Abstract
Purpose
The time at which embryo developmental velocity variations occur remains unknown. Therefore, we analysed the morphokinetic trajectory of human early fertilisation from the extrusion of the second polar body (PB2) to pronuclear breakdown (PNBD) and retrospectively compared its alterations during the fertilisation of day 4–7 blastocysts.
Methods
Patients (n = 905) who underwent oocyte retrieval and blastocyst expansion between October 2019 and December 2020 were included. This study involved time-lapse technology monitoring of day 4 (n = 29), 5 (n = 1181), 6 (n = 244), and 7 (n = 12) blastocysts generated over 1009 intracytoplasmic sperm injection cycles. Morphokinetics during fertilisation were compared among the day 4–7 blastocyst groups.
Results
The timing of fertilisation events in the cytoplasm and pronuclei (PN) differed among the day 4–7 blastocyst groups from the outset. PB2 extrusion occurred significantly earlier in day 4 blastocysts than in day 5 blastocysts. The duration of the cytoplasmic halo was shortened in day 4 blastocysts and prolonged in day 6 blastocysts compared with that in day 5 blastocysts. Paternal PN appeared earlier in day 4 blastocysts than in day 5 blastocysts, whereas their appearance was delayed in day 6 blastocysts. In paternal and maternal PN, the time interval from PN appearance to PNBD was shortened in day 4 blastocysts but was prolonged in day 6 and 7 blastocysts.
Conclusions
Embryos that reached the blastocyst stage between days 4 and 7 displayed different morphokinetic speeds throughout fertilisation and as early as PB2 extrusion. Aberrant cytoplasmic granules, organelles, and PN rearrangement may contribute to these differences.
Supplementary information
The online version contains supplementary material available at 10.1007/s10815-025-03476-8.
Keywords: Blastocyst, Cytoplasmic halo, Fertilisation, Morphokinetics, Pronuclei, Second polar body
Introduction
In human in vitro fertilisation (IVF), the practice of extended embryo culture and blastocyst transfer has progressively become widespread owing to the need for elective single embryo transfer policies [1], freeze-all strategies [2], and pre-implantation genetic testing cycles [3]. Static observation–based morphological grading is the most used method to assess blastocyst quality in daily IVF practice [4–6]. However, the widespread use of time-lapse technology (TLT) over the last decade has enabled the continuous monitoring of human embryos, revealing developmental variations, including changes in cell cycle duration and abnormal features and behaviours, which are predictive indicators of embryo developmental competence, ploidy status, and pregnancy outcomes [7–11].
The speed of development is one of such variations. Human zygotes can develop into blastocysts between days 4 and 7 post-insemination [12–15]. The day of blastocyst development is considered a robust predictor of pregnancy outcomes after blastocyst transfer; specifically, clinical outcomes progressively worsen as the time of development to the blastocyst stage increases [16–18]. Most clinical and laboratory evidence is focused on day 5 and 6 blastocysts, while day 4 and 7 blastocysts remain less thoroughly investigated. Studies directly comparing the developmental patterns and trajectories of day 4–7 blastocysts are limited. A recent study demonstrated that the morphokinetic trajectories of day 4–7 blastocysts already diverge at the time of pronuclear (PN) breakdown (PNBD) [19]. These differences further increased during the final stage of fertilisation, from PNBD to first cytokinesis. Interestingly, the delay in this process is associated with trichotomous and early developmental arrest [19, 20]. However, whether and how differences among such embryos emerge during earlier stages of fertilisation remain entirely unresolved. The acquisition of such knowledge would significantly contribute to understanding the relative impact of intrinsic and extrinsic factors on embryo competence and shed further light on the foundational role of fertilisation in development. Therefore, this study retrospectively compared the multiple morphokinetics of day 4–7 blastocysts throughout fertilisation. To this end, we obtained not only information on conventional morphokinetic parameters but also described the kinetics of rarely reported and yet highly relevant morphological manifestations, such as the cytoplasmic wave and patterning of nucleolar precursor bodies.
Materials and methods
Ethics statements
Our Institutional Review Board approved this study on 30 March 2021 (approval number: 21–21). Written informed consent was obtained from all patients for the analysis of de-identified data.
