The Onco Fertility Journal

: 2019  |  Volume : 2  |  Issue : 1  |  Page : 41--45

Should we discard cryopreserved embryos with poor post-thaw survival? – Report of a successful pregnancy with <50% blastomere survival

Surleen Kaur1, Snigdha Pathak2, Nidhi Sehrawet2, Manika Saxena1,  
1 Reproductive Medicine Unit, Ferticity Fertility Clinics, New Delhi, India
2 Department of Reproductive Medicine, Mother and Child Hospital, New Delhi, India

Correspondence Address:
Dr. Surleen Kaur
Ferticity Fertility Clinics, 12, Navjeevan Vihar, Malviya Nagar, Delhi - 110 017


The objective of the present study is to report a case of successful ongoing pregnancy following transfer of two cleavage-stage day 3 embryos with one-two blastomeres survival. The study was conducted at a private clinical-assisted reproductive technology center. A 31-year-old female with a 3-year history of primary infertility underwent an in vitro fertilization cycle followed by a frozen embryo transfer cycle. The main outcome measure was clinical pregnancy. Ultrasonographic examination at 6 weeks revealed a single gestational sac with positive cardiac activity. Follow-up scans at 10, 12, and 14 weeks of pregnancy showed fetal age parameters corresponding to gestational age and normal value of nuchal translucency. Noninvasive prenatal screening test confirmed a euploid fetal development. Transfer of frozen-thawed embryos with only one–two blastomeres survival resulted in an ongoing pregnancy with euploid fetus development; therefore, we suggest that embryo with <50% post-thaw survival should not be discarded and considered as a viable embryo.

How to cite this article:
Kaur S, Pathak S, Sehrawet N, Saxena M. Should we discard cryopreserved embryos with poor post-thaw survival? – Report of a successful pregnancy with <50% blastomere survival.Onco Fertil J 2019;2:41-45

How to cite this URL:
Kaur S, Pathak S, Sehrawet N, Saxena M. Should we discard cryopreserved embryos with poor post-thaw survival? – Report of a successful pregnancy with <50% blastomere survival. Onco Fertil J [serial online] 2019 [cited 2022 May 28 ];2:41-45
Available from:

Full Text


Cryopreservation of embryos in assisted reproductive technology treatment cycles opens the opportunity to either freeze supernumerary embryos or apply freeze all strategy to the cleavage stage (day 2 or 3) or blastocyst stage (day 5 or 6) embryos. Subsequently, these embryos are thawed and transferred in frozen embryo transfer (FET) cycles. The first successful pregnancy from a FET was reported in 1984,[1] and since then, the number of FET cycles has dramatically risen.[2] The main reason underlying the increasing trend for FET cycles is that it results in improved cumulative pregnancy rates, eliminates the risk of ovarian hyperstimulation syndrome (OHSS), reduce multiple gestations, and allow window for preimplantation genetic testing.[3] However, one of the major challenges in FET cycles with cleavage-stage embryos is cryopreservation-related blastomere loss.[4] Blastomere lysis is commonly seen due to the formation of ice crystals and exposure to the hyperosmotic environment.[5] This leads to loss of genetic material, disruption of cell-to-cell communication, and metabolic derangements by the induction of possible toxic effect of lysed blastomeres on the remaining viable blastomeres, thus decreasing the overall developmental potential of the embryo.

In FET cycles, higher pregnancy and implantation rates are achieved when embryos are of high quality and exhibit complete post-thaw survival. The primary objective of embryo cryopreservation, therefore, is to obtain the highest possible post-thaw survival rates. There are only a few reports that state clinical pregnancy after transfer of frozen-thawed embryos with <50% survival of their original blastomeres.[6],[7] Blastomere loss may be associated with impaired post-thaw in vitro development and thus resulting in decreased implantation rates.[4],[8],[9] Overall, the number of viable blastomeres at cleavage-stage embryos is thought to be important markers of pregnancy and implantation; however, they do not predict the developmental potential of the embryo. Further, the implantation of partially damaged embryo also raises concerns regarding normal fetal development.

The present report describes for the first time an ongoing pregnancy with euploid fetal development following the transfer of two day 3 cleavage-stage embryos with only one-two blastomeres survival in a patient with primary infertility.

 Case Report

A 31-year-old patient with endometriosis, polycystic ovarian syndrome, and hypothyroidism presented to our clinic with primary infertility for 3 years. She had been operated for endometriosis earlier and was started on treatment for hypothyroidism. Hysterosalpingography revealed patent tubes with normal course and caliber. Semen analysis showed a normal volume of 3.5 ml, sperm concentration of 75 million/mL, and 35% motility with forward progression. She failed to conceive following two cycles of ovulation induction with timed intercourse and one cycle of controlled ovarian stimulation with intrauterine insemination. She opted to go ahead with in vitro fertilization (IVF) cycle.

