|Year : 2020 | Volume
| Issue : 1 | Page : 26-31
Comparative analysis of outcome of morula versus blastocyst transfer
Himanshu Bavishi1, Sonali Tawde2, Falguni Bavishi1, Parth Bavishi1
1 Department of ART, Bavishi Fertility Institute, Ahmedabad, Gujarat, India
2 Department of ART, Bavishi Fertility Institute, Mumbai, Maharashtra, India
|Date of Submission||30-Nov-2019|
|Date of Acceptance||07-Jul-2020|
|Date of Web Publication||30-Jan-2021|
Dr. Sonali Tawde
B 202, Asha Nagar, Phase 2, Nirmal Group, P. K. Road, Mulund West, Mumbai - 400 080, Maharashtra
Source of Support: None, Conflict of Interest: None
Introduction: The human embryonic genome is not activated before 4–8 cell stage, so morphological grading on D2/3 does not predict the pregnancy potential of embryo. Sequential media and better culture conditions have made D4/5 embryo transfers (ETs) feasible. Embryos enter the uterus as morulae, which is the rationale behind D4 ET.
Aims and Objectives: The aims and objectives of this study were to compare the in vitro fertilization (IVF) outcomes of morula versus blastocyst transfers in fresh IVF intracytoplasmic sperm injection (ICSI) cycles in terms of implantation rates, clinical pregnancy rates (CPRs), and live birth rates (LBRs).
Materials and Methods: This is a retrospective study conducted in Bavishi Fertility Institute, Mumbai and Ahmedabad, from January 2017 to September 2018; a total of 349 in vitro fertilization (IVF)-intracytoplasmic sperm injection (ICSI) cycles in females <37 years old, were analyzed, among which 53 were in morula group and 296 in blastocyst group. Cases with severe endometriosis, huge fibroids, severe male factor infertility, and TESA/PESA ICSI were excluded. Outcomes were analyzed statistically in terms of implantation rate, clinical pregnancy rate, and live birth rates.
Statistics: Chi-square test (with Yates correction, if required) and unpaired t-test were used for statistical analysis.
Results: The implantation rate, CPR, and LBR in morula versus blastocyst group were 34.54% versus 37.79%, 45.28% versus 51.35% and 37.73% versus 45.6%, respectively.
Conclusion: The outcome in terms of implantation rate, CPR, and LBR of blastocyst transfer was marginally better than that of morula transfer, but statistically did not reach any significance. Morula transfers done in selective situations offer effective alternative to blastocyst ET without compromising results.
Keywords: Blastocyst, clinical pregnancy, implantation, live birth, morula
|How to cite this article:|
Bavishi H, Tawde S, Bavishi F, Bavishi P. Comparative analysis of outcome of morula versus blastocyst transfer. Onco Fertil J 2020;3:26-31
| Introduction|| |
Over the last decade, with the advent of better culture media and better culture conditions, day-5 embryo transfers (ETs) have become more feasible and practical. Blastocyst transfer offers advantage of selecting developmentally most competent embryos and reducing the number of transferred embryos, thereby limiting high order multiple, pregnancies while maintaining an acceptable pregnancy rate. Selection of embryos is one of the key determinants of success in assisted reproductive technology (ART). Morphological grading for cleavage-stage embryos has many drawbacks. Human embryonic genome is known to get activated beyond 4-cell stage, and embryo selection on D2/3 has its limitations. Embryos complete compaction and blastulation on day 4/5, hence selection is better on D4/5. Blastocyst transfer also permits advanced techniques such as Pre-implantation genetic testing (PGT) more easily and effectively, as the perivitelline space is more and the risk of loss of cells during biopsy is less. The rationale behind day-4 ET is, in nature, embryos enter the uterine cavity as morulae, so being more physiological. Morula transfers have not gained wide acceptance so far, and the main reason behind this could be that a standard classification for day-4 embryos is not available as of now. In the last few years, some studies have come up with classification for day-4 embryos, based on compaction and blastulation.,
The aim of this study is to evaluate and compare the outcome of morula versus blastocyst transfer in self-egg fresh IVF-ICSI cycles in terms of implantation rate, clinical pregnancy rate (CPR), and live birth rate (LBR).
| Materials and Methods|| |
This is a retrospective study conducted from January 2017 to September 2018. A total of 349 cycles have been studied, of which 296 are blastocyst transfers and 53 cycles are of morula transfer.