Study design and patients
We reviewed the treatment cycle records of women who underwent clomiphene citrate (CC)-based minimal stimulation cycles followed by oocyte retrieval and intracytoplasmic sperm injection (ICSI) at the local clinic between October 2019 and December 2020 in this retrospective, single-centre cohort study (Fig. 1). Patients undergoing procedures involving cryopreserved gametes or surgically retrieved sperm were excluded since such factors can independently affect embryo morphokinetics and represent possible sources of bias [21, 22]. Those who underwent pre-implantation genetic testing were not included in this study.
Fig. 1.
Inclusion and exclusion criteria. ICSI, intracytoplasmic sperm injection; TESE, testicular sperm extraction; PESA; percutaneous epididymal sperm aspiration
CC-based minimal ovarian stimulation cycle in-vitro fertilisation
A detailed protocol for minimal stimulation with CC has been previously reported [23, 24]. Briefly, CC (50–100 mg/day; Fuji Pharma Co., Ltd., Tokyo, Japan) was administered according to an extended regimen from day 3 of the cycle until the induction of final oocyte maturation. Ultrasound and hormonal monitoring were initiated on day 8 and performed daily until ovulation was triggered. A minimal dose of human menopausal gonadotropin (Ferring Pharmaceuticals, Saint-Prex, Switzerland) or recombinant follicle-stimulating hormone (Merck & Co., Kenilworth, NJ, USA) was administered on days 8 and 10 to induce final follicular growth as previously described [23]. When the dominant follicle developed to a size > 18 mm, ovulation was triggered by the gonadotropin-releasing hormone agonist buserelin via the nasal route (Suprecur; Mochida Pharmaceutical Co., Ltd., Tokyo, Japan or Buserecur; Fuji Pharma Co., Ltd.).
Oocyte retrieval was performed 34 and 36 h after the trigger using a 21-gauge needle (Kitazato Corporation, Shizuoka, Japan). Cumulus-oocyte complexes were collected, washed, and cultured in human tubal fluid (HTF) medium (Kitazato Corporation) with paraffin oil at 37 °C (gas phase 5% O2, 5% CO2, and 90% N2) until denudation before ICSI [25].
Sperm samples were obtained through masturbation and examined according to the World Health Organisation’s laboratory manual for the examination and processing of human semen, 6 th edition [26]. The proportion of sperm with abnormal morphology of the head, neck/midpiece, and tail was calculated as the abnormal rate. Sperm samples were subsequently washed via centrifugation (600 g, 15 min) using 70% and 90% density gradients (Isolate; Irvine Scientific, Santa Ana, CA, USA). Finally, the prepared sperm was cultured in an HTF medium at 37 °C (gas phase 5% O2, 5% CO2, and 90% N2) until use.
Intracytoplasmic sperm injection and embryo culture
ICSI-inseminated oocytes were placed in N-hydroxyethylpiperazine-N-ethanesulfonate-buffered HTF medium [27]. Next, the injected oocytes were immediately placed on EmbryoSlides (Vitrolife Inc., Göteborg, Sweden) and cultured individually in a 180-µL medium (SAGE 1-Step; CooperSurgical, Inc., Måløv, Denmark) under paraffin oil. Embryos were cultured at 37 °C (gas phase 5% O2, 5% CO2, and 90% N2) in an Embryoscope + time-lapse incubator (Vitrolife Inc.) for 4–7 days [28]. Fertilisation and embryo development were observed using a TLT system (EmbryoViewer; Vitrolife Inc.). The inner cell mass (ICM) and trophectoderm (TE) of the blastocysts were graded morphologically according to Gardner’s criteria [29]. Furthermore, the quality of the blastocyst was classified into three groups as follows: excellent (AA), good (AB, BA, and BB), and poor (AC, BC, CA, CB, and CC).