In her IVF cycle, ovarian stimulation was started on day 2 of the menstrual cycle using 225 IU recombinant FSH (Gonal-F, Merck, Modugno, Italy) for 6 days and followed by 300 IU purified human menopausal gonadotropin (Menopur, Ferring Pharmaceuticals, Germany) for 4 days. To prevent premature luteinizing hormone surge, gonadotropin-releasing hormone (GnRH) antagonist injection, cetrorelix 0.25 mg s.c (Ciscure, Emcure Pharmaceuticals, Pune, India) was started on the 5th day of stimulation and continued till the day of the trigger. GnRH agonist trigger injection, 0.2 mg s.c triptorelin (Decapeptyl, Ferring Pharmaceuticals, Kiel, Germany) was given. Thirty-five hours later, transvaginal ultrasound-guided oocyte retrieval was performed, yielding 23 cumulus-oocyte complexes (COCs). All COCs were transferred to G-IVF, fertilization media (Vitrolife, Kungsbacka, Sweden) for culture at 37°C and 6% CO2 for 3–4 h. Oocytes were denuded with the use of enzymatic (HYASE™-10x; Vitrolife, Kungsbacka, Sweden) and mechanical processes. Nineteen oocytes were found to be mature (metaphase 2), and four were immature (two at metaphase 1 and two with visible germinal vesicles). Among the mature oocytes, nine showed normal morphology, six had abnormal shapes (elongated oocyte/zona pellucida), and four had big central smooth endoplasmic reticulum. All mature oocytes were used for intracytoplasmic sperm injection (ICSI). The semen sample, obtained on the day of oocyte retrieval, showed a normal volume of 1.6 ml, sperm concentration of 74 million/ml, and forward progressive motility of 45%. The morphology of the spermatozoa selected for ICSI was normal. There were 13 resultant normally fertilized zygotes (2PN). The zygotes were placed in G1 Plus media supplemented with HSA (Vitrolife, Kungsbacka, Sweden), under oil (Irvine Scientific, Santa Ana, CA) and left in culture until day 3. Ten day 3 embryos were developed, of which five embryos (one seven cells, three eight cells, and one 12 cells) were of Grade I and rest five embryos (one six cells, two seven cells, one nine cells, and one 12 cells) were of Grade II quality. All day 3 embryos were vitrified using commercially available vitrification solution kit and cryotop device (Kitazato BioPharma, Tokyo, Japan) instead of possible risk of OHSS in the patient.

For her subsequent FET cycle, she was downregulated with a GnRH agonist leuprolide 3.75 mg i.m (Lupride Depot, Sun Pharmaceuticals, India) followed by hormonal replacement therapy cycle for building endometrial lining. She was given estradiol valerate (Progynova, Bayer HealthCare, Germany) 4 mg for 4 days, which was then increased to 6 mg/day. After obtaining adequate endometrial thickness, vaginal micronized progesterone 400 mg once a day (Gestone, Ferring Pharmaceuticals, India) and injectable 50 mg i.m daily were started (Uterone, Jagsonpal Pharmaceuticals, India). After 3 days of progesterone, the patient was called for FET. On the day of transfer, two eight-cell day 3 embryos were thawed using commercially available thawing solution kit (Kitazato BioPharma, Tokyo, Japan). Immediately after thawing, ≥50% of blastomeres were found to be degenerated in both the embryos. Following 2 h of incubation, it was observed that only one blastomere survived in one embryo and two blastomeres survived in the other embryo [Figure 1]. Fearing that there may be a problem with the thawing media, we decided not to thaw any more embryos and to cancel the transfer cycle. After detailed discussion, the patient was agreeable that no more embryos should be thawed but wanted to take a chance with these embryos. Thus, both the embryos were transferred using a Cook catheter (Sydney IVF Embryo Transfer Set, Cook, Bloomington, IN) and the procedure was uneventful.{Figure 1}

Fourteen days later, the patient had a positive urine pregnancy test. Serum beta-human chorionic gonadotropin levels were found to be 57.9, 420.7, and 2363 mIU/mL on 14, 18, and 22 days after embryo transfer. At 6 weeks of gestation, an ultrasound examination revealed a single live intrauterine gestational sac with positive cardiac activity. A follow-up scan at 10 weeks showed fetal age parameters corresponding to gestational age. Ultrasound at 12 weeks showed normal nuchal translucency, presence of nasal bone, and no gross anomalies. In addition, noninvasive prenatal screening test confirmed a euploid fetus. The pregnancy is ongoing.


Cryopreservation-related blastomere loss has a direct impact on the outcome of FET cycles. Understanding the impact of such loss on the implantation potential and FET outcome can assist in making clinical decisions on how best to utilize the embryos with poor survival. In the present case, we describe an ongoing clinical pregnancy following transfer of two cleavage-stage day 3 embryos with <50% blastomere survival. In addition, this is the first case report where the transfer of day 3 embryos with one-two blastomeres survival resulted in a euploid fetal development.