- Autologous IVF-ICSI cycles, where fresh ET was done
- Agonist/antagonist protocol
- Cycles in which four or more embryos were of Grade 1 on D2
- Cycles with two blastocysts or morulae were transferred.
- Women above the age of 37 years, due to established lower reproductive performance
- Severe oligoasthenospermia/TESA or PESA cycles
- Huge fibroids/severe endometriosis
- Poor responders/genetic factors in either partner
- Very thin ET/history of genital Koch's/Asherman's syndrome
- Bad obstetric history, antiphospholipid antibody-positive cases
- Cycles with sequential transfer.
Patients' records were collected in terms of anti-Müllerian hormone (AMH), causes of infertility, dose and duration of gonadotropins used, number of retrieved oocytes, M2 oocytes, fertilization, rate and embryo grading.
Stimulation protocol, either antagonist or long agonist, was used depending on age, AMH, antral follicle count, and previous stimulation response. Recombinant FSH or human menopausal gonadotropins were started at a dose of 225 IU, and if required stepped up depending on follicle growth rate and serum estradiol (E2) levels. Cases with severe polycystic ovarian syndrome were started with a lower dose of 150 IU. The follicular growth was monitored by serial vaginal ultrasound scans. Those in agonist protocol received leuprolide depot injection (half of 3.75 mg) in luteal phase of previous cycle. In antagonist protocol, 0.25-mg injection cetrorelix was started when the largest follicle was 13 mm. When three or more leading follicles reached the size of 18 mm, ovulation was triggered by urinary human chorionic gonadotropin (HCG) 10,000 IU. Ovum pickup was performed 34–36 h after the HCG trigger.
All patients underwent ICSI, and fertilization was confirmed 16–18 h later. Laboratory protocols, culture media, and incubators remained same throughout the study period. We have used sequential media (Vitrolife Sweden AB, V. Frolunda, Sweden) and Trigas incubator (6% O2, 6% CO2, and 88% N2) during the study period.
Grading of embryos
For blastocysts, Gardner's classification was followed for embryo grading. The grading which was followed for day-4 embryos is in accordance with the ESHRE 2011 consensus. Day-4 embryos, which have lost all of the boundaries of the blastomere, are defined to achieve complete compaction, and were classified as Grade 1 [Table 1]. Embryos with <100% but >50% of compaction were rated as Grade 2 and embryos with <50% compaction as Grade 3. Embryos without compaction but possessing at least 8 blastomeres (8 cells) were classified as Grade 4. And, those without compaction but with <8 blastomeres (<8 cells) were rated as Grade 5.
Grade 1 was further divided into Grade 1a and Grade 1b according to the formation of blastocele, also called early cavitation. Embryos with full compaction and early cavitation were defined as Grade 1a, whereas those with full compaction but without early cavitation were placed in Grade 1b. We have not included Grade 4 and 5 morula transfer cycles in this study.
The number of embryos transferred on day 4 or 5 was two; the transfer was done under ultrasound guidance (transabdominal sonography). Luteal-phase support was given by vaginal progesterone (400-mg capsule 1 TDS or 8% gel twice daily), for 14 days post-ET. Pregnancy was confirmed by serum beta-HCG on day 12.
The outcome was analyzed in terms of implantation rate, CPR, LBR, and miscarriage rate. The implantation rate was calculated as number of gestational sacs seen on sonography divided by the number of embryos transferred. Clinical pregnancy was defined as presence of at least one intrauterine gestational sac on ultrasound at 5–6 weeks. Any pregnancy loss up to 20 weeks of gestation was termed as miscarriage. Live birth was defined as delivery beyond 28 weeks of gestation.
Statistical analysis was done with Chi-square test (with Yates correction, if required) and unpaired t-test. P < 0.005 was considered statistically significant.
| Results and Observations|| |
The morula group had 53 cases, whereas the blastocyst group included 296 cases.
The mean age in both groups was similar. Other demographic characteristics, AMH levels, and the number of previous failed IVF cycles did not differ in both groups statistically [Table 2].
The causes of infertility were similar in both groups [Table 3].
The mean endometrial thickness was similar in both groups [Table 4]. The mean number of M2 oocytes and fertilized oocytes was marginally higher in the blastocyst group, and the difference was statistically significant. The possibility that it would have affected our results is almost negligible because the number of embryos transferred was same in both groups.