Annotation and assessment of embryo development
Images were captured every 10 min in 11 focal planes over 4–7 days of culture. The time points for the onset of the following events were recorded and analysed: second polar body extrusion (tPB2), cytoplasmic wave (tCW), appearance of female and male pronuclei (tPNa), PN juxtaposition (tPNj), PNBD (tPNBD), and nucleolus precursor body (tNPB) alignment in the PN. Female and male PN were differentiated based on their mutual positions relative to the second polar body (PB2) and positional association with the CW [8, 27]. The presence and timing of the cytoplasmic halo during fertilisation were also annotated, as previously reported [25, 29]. Briefly, the time of halo appearance (tHa) was first identified by the cytoplasmic granule’s movement towards the oocyte. The end time of the centripetal movement was recorded as tHc. After central repositioning, the granules began to move back into the cortex. We recorded tHr and tHd as the times when the granules started redistributing and when the halo phenomenon disappeared, respectively. PNj and PNBD synchrony were annotated as previously reported [30]. All annotations in the Embryo Viewer software were performed by nine operators, each with > 5 years of embryology experience, who were blinded to the patient’s characteristics and clinical outcomes.
Blastocyst vitrification and warming
Cryotop (Kitazato Corporation, Shizuoka, Japan) was used for embryo vitrification, as previously described [31]. Briefly, the blastocysts were immersed in an equilibration solution composed of 7.5% (v/v) ethylene glycol and 7.5% (v/v) dimethylsulfoxide for 15 min. The blastocysts were subsequently transferred to a vitrification solution containing 15% (v/v) ethylene glycol, 15% (v/v) dimethylsulfoxide, and 0.5 M sucrose for 1.5 min. Next, the blastocysts were placed on the Cryotop (Kitazato Corporation) and immediately plunged into liquid nitrogen.
Blastocyst warming was performed as previously described [31]. The Cryotop (Kitazato Corporation) was briefly placed into a warming solution of 1.0 M sucrose at 37 °C for 1 min. Subsequently, the blastocysts were removed from the warming solution and transferred to a diluent solution of 0.5 M sucrose at room temperature. After 3 min, they were moved to the washing solution without sucrose. The blastocysts were placed in the washing solution for 1 min for the final dilution.
Blastocyst transfer
Monitoring involved transvaginal ultrasonography and blood hormone testing [32]. Ovulation was triggered using buserelin (Suprecur; Mochida Pharmaceutical Co., Ltd., Tokyo, Japan or Buserecur; Fuji Pharma Co., Ltd.) after confirming the initiation of the luteinizing hormone surge. Single vitrified-warmed blastocyst transfers (SVBTs) were conducted on day 7 following the ovulation trigger. Dydrogesterone (30 mg/day) was administered orally during the early luteal phase after SVBT. In cases of insufficient luteal function (progesterone level on the day of SVBT < 11 ng/mL), progesterone was administered intravaginally (Lutinus, Ferring Pharmaceuticals, Saint Prex, Switzerland) until the ninth week of pregnancy. Implantation was defined by a serum human chorionic gonadotropin (hCG) level (> 20 IU/mL) according to a previous study [33]. The clinical and ongoing pregnancy rates were defined based on ultrasonographic observation of a gestational sac 3 weeks after SVBTs and the detection of a foetal heartbeat 5 weeks after SVBTs, respectively. Live birth was defined as delivery at or beyond 22 weeks of pregnancy. Early pregnancy loss, also known as a chemical pregnancy, was defined by the absence of a gestational sac after implantation (serum hCG level > 20 IU/mL) [34]. Miscarriage and stillbirth are defined based on the absence of a foetal heartbeat by ultrasound for a previously documented live pregnancy, occurring before and after 22 weeks of gestational age, respectively.
Statistical analyses
Proportion data were analysed using the chi-square and Fisher exact tests. Continuous parameters were compared using the Mann–Whitney U test. Multivariate linear regression analysis was performed to adjust for bias using confounders and to verify statistical significance. The regression coefficients are reported with 95% confidence intervals. All statistical analyses were performed using the JMP software (SAS, Cary, NC, USA), and statistical significance was set at P < 0.05.
Results
Population and blastocyst formation.
Table 1 presents the patient characteristics of the study cohort. This study included 905 couples treated for diverse causes of infertility. The proportion of male factor cases was substantial (36.7%). Following ovarian stimulation achieved using a CC regimen with or without gonadotropin, we retrieved an average of 2.7 oocytes per cycle, of which an average of 2.0 oocytes were mature and suitable for treatment. A total of 1,936 oocytes were fertilised after the injection, and 1466 blastocysts (day 4, n = 29; day 5, n = 1181; day 6, n = 244; and day 7, n = 12) were obtained. Table 2 presents the patient characteristics stratified by day 4–7 blastocysts. Maternal age was higher in day 6 and 7 blastocysts than in day 4 and 5 blastocysts. Similarly, paternal age was older in day 6 blastocysts than in day 5 blastocysts. No correlation was found between semen quality and duration of blastocyst development.