In the FET cycle, an embryo is considered to be a survivor when it possesses ≥50% viable blastomeres postthaw.[10] In accordance with this, no significant difference has been demonstrated between the implantation rates associated with transferring post-thaw embryos with <25% blastomere lysis and embryos with no damage.[11] However, conflicting results are reported following the transfer of embryos with ≥50% blastomere loss with regard to pregnancy outcome. In case of transfer of embryos with upto 50% blastomere loss, studies have reported significantly poorer outcomes with respect to implantation, pregnancy, and live birth rates as compared to the transfer of fully intact embryos.[12],[13],[14] In addition, a study correlated the pregnancy rate for FET cycles with the number of viable blastomeres rather than of number of blastomeres loss post-thaw.[15] It was demonstrated that embryos with survival of <6 blastomeres show significantly low potential for pregnancy. To the best of our knowledge, there is only one study that has reported clinical pregnancy following transfer of embryo with ≥50% blastomere loss and only one intact blastomere from frozen-thawed day 2 cleavage-stage embryos.[6]

Several attempts have been taken to identify the parameters that are related to intactness and development potential in embryos with poor post-thaw survival to improve the pregnancy outcome. There are studies in which the viability of partially damaged embryos was checked before transfer by assessing the mitosis resumption in either overnight culture or extended culture to the blastocyst stage.[16],[17],[18],[19] The cleavage stage cells are flexible, and the embryo has an intrinsic capacity to overcome cell loss. Culture of these embryos to blastocyst stage, therefore, results in either survival or complete degeneration. This allows the better selection of embryos with development and implantation potential. Another important aspect is the "toxic effect" from the damaged blastomeres on the further development of the intact ones.[5],[8] It has been reported that removal of lysed blastomeres significantly increases the rate of implantation of partially damaged frozen-thawed embryos.[7],[20],[21]

During embryo development, cleavage-stage blastomere cells are suggested to be totipotent. A case report describing the birth of a child after the transfer of a day 2 cryopreserved embryo, of which only one out of four cells had survived postthaw, provided evidence that at least one of the blastomeres at the 4-cell stage is totipotent.[6] Thus, even if a part of the embryo is lost due to freeze thaw, the cleavage-stage embryo remains capable of producing an intact individual. However, it is still not clear why blastomere loss is associated with reduced implantation potential and poor cycle outcome. One possible explanation could be that there is the differential inheritance of totipotency-relevant components between sister blastomeres and not all the sibling blastomeres are totipotent.[22]

It has been suggested that embryos with ≥50% blastomere loss have developmental potential lower than that of intact embryos and therefore, such embryos should not be transferred. However, cryopreserved embryos have similar implantation potential if further cleavage occurs overnight, regardless of the number of cells lost.[17],[19] Further, concerns have been raised regarding the safety of FET, particularly in terms of pregnancy and perinatal outcomes. Recently, it was demonstrated by Yu et al. that in embryos with <50% blastomere survival being cultured to fully expanded blastocysts had normal chromosomal status and therefore could be considered for transfer.[23] A multicenter prospective cohort study with large sample size reported that once the damaged embryos have implanted, pregnancies appear to have the same probability of progressing to live birth.[13] There was no report of increased risk of preterm birth, perinatal mortality, or congenital anomalies in association with blastomere loss, thereby suggesting the safety of such embryos.[13],[19] Overall, these studies suggest that embryos with poor post-thaw blastomere survival can be considered for transfer because these embryos will implant only if they have the developmental potential.

The present case study supports the theory of blastomere totipotency indicating that even one-two blastomeres till day 3 of culture have the potential to develop to a euploid fetus and therefore, the implantation potential of such embryos should not be underestimated. However, additional studies are required to evaluate the possibility of using the embryos with poor post-thaw survival in FET cycles.

Declaration of patient consent

The authors certify that they have obtained all appropriate patient consent forms. In the form the patient(s) has/have given his/her/their consent for his/her/their images and other clinical information to be reported in the journal. The patients understand that their names and initials will not be published and due efforts will be made to conceal their identity, but anonymity cannot be guaranteed.

Financial support and sponsorship


Conflicts of interest

There are no conflicts of interest.