The implantation rates in the study (D4) versus control (D5) groups were 34.54% versus 37.79%, respectively (P = 0.51). The CPRs were 45.28% versus 51.35% (P = 0.41) and the LBRs were 37.73% versus 45.6% (P = 0.28) of the study and control groups, respectively [Table 5]. The miscarriage rates were 16.66% versus 11.18%, (P = 0.44) and the multiple pregnancy rates were 33.33% versus 30.20% (P = 0.76) of the study and control groups, respectively. There was one case of ectopic pregnancy in the control group. The number of embryos frozen in both groups was similar (2.0 ± 0.2 vs. 1.8 ± 0.2).
Overall, the pregnancy outcome did not have any statistically significant difference in both groups.
| Discussion|| |
Human genome activation occurs at 4–8 cell stage. Extension of embryo culture beyond the point of genome activation permits discrimination of embryos with a slower or arrested cleavage stage from those undergoing regular cell division, potentially enhancing the capacity for implantation.,, After activation of the embryonic genome, the apoptosis system and checkpoints of cell cycles might be activated and may result in reduction of mosaicism from day-4 embryos. Hence, the embryos with genetic abnormalities find it more difficult to grow beyond the cleavage stage.
Harper reported that the embryo travels to the uterine cavity about 3–4 days after fertilization in mammals. Hence, the synchronization is better between the uterine environment and embryo on day 4.
In morula transfer, reduced exposure in vitro reduces the susceptibility to the interruption of genetic/epigenetic regulatory mechanism, risk of fetal malformations, monozygotic twinning, and preterm delivery, which is associated with blastocyst culture.
There are certain situations in which morula transfer is practically useful such as:
- Suboptimal culture conditions, due to overload on IVF laboratory (batch IVF programs)
- Cases of Pre-implantation Genetic screening (PGS) done on day-3 embryos
- When the patient or the embryologist or clinician is not available for day-5 ET
- At centers where large number of cycles are being done, excessive load for incubators can lead to excessive opening of the incubator door and increased time to reach an optimal pH and temperature, hence suboptimal culture conditions. Morula transfers can be considered to avoid ET cancelations in such circumstances with acceptable pregnancy rates for those with enough number of good-quality embryos on day 3
- It offers some advantages such as:
- Embryos return to the uterus in an environment where it would normally reside
- Uterine contractility is minimal, hence we can expect optimum implantation potential
- Embryo selection is better than cleavage-stage embryos
- In vitro exposure is reduced than blastocysts
- Provides more flexibility to patient and clinicians.
Huisman reported the very first pregnancy achieved by day-4 embryos in the late 1990s. Later Grifo et al. and Gianaroli et al. both reported successful pregnancy after transferring day-4 morula embryos which received Pre-implantation Genetic diagnosis (PGD) on day 3. Gianaroli et al. also concluded that day-4 morula ET provided more convenient environment for patients and clinical staff because working and waiting until late in the evening of day 3 for ET was no more mandatory.
Tao et al. studied the outcome of day-3 versus day-4 embryos retrospectively and reported pregnancy from morula transfer, in 2001. The number of embryos transferred in this study was 2.35 ± 0.6 in day-4 ET (at least two good morula ET [METs]) and 4.07 ± 0.9 in day-3 ET (at least two good cleavage-stage ETs). The ongoing pregnancy rate was higher in day-4 ET than in day-3 ET (63.6% vs. 44.9%, respectively, P < 0.05). They concluded that implantation rates of day-4 embryos were similar or higher than that of day-3. Other studies have also proven that pregnancy outcomes are better in day-4 ETs than that of cleavage-stage transfers.
One of the most recent studies published in 2018 by Li et al. concluded that the term pregnancy rate was higher in day-4 good MET (morula ET) than that in day-5 good blastocyst ET (P = 0.025). Morula ET on day 4 might grant a success rate equal to blastocyst ET on day 5, and furthermore, the pregnancy after day-4 ET will result in term delivery. Kang et al. in 2013 concluded that the preterm pregnancy rate was lower in the elective single morula embryo transfer (eSMET) group than that in the elective single blastocyst embryo transfer (eSBET) group (0% vs. 6.4%, respectively). This might be due to reduction of in vitro exposure of embryos.
During our study period, patients were offered day-4 ET after extensive counseling about the outcome. All patients had four or more good-quality embryos on day 3. Indications for day-4 ET, such as suboptimal culture conditions, do not apply to our study, as it was not the indication for morula transfer. We have not included morula and blastocyst formation rates in our study.
