Comparison of pregnancy outcomes between 4th day morula and 5th day blastocyst after embryo transfer: a retrospective cohort study

Abstract

Background: This study was designed to evaluate pregnancy outcomes between morulae transferred on day 4 (D4) and blastocysts transferred on day 5 (D5).

Methods: From September 2017 to September 2020, 1963 fresh transfer cycles underwent early follicular phase extra-long protocol for assisted conception in our fertility center were divided into D4 (324 cases) and D5 (1639 cases) groups, and the general situation and other differences of patients in both groups were compared. To compare the differences in pregnancy outcomes, the D4 and D5 groups were further divided into groups A and B based on single and double embryo transfers. Furthermore, the cohort was divided into two groups: those with live births (1116 cases) and those without (847 cases), enabling a deeper evaluation of the effects of D4 or D5 transplantation on assisted reproductive outcomes.

Results: In single embryo transfer, there was no significant difference between groups D4A and D5A (P > 0.05). In double embryo transfer, group D4B had a lower newborn birthweight and a larger proportion of low birthweight infants (P < 0.05). The preterm delivery rate, twin delivery rate, cesarean delivery rate, and percentage of low birthweight infants were lower in the D5A group than in the D5B group (P < 0.05). Analysis of factors influencing live birth outcomes further confirmed the absence of a significant difference between D4 and D5 transplantation in achieving live birth (P > 0.05).

Conclusion: When factors such as working life and hospital holidays are being considered, D4 morula transfer may be a good alternative to D5 blastocyst transfer. Given the in vitro fertilization/intracytoplasmic sperm injection (IVF/ICSI) success rate and risk of twin pregnancy, D4 morula transfer requires an adapted decision between single and double embryo transfer, although a single blastocyst transfer is recommended for the D5 transfer in order to decrease the twin pregnancy rate. In addition, age, endometrial thickness and other factors need to be taken into account to personalize the IVF program and optimize pregnancy outcomes.

Keywords: Assisted reproduction; Blastocyst; Early follicular phase prolonged protocol; IVF/ICSI outcome; Morula.

Fig. 1

Grouping of morulae on day 4 and blastocysts on day 5 after fertilization using an early follicular phase prolonged protocol. D4, the fourth day after fertilization; D5, the fifth day after fertilization; D4A, single morula was transplanted; D4B, double morulae were transplanted; D5A, single blastocyst was transplanted; D5B, double blastocysts were transplanted

Fig. 2

Photomicrographs of different grades of D3 cleavage embryo, D4 morula, and D5 blastocyst. a Inferior D3 cleavage embryo. The embryo had uneven surfaces, irregular shapes, and was filled with more fragments and visible cell granules. b Inferior D4 morula. Only some blastomeres underwent compaction, that was partial compaction, and other cells remained in a separated status, then a smaller compacted multicellular mass would form. c Inferior D5 blastocyst. The blastocyst cavity was not significantly dilated, the number of ICM cells was low, and the TE cells were sparse. d High quality D3 cleavage embryo. The embryo was of uniform size, regular shape, full hyaline band, uniform cell quality, no granularity and few fragments. e High quality D4 morula. The embryo was nearly fully compacted with all blastomeres undergoing the compact process, and a large multicellular mass was expected. f High quality D5 blastocyst. The blastocyst cavity was significantly enlarged in volume, the zona pellucid was thinned, the number of ICM cells was high and tightly arranged, and the number of TE cells was high, forming a tight epithelium. Original magnification: x200.

Fig. 3

Comparison of single and double embryo transfer assisted pregnancy outcomes in the D4 group. D4A, single morula was transplanted; D4B, double morulae were transplanted; CPR, Clinical pregnancy rate; LBR, Live birth rate; PBR, Preterm birth rate; TBR, Twin birth rate; LBIR, Low birthweight infant rate; np> 0.05, * p < 0.05, ** p < 0.01

Fig. 4

Comparison of single and double embryo transfer assisted pregnancy outcomes in the D5 group. D5A, single blastocyst was transplanted; D5B, double blastocysts were transplanted. PBR, Preterm birth rate; TBR, Twin birth rate; NBR, Natural birth rate; CR, Cesarean rate; NB, Newborn birthweight; LBIR, Low birthweight infant rate; ** p < 0.01

Fig. 5

a ROC curve of Age. b ROC curve of Endometrial thickness