health of the newborn. Storage time did NOT lead to a difference in birth defects, being born prematurely, or having an unhealthy birth weight. Notably, when assessing newborn outcomes, the authors did NOT include twin births, which may have skewed the results.

Authors’ Thoughts

• Recognized that women with diminished ovarian reserve (aka poor ovarian reserve) tended to store embryos longer

• Acknowledged main limitations of study were retrospective nature and lack of long-term follow-up of newborns

This Pharmacist’s Thoughts

• Study Strengths

  • Large enough to detect differences if differences exist

  • Well written article

    • Authors clearly understood previous research

    • Process of vitrification explained well

• Study Weaknesses

  • Did variations within the study impact the results?

    • What were the doses and formulations of the progesterone supplements? Did they vary based on patient factors or clinician preference?

    • Natural vs. artificial cycles to prepare for transfer

    • Transfer of one vs. two embryos

      • 87.6% Group 1 vs. 68.7% Group 4 had two embryos transferred

      • twins born were excluded from neonatal outcomes

    • Transfer of blastocyst vs. cleavage-stage embryo

      • Included more cleavage-stage embryos though blastocysts tend to thaw better

      • Not generalizable to other IVF centers based on variations at this single IVF center

  • Women with low ovarian reserve tended to store embryos longer - 3.8% in Group 1 vs. 13.25% in Group 4 - how did this impact the results?

Time will tell how these results compare to those at other reproductive centers. In progress is a randomized controlled trial that involves more reproductive centers and it includes embryos frozen more recently (2015-2020).

Resources

Alikani M. Cryostorage of human gametes and embryos: a reckoning. Reprod Biomed Online. 2018;37(1):1-3. doi:10.1016/j.rbmo.2018.05.004

Maheshwari A, Bhattacharya S, Bowler U, et al. Study protocol: E-freeze - freezing of embryos in assisted conception: a randomised controlled trial evaluating the clinical and cost effectiveness of a policy of freezing embryos followed by thawed frozen embryo transfer compared with a policy of fresh embryo transfer, in women undergoing in vitro fertilisation. Reprod Health. 2019;16(1):81. Published 2019 Jun 13. doi:10.1186/s12978-019-0737-2

Nagy ZP, Shapiro D, Chang CC. Vitrification of the human embryo: a more efficient and safer in vitro fertilization treatment. Fertil Steril. 2020;113(2):241-247. doi:10.1016/j.fertnstert.2019.12.009

Rezazadeh Valojerdi M, Eftekhari-Yazdi P, Karimian L, Hassani F, Movaghar B. Vitrification versus slow freezing gives excellent survival, post warming embryo morphology and pregnancy outcomes for human cleaved embryos. J Assist Reprod Genet. 2009;26(6):347-354. doi:10.1007/s10815-009-9318-6