Some of the centers have been routinely doing ETs on day 4, while some have been doing it regularly in cases of PGD and have reported successful pregnancy outcomes with morula transfers since 1995. The study by Sun-Hee Lee, in 2013, claimed to have better pregnancy rates with day-4 ET than day-5 ET, in spite of three blastocysts being transferred. He compared embryos which did not develop into morula with compaction on day 4 and ones which did not develop into blastocyst on day 5. Interestingly, he found 4.4% implantation rate and 14.2% CPR even if there were no morula- and compaction-stage embryos on day 4. On the other hand, morula- and compaction-stage ET cycles at day 5 showed no implantation or clinical pregnancy.
Many studies have reported high cancellation rates in blastocyst ET, when there are no embryos for ET on day 5. Montag suggested that extended embryo culture is not beneficial when the option for embryo selection at later stages of development is not available. For slow-growing embryos, if there is no compaction on day 4, it is more logical to transfer such embryos on day 4 rather than waiting till day 5 so as to not lose the implantation potential of such embryos.
According to previous studies, 59.2% of good-quality embryos on day 3 developed to good-quality embryos on day 4, whereas 47% of good-quality embryos on day 3 developed to blastocyst on day 5. These results suggest that the rate of ET cancelation is lower after morula-stage ET than blastocyst-stage ET. A study on morula-stage ET cancelation has not been conducted, and a prospective, randomized study should be performed in future.
In our study, CPRs and LBRs were marginally better in the blastocyst group, without reaching statistical significance. The higher miscarriage rates (16.66% vs. 11.18%) could be one of the reasons for lower ongoing pregnancy rates in the morula group. The rate of multiple pregnancies was 33.33% versus 30.20%, marginally higher in the morula group. We had all twin pregnancies in the multiple pregnancy group, with none of these being monozygotic ones.
Ectopic pregnancy has been reported to occur in approximately 1%–2% of clinical pregnancy after IVF-ET, and the incidence varies in some studies. It has been known that ectopic pregnancy is related to stimulation due to oocyte retrieval, amount of transfer medium injected for transferring embryos, technique of transferring, and uterine contractions. It has been suggested that the incidence of ectopic pregnancy could be reduced by blastocyst transfer compared to cleavage-stage embryos because of the decreased uterine contractility and larger diameter of the blastocyst. We had one case of ectopic pregnancy in the control group.
For embryo selection, there is insufficient data regarding PGS without indication, such as parental chromosomal abnormalities, repeated implantation failure, or habitual abortion. Kinetic embryo grading (embryo scope) has not gained much popularity, mainly because of the inconsistent data, need of manual review, and increased cost of the treatment. Day-4 or day-5 ET provides efficient mode of embryo selection in such scenario.
The only possible disadvantage of morula ET is no standard grading system is available. It is observer dependent. The drawback of our study is that it is not randomized and the sample size in the study group is small. This being a retrospective study, we propose to conduct a prospective study to further evaluate and compare the outcome of morula transfer.
Morula transfer has rarely been attempted by ART specialists, even if embryos that have undergone compaction and reached the morula stage may have certain advantages. Now, the shift is more toward reducing multiple pregnancies and improving pregnancy outcome, hence embryo selection is the key to reduce the number of embryos transferred. Morula transfer holds the promise to enable us to select better-quality embryos as compared to cleavage-stage embryos and provide effective alternative for blastocyst transfer.
| Conclusion|| |
Till date, blastocyst transfer is considered to be better in terms of pregnancy outcome for single IVF/ICSI cycle. This study suggests that morula transfer offers viable and effective alternative to blastocyst transfer with comparable pregnancy rates in selective situations. Blastocyst culture has the potential risk of developmental arrest, reduced embryo quality in vitro especially in batch IVF, and risk of genetic/epigenetic changes due to prolonged in vitro exposure. Morula transfer offers more flexibility for clinicians. It has the added advantage of embryo selection over cleavage-stage embryos, and at the same time minimizes potential risks involved in blastocyst culture.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
| References|| |
Braude P, Bolton V, Moore S. Human gene expression first occurs between the 4- and 8-cell stages of preimplantation development. Nature 1988;332:459-61.
Speroff L, Fritz M. Clinical Gynecologic Endocrinology and Infertility. 7th
ed.. Philadelphia: Lippincott Williams & Wilkins; 2005. p. 246.