Table 1.
Patient and cycle characteristics
| No. of patients | 905 |
|---|---|
| Maternal age (years) | 38.3 ± 0.1 |
| Paternal age (years) | 40.6 ± 0.2 |
| No. of previous oocyte retrieval cycles | 1.9 ± 0.0 |
| No. of previous embryo transfer cycles | 1.4 ± 0.0 |
| Body mass index (kg/m2) | 21.5 ± 0.1 |
| Infertility causes | |
| Ovulation, n (%) | 16 (1.8) |
| Tubal factor, n (%) | 34 (3.8) |
| Endometriosis, n (%) | 28 (3.1) |
| Endometrial factor, n (%) | 32 (3.5) |
| Male factor, n (%) | 332 (36.7) |
| Combined, n (%) | 148 (16.4) |
| Unexplained, n (%) | 315 (34.8) |
| No. of cycles | 1009 |
| Clomiphene citrate only, n (%) | 856 (84.8) |
| Clomiphene citrate with gonadotropin, n (%) | 153 (15.2) |
| No. of oocytes retrieved | 2.7 ± 0.1 |
| No. of oocytes matured at the retrieval | 2.0 ± 0.0 |
| Semen analysis | 2,019 |
| Sperm concentration (M/mL) | 52.1 ± 1.4 |
| Total sperm number (M) | 136.4 ± 4.0 |
| Motility rate (%) | 39.3 ± 0.5 |
| Abnormal rate (%) | 95.7 ± 0.0 |
| No. of oocytes inseminated, n | 2019 |
| No. of oocytes fertilised, n | 1936 |
| No. of blastocysts, n | 1466 |
| Day 4, n (%) | 29 (2.0) |
| Day 5, n (%) | 1181 (80.6) |
| Day 6, n (%) | 244 (16.6) |
| Day 7, n (%) | 12 (0.8) |
Values are presented as mean ± SEM or n (%)
No. number, SEM standard error of mean
Table 2.
Patient characteristics stratified by days 4–7 blastocysts
| Day 4 | Day 5 | Day 6 | Day 7 | P value | |
|---|---|---|---|---|---|
| No. of zygotes, n | 29 | 1,181 | 244 | 12 | |
| Maternal age (years) | 37.0 ± 0.8a | 37.7 ± 0.1a | 39.2 ± 0.2b | 40.2 ± 0.9b | < 0.0001 |
| Paternal age (years) | 39.7 ± 1.0a, b | 40.0 ± 0.2a | 41.4 ± 0.4b | 41.8 ± 1.7a, b | 0.0058 |
| No. of previous oocyte retrieval cycles | 2.0 ± 0.1 | 2.0 ± 0.0 | 2.0 ± 0.0 | 2.2 ± 0.3 | 0.8555 |
| No. of previous embryo transfer cycles | 1.7 ± 0.1 | 1.5 ± 0.0 | 1.4 ± 0.1 | 1.3 ± 0.3 | 0.1452 |
| Body mass index (kg/m2) | 21.6 ± 0.5 | 21.5 ± 0.1 | 21.6 ± 0.2 | 21.9 ± 0.7 | 0.9201 |
| Serum anti-Müllerian hormone level (ng/mL) | 2.5 ± 0.5 | 2.6 ± 0.1 | 2.3 ± 0.1 | 1.4 ± 0.3 | 0.0986 |
| Infertility causes | 0.5187 | ||||
| Ovulation, n (%) | 2 (6.9) | 23 (2.0) | 3 (10.7) | 0 (0) | |
| Tubal factor, n (%) | 0 (0) | 44 (3.7) | 9 (3.7) | 0 (0) | |
| Endometriosis, n (%) | 0 (0) | 32 (2.7) | 6 (2.5) | 1 (8.3) | |
| Endometrial factor, n (%) | 1 (3.5) | 40 (3.4) | 8 (3.3) | 1 (8.3) | |
| Male factor, n (%) | 15 (51.7) | 446 (37.8) | 108 (44.3) | 5 (41.7) | |
| Combined, n (%) | 2 (6.9) | 188 (15.9) | 35 (14.3) | 3 (25.0) | |
| Unexplained, n (%) | 9 (31.0) | 408 (34.6) | 75 (30.7) | 2 (16.7) | |
| Semen analysis | |||||
| Total sperm number | 125.8 ± 30.2 | 136.2 ± 4.4 | 138.2 ± 9.8 | 142.7 ± 35.9 | 0.9784 |
| Sperm concentration (M/mL) | 47.0 ± 11.0 | 52.1 ± 1.5 | 52.5 ± 3.5 | 55.5 ± 15.6 | 0.9538 |
| Motility rate (%) | 34.3 ± 3.5 | 39.4 ± 0.5 | 39.3 ± 1.2 | 41.4 ± 6.3 | 0.5044 |
| Abnormal rate (%) | 95.4 ± 0.3 | 95.7 ± 0.0 | 95.7 ± 0.1 | 95.7 ± 0.3 | 0.8223 |
Values are presented as mean ± SEM or n (%). (a–b) Different letters indicate a significant difference at P < 0.05