1Zeilmaker GH, Alberda AT, van Gent I, Rijkmans CM, Drogendijk AC. Two pregnancies following transfer of intact frozen-thawed embryos. Fertil Steril 1984;42:293-6.
2Pereira N, Rosenwaks Z. A fresh (er) perspective on frozen embryo transfers. Fertil Steril 2016;106:257-8.
3Zhang W, Xiao X, Zhang J, Wang W, Wu J, Peng L, et al. Clinical outcomes of frozen embryo versus fresh embryo transfer following in vitro fertilization: a meta-analysis of randomized controlled trials. Arch Gynecol Obstet 2018;298:259-72.
4Edgar DH, Bourne H, Speirs AL, McBain JC. A quantitative analysis of the impact of cryopreservation on the implantation potential of human early cleavage stage embryos. Hum Reprod 2000;15:175-9.
5Dulioust E, Toyama K, Busnel MC, Moutier R, Carlier M, Marchaland C, et al. Long-term effects of embryo freezing in mice. Proc Natl Acad Sci U S A 1995;92:589-93.
6Veiga A, Calderon G, Barri PN, Coroleu B. Pregnancy after the replacement of a frozen-thawed embryo with less than 50% intact blastomeres. Hum Reprod 1987;2:321-3.
7Nagy ZP, Taylor T, Elliott T, Massey JB, Kort HI, Shapiro DB. Removal of lysed blastomeres from frozen-thawed embryos improves implantation and pregnancy rates in frozen embryo transfer cycles. Fertil Steril 2005;84:1606-12.
8Van den Abbeel E, Camus M, Van Waesberghe L, Devroey P, Van Steirteghem AC. Viability of partially damaged human embryos after cryopreservation. Hum Reprod 1997;12:2006-10.
9Archer J, Gook DA, Edgar DH. Blastocyst formation and cell numbers in human frozen-thawed embryos following extended culture. Hum Reprod 2003;18:1669-73.
10Gardner DK, Weissman A, Howles CM, Shoham Z. Textbook of Assisted Reproductive Techniques: Laboratory and Clinical Perspectives. 3rd ed. U.K: Informa Healthcare; 2012.
11Zheng X, Liu P, Chen G, Qiao J, Wu Y, Fan M. Viability of frozen-thawed human embryos with one-two blastomeres lysis. J Assist Reprod Genet 2008;25:281-5.
12El-Toukhy T, Khalaf Y, Al-Darazi K, Andritsos V, Taylor A, Braude P. Effect of blastomere loss on the outcome of frozen embryo replacement cycles. Fertil Steril 2003;79:1106-11.
13Wu YT, Li C, Zhu YM, Zou SH, Wu QF, Wang LP, et al. Outcomes of neonates born following transfers of frozen-thawed cleavage-stage embryos with blastomere loss: A prospective, multicenter, cohort study. BMC Med 2018;16:96.
14Tang R, Catt J, Howlett D. Towards defining parameters for a successful single embryo transfer in frozen cycles. Hum Reprod 2006;21:1179-83.
15Zhang S, Lu C, Lin G, Gong F, Lu G. The number of blastomeres in post-thawing embryos affects the rates of pregnancy and delivery in freeze-embryo-transfer cycles. J Assist Reprod Genet 2009;26:569-73.
16Fernandez Gallardo E, Spiessens C, D'Hooghe T, Debrock S. Effect of embryo morphology and morphometrics on implantation of vitrified day 3 embryos after warming: A retrospective cohort study. Reprod Biol Endocrinol 2016;14:40.
17Van Landuyt L, Van de Velde H, De Vos A, Haentjens P, Blockeel C, Tournaye H, et al. Influence of cell loss after vitrification or slow-freezing on further in vitro development and implantation of human Day 3 embryos. Hum Reprod 2013;28:2943-9.
18Solé M, Santaló J, Rodríguez I, Boada M, Coroleu B, Barri PN, et al. Correlation between embryological factors and pregnancy rate: Development of an embryo score in a cryopreservation programme. J Assist Reprod Genet 2011;28:129-36.
19O'Shea LC, Hughes C, Kirkham C, Mocanu EV. The impact of blastomere survival rates on developmental competence of cryo-thawed day 2 embryos. Eur J Obstet Gynecol Reprod Biol 2016;197:98-102.
20Elliott TA, Colturato LF, Taylor TH, Wright G, Kort HI, Nagy ZP. Lysed cell removal promotes frozen-thawed embryo development. Fertil Steril 2007;87:1444-9.
21Rienzi L, Ubaldi F, Iacobelli M, Minasi MG, Romano S, Ferrero S, et al. Developmental potential of fully intact and partially damaged cryopreserved embryos after laser-assisted removal of necrotic blastomeres and post-thaw culture selection. Fertil Steril 2005;84:888-94.
22Boiani M, Casser E, Fuellen G, Christians ES. Totipotency continuity from zygote to early blastomeres – A model under revision. Reproduction 2019. pii: REP-18-0462.R2.
23Yu L, Jia C, Lan Y, Song R, Zhou L, Li Y, et al. Analysis of embryo intactness and developmental potential following slow freezing and vitrification. Syst Biol Reprod Med 2017;63:285-93.