Alpha Scientists in Reproductive M, Embryology ESIGo. The Istanbul consensus workshop on embryo assessment: Proceedings of an expert meeting. Hum Reprod 2011;26:1270-83.
Tao J, Tamis R, Fink K, Williams B, Nelson-White T, Craig R. The neglected morula/compact stage embryo transfer. Hum Reprod 2002;17:1513-8.
Menezo Y, Hazout A, Dumont M, Herbaut N, Nicollet B. Co-culture of embryos on Vero cells and transfer of blastocyst in humans. Hum Reprod 1992;7:101-6.
Dawson KJ, Conaghan J, Ostera GR, Winston RM, Hardy K. Delaying transfer to the third day post-insemination to select non arrested embryos, increases development to the fetal heart stage. Hum Reprod 1995;10:177-82.
Jones GM, Trounson AO, Gardner DK, Kausche A, Lolatgis N, Wood C. Evolution of a culture protocol for successful blastocyst development and pregnancy. Hum Reprod 1998;13:169-77.
Magli MC, Jones GM, Gras L, Gianaroli L, Korman I, Trounson AO. Chromosome mosaicism in day 3 aneuploid embryos that develop to morphologically normal blastocysts in vitro
. Hum Reprod 2000;15:1781-6.
Harper M. Gamete and zygote transport. In: Knobil E, Neill J, editors. The Physiology of Reproduction. New York: Raven; 1994. p. 123-88.
Huisman GJ, Alberda AT, Leerentveld RA, Verhoeff A, Zeilmaker GH. A comparison of in vitro
fertilization results after embryo transfer after 2, 3, and 4 days of embryo culture. Fertil Steril 1994;61:970-1.
Grifo JA, Giatras K, Tang YX, Krey LC. Successful outcome with day 4 embryo transfer after preimplantation diagnosis for genetically transmitted diseases. Hum Reprod 1998;13:1656-9.
Gianaroli L, Magli MC, Munné S, Fortini D, Ferraretti AP. Advantages of day 4 embryo transfer in patients undergoing preimplantation genetic diagnosis of aneuploidy. J Assist Reprod Genet 1999;16:170-5.
Kang SM, Lee SW, Jeong HJ, Yoon SH, Koh MW, Lim JH, et al
. Clinical outcomes of elective single morula embryo transfer versus elective single blastocyst embryo transfer in IVF-ET. J Assist Reprod Genet 2012;29:423-8.
Li RS, Hwu YM, Kuo-Kuang Lee R, Li SH, Lin MH. Day 4 good morula embryo transfer provided compatible live birth rate with day 5 blastocyst embryo in fresh IVF/ET cycles. Taiwanese J Obstet Gynecol 2018;57:52-7.
Lee SH, Lee HS, Lim CK, Park YS, Yang KM, Park DW. Comparison of the clinical outcomes of day 4 and 5 embryo transfer cycles. Clin Exp Reprod Med 2013;40:122-5.
Montag M, van der Ven K, Dorn C, van der Ven H. Extended embryo culture reduces the implantation rate on day 4 and day 5 when only a maximum of three embryos are cultured beyond the pronuclear stage. Eur J Obstet Gynecol Reprod Biol 2006;124:65-9.
Rijnders PM, Jansen CA. The predictive value of day 3 embryo morphology regarding blastocyst formation, pregnancy and implantation rate after day 5 transfer following in vitro
fertilization or intracytoplasmic sperm injection. Hum Reprod 1998;13:2869-73.
Milki AA, Jun SH. Ectopic pregnancy rates with day 3 versus day 5 embryo transfer: a retrospective analysis. BMC Pregnancy Childbirth 2003;3:7.
Fanchin R, Ayoubi JM, Righini C, Olivennes F, Schönauer LC, Frydman R. Uterine contractility decreases at the time of blastocysts transfers. Hum Reprod 2001;16:1115-9.
Schoolcraft WB, Surrey ES, Gardner DK. Embryo transfer: Techniques and variables affecting success. Fertil Steril 2001;76:863-70.
Pantos K, Makrakis E, Chronopoulou M, Biba M, Perdikaris A, Dafereras A. Day 4 versus day 3 embryo transfer: A prospective study of clinical outcomes. Fertil Steril 2008;89:573-7.
[Table 1], [Table 2], [Table 3], [Table 4], [Table 5]