No. number, SEM standard error of mean
Morphokinetics
Table 3 presents the timing of each event during fertilisation. Regarding the cytoplasm, tPB2 in day 4 blastocysts was earlier than that in day 5 and 6 blastocysts. A similar trend was observed in tCW and tHa in day 4 blastocysts, which were earlier than those in day 5–7 blastocysts. The delay in tHc, tHr, and tHd was associated with prolonged blastocyst development time. Specifically, the timings of these events were significantly earlier in day 4 blastocysts than in day 5 blastocysts, which were also earlier than in day 6 and 7 blastocysts. Regarding the pronucleus and nucleolus precursor body, the time of paternal PNa (tpPNa) was earlier in day 4 blastocysts than in day 5 and 6 blastocysts, whereas that of maternal PNa (tmPNa) was comparable between the groups. The delay in tPNj was associated with prolonged blastocyst development time. tPNj was significantly earlier in day 4 blastocysts than in day 5 blastocysts, whereas it was slower in day 7 blastocysts than in day 5 blastocysts. tNPB in maternal PN (tmNPB) was comparable between the groups; however, tNPB in paternal PN (tpNPB) was earlier in day 4 and 5 blastocysts than in day 6 and 7 blastocysts. tPNBD in maternal and paternal PNs (tmPNBD and tpPNBD, respectively) in day 4–6 blastocysts increased with the number of days of blastocyst development.
Table 3.
Timings of each event during fertilisation stratified by days 4–7 blastocysts
| Day 4 | Day 5 | Day 6 | Day 7 | P value | |
|---|---|---|---|---|---|
| No. of zygotes, n | 29 | 1,181 | 244 | 12 | |
| tPB2 (h) | 2.6 ± 0.1a | 3.0 ± 0.0b | 3.0 ± 0.0b | 3.0 ± 0.2a, b | 0.0226 |
| tCW (h) | 3.8 ± 0.1a | 4.5 ± 0.0b | 4.6 ± 0.1b | 4.5 ± 0.2b | 0.0005 |
| tpPNa (h) | 4.2 ± 0.2a | 4.8 ± 0.0b | 4.9 ± 0.1b | 4.9 ± 0.3a, b | 0.0121 |
| tmPNa (h) | 4.6 ± 0.2 | 5.0 ± 0.0 | 5.1 ± 0.1 | 5.1 ± 0.4 | 0.1670 |
| tHa (h) | 7.2 ± 0.3a | 7.9 ± 0.0b | 7.9 ± 0.1b | 8.2 ± 0.4b | 0.0399 |
| tPNj (h) | 8.1 ± 0.5a | 8.6 ± 0.1b | 9.3 ± 0.2b, c | 12.0 ± 1.6c | 0.0007 |
| tmNPB (h) | 10.4 ± 0.6 | 12.2 ± 0.2 | 12.7 ± 0.3 | 11.0 ± 2.3 | 0.1084 |
| tHc (h) | 11.3 ± 0.3a | 12.4 ± 0.1b | 12.8 ± 0.2c | 14.4 ± 1.0c | 0.0011 |
| tpNPB (h) | 13.5 ± 1.2a | 15.9 ± 0.2a | 17.5 ± 0.4b | 17.9 ± 6.9b | 0.0003 |
| tHr (h) | 18.0 ± 0.3a | 19.9 ± 0.1b | 20.6 ± 0.2c | 22.0 ± 0.9c | < 0.0001 |
| tHd (h) | 19.6 ± 0.3a | 22.3 ± 0.1b | 23.3 ± 0.2c | 24.1 ± 0.7c | < 0.0001 |
| tmPNBD (h) | 19.7 ± 0.3a | 22.2 ± 0.1b | 23.4 ± 0.2c | 23.8 ± 0.6c | < 0.0001 |
| tpPNBD (h) | 19.7 ± 0.3a | 22.3 ± 0.1b | 23.4 ± 0.2c | 23.8 ± 0.7c | < 0.0001 |
Values are presented as mean ± SEM or n (%). (a–c) Different letters indicate a significant difference at P < 0.05
tPB2 time of second polar body extrusion, tCW time of cytoplasmic wave, tpPNa time of appearance of paternal pronucleus (PN), tmPNa time of appearance of maternal PN, tHa time of halo appearance, tPNj time of PN juxtaposition, tmNPB time of nucleolus precursor body (NPB) alignment in maternal PN, tHc time of halo centring, tpNPB time of NPB alignment in paternal PN, tHr time of halo redistribution, tHd time of halo disappearance, tmPNBD time of maternal PN breakdown, tpPNBD time of paternal PN breakdown, No. number, SEM standard error of mean
Table 4 presents the incidence and time intervals (TIs) of the cytoplasmic waves and halos. CW was observed in almost all fertilised oocytes (days 4, 6, and 7, 100%; day 5, 99.8%). The cytoplasmic halo incidence differed among the groups, and its absence was associated with prolonged blastocyst developmental time. Additionally, the TI from tPB2 to tCW was shorter in day 4 blastocysts than in day 5 and 6 blastocysts. The TIs from tHr to tHd and from tHa to tHd in day 4 blastocysts were shorter than those in day 5 blastocysts, which were also shorter than those in day 6 and 7 blastocysts.
Table 4.
Cytoplasmic wave and halo stratified by days 4–7 blastocysts
| Day 4 | Day 5 | Day 6 | Day 7 | P value | |
|---|---|---|---|---|---|
| Zygote with cytoplasmic wave, n (%) | 29 (100) | 1,179 (99.8) | 244 (100) | 12 (100) | 0.9225 |
| Zygotes with cytoplasmic halo, n (%) | 29 (100)a, b | 1,163 (98.5)a | 235 (96.3)b | 10 (83.3)c | 0.0003 |
| Position of cytoplasmic halo | 0.1949 | ||||
| Symmetric, n (%) | 13 (44.8) | 398 (34.2) | 69 (29.4) | 2 (20.0) | |
| Asymmetric, n (%) | 16 (55.2) | 745 (64.1) | 69 (68.9) | 7 (70.0) | |
| Unstable, n (%) | 0 (0) | 20 (1.7) | 4 (1.7) | 1 (10.0) | |
| Time interval from tPB2 to tCW (h) | 1.2 ± 0.1a | 1.5 ± 0.0b | 1.6 ± 0.1b | 1.5 ± 0.2a, b | 0.0446 |
| Time interval from tCW to tHa (h) | 3.4 ± 0.3 | 3.4 ± 0.0 | 3.4 ± 0.1 | 3.7 ± 0.3 | 0.7762 |
| Time interval from tHa to tHc (h) | 4.2 ± 0.3 | 4.5 ± 0.1 | 4.8 ± 0.2 | 6.1 ± 1.0 | 0.0863 |
| Time interval from tHc to tHr (h) | 6.7 ± 0.4 | 7.5 ± 0.1 | 7.8 ± 0.3 | 7.6 ± 1.0 | 0.3218 |
| Time interval from tHr to tHd (h) | 1.6 ± 0.1a | 2.6 ± 0.1b | 2.9 ± 0.2c | 2.2 ± 0.3a, b, c | 0.0450 |
| Time interval from tHa to tHd (h) | 12.4 ± 0.4a | 14.3 ± 0.1b | 15.3 ± 0.2c | 15.9 ± 1.0c | < 0.0001 |
Values are presented as mean ± SEM or n (%). (a–c) Different letters indicate a significant difference at P < 0.05
tPB2 time of second polar body extrusion, tCW time of cytoplasmic wave, tHa time of halo appearance, tHc time of halo centring, tHr time of halo redistribution, tHd time of halo disappearance, SEM standard error of mean
Table 5 presents the characteristics of PN dynamics. The TI from tpPNa to tPNj in day 7 blastocysts was longer than that in day 4 and 5 blastocysts. Although the TI from tPNj to tpNPB in day 6 blastocysts was prolonged compared with that in day 5 blastocysts, it was shorter in day 7 blastocysts than in day 5 and 6 blastocysts. The TI from tpPNa to tpPNBD in day 4 blastocysts was shorter than that in day 5 blastocysts, which were also shorter than that in day 6 and 7 blastocysts. Additionally, the same TIs in maternal PN were examined, and they showed a similar trend to those of paternal PN; however, the TI from tPNj to tmNPB was comparable between the groups.
Table 5.
Behaviour of pronuclei and nucleolus precursor bodies stratified by days 4–7 blastocysts
| Day 4 | Day 5 | Day 6 | Day 7 | P value | |
|---|---|---|---|---|---|
| Area of maternal PN (µm2) | 553.5 ± 14.2 | 533.1 ± 2.1 | 530.0 ± 5.4 | 522.7 ± 14.7 | 0.4466 |
| Area of paternal PN (µm2) | 633.0 ± 20.6 | 605.2 ± 2.8 | 612.0 ± 6.3 | 584.3 ± 16.7 | 0.2798 |
| Volume of maternal PN (103 × µm3) | 9.9 ± 0.4 | 9.3 ± 0.1 | 9.3 ± 0.1 | 9.0 ± 0.4 | 0.4640 |
| Volume of paternal PN (103 × µm3) | 12.1 ± 0.6 | 11.3 ± 0.1 | 11.5 ± 0.2 | 10.7 ± 0.4 | 0.2563 |
| Zygote with PN juxtaposition, n (%) | 29 (100) | 1,177 (99.7) | 241 (98.8) | 12 (100) | 0.3106 |
| Zygotes with NPB alignment in maternal PN, n (%) | 19 (65.5) | 822 (69.6) | 168 (68.9) | 6 (50.0) | 0.5004 |
| Zygotes with NPB alignment in paternal PN, n (%) | 12 (41.4) | 519 (44.0) | 119 (48.8) | 2 (16.7) | 0.1203 |
| Zygote with synchronous PNBD, n (%) | 29 (100) | 1,173 (99.3) | 241 (98.8) | 12 (100) | 0.7658 |
| Paternal PN | |||||
| Time interval from tpPNa to tPNj (h) | 3.9 ± 0.5a | 4.1 ± 0.1a | 4.4 ± 0.2a, b | 7.3 ± 1.6b | 0.0021 |
| Time interval from tPNj to tpNPB (h) | 5.6 ± 1.5a, b, c | 6.7 ± 0.2a | 8.1 ± 0.6b | − 2.64 ± 2.6c | 0.0060 |
| Time interval from tpNPB to tpPNBD (h) | 6.9 ± 1.6 | 6.8 ± 0.2 | 6.3 ± 0.4 | 6.7 ± 6.0 | 0.6586 |
| Time interval from tpPNa to tpPNBD (h) | 15.5 ± 0.3a | 17.5 ± 0.1b | 18.5 ± 0.2c | 19.1 ± 0.7c | < 0.0001 |
| Maternal PN | |||||
| Time interval from tmPNa to tPNj (h) | 2.6 ± 0.5a | 3.9 ± 0.1a | 4.2 ± 0.2a, b | 7.0 ± 1.5b | 0.0019 |
| Time interval from tPNj to tmNPB (h) | 1.9 ± 1.0 | 3.1 ± 0.2 | 3.6 ± 0.5 | − 2.2 ± 2.2 | 0.0550 |
| Time interval from tmNPB to tmPNBD (h) | 5.7 ± 0.6 | 7.2 ± 0.2 | 7.8 ± 0.3 | 5.9 ± 1.9 | 0.1326 |
| Time interval from tmPNa to tmPNBD (h) | 15.1 ± 0.3a | 17.2 ± 0.1b | 18.3 ± 0.2c | 18.8 ± 0.7c | < 0.0001 |
Values are presented as mean ± SEM or n (%). (a–c) Different letters indicate a significant difference at P < 0.05
tpPNa time of appearance of paternal pronucleus (PN), tmPNa time of appearance of maternal PN, tPNj time of PN juxtaposition, tmNPB time of nucleolus precursor body (NPB) alignment in maternal PN, tpNPB time of NPB alignment in paternal PN, tmPNBD time of maternal PN breakdown (PNBD), tpPNBD time of paternal PNBD, SEM standard error of mean
Multivariate linear regression analysis demonstrated that tPB2 and tpPNa were significantly earlier in day 4 blastocysts than in day 5 blastocysts, whereas tpPNa was delayed in day 6 blastocysts compared with day 5 blastocysts (Table 6). The TI from tHr to tHd was prolonged in day 6 blastocysts compared with day 5. However, the TI from tHa to tHd was shortened in day 4 blastocysts and prolonged in day 6 blastocysts compared with day 5 blastocysts. The TI from tpNa to tPNj was prolonged in day 6 and 7 blastocysts, and that from tPNj to tpNPB was prolonged in day 6 blastocysts; however, it was shortened in day 7 blastocysts compared with day 5 blastocysts. Additionally, the TI from tPNa to tpPNBD was shortened in day 4 blastocysts and prolonged in day 6 and 7 blastocysts. A similar trend was observed in the TI from tmPNa to tPNj and that from tmPNa to tmPNBD.
Table 6.
Multivariate linear regression analysis for the time required for blastocyst expansion
| Coefficient of regression (95% confidential intervals) | Standard error | T score | P value | ||
|---|---|---|---|---|---|
| tPB2 | Day 4 | − 2.03 (− 0.552 to − 0.008) | 0.138 | − 2.03 | 0.0430 |
| Day 5 | Reference | – | – | – | |
| Day 6 | 0.08 (− 0.077 to 0.242) | 0.008 | 1.01 | 0.3114 | |
| Day 7 | 0.120 (− 0.234 to 0.476) | 0.181 | 0.67 | 0.5050 | |
| tpPNa | Day 4 | − 0.405 (− 0.793 to − 0.017) | 0.197 | − 2.05 | 0.0406 |
| Day 5 | Reference | – | – | – | |
| Day 6 | 0.234 (0.005–0.462) | 0.116 | 2.01 | 0.0445 | |
| Day 7 | 0.117 (− 0.390 to 0.625) | 0.258 | 0.45 | 0.6501 | |
| Time interval from tPB2 to tCW | Day 4 | − 0.232 (− 0.524 to 0.060) | 0.149 | − 1.56 | 0.1197 |
| Day 5 | Reference | – | – | – | |
| Day 6 | 0.166 (− 0.005 to 0.339) | 0.087 | 1.90 | 0.0577 | |
| Day 7 | 0.004 (− 0.378 to 0.387) | 0.195 | 0.02 | 0.9802 | |
| Time interval from tHr to tHd | Day 4 | − 0.778 (− 1.842 to − 0.286) | 0.542 | − 1.43 | 0.1516 |
| Day 5 | Reference | – | – | – | |
| Day 6 | 0.727 (0.073–1.381) | 0.333 | 2.18 | 0.0293 | |
| Day 7 | − 0.249 (− 1.763 to 1.265) | 0.772 | − 0.32 | 0.7469 | |
| Time interval from tHa to tHd | Day 4 | − 2.008 (− 3.009 to − 1.007) | 0.510 | − 3.94 | < 0.0001 |
| Day 5 | Reference | – | – | – | |
| Day 6 | 0.946 (0.343–1.550) | 0.307 | 3.08 | 0.0021 | |
| Day 7 | 1.222 (− 0.140 to 2.585) | 0.694 | 1.76 | 0.0787 | |
| Time interval from tpPNa to tPNj | Day 4 | − 1.089 (− 2.199 to 0.020) | 0.565 | − 1.93 | 0.0544 |
| Day 5 | Reference | – | – | – | |
| Day 6 | 0.659 (0.004–1.313) | 0.333 | 1.98 | 0.0484 | |
| Day 7 | 2.757 (1.304–4.210) | 0.740 | 3.72 | 0.0002 | |
| Time interval from tPNj to tpNPB | Day 4 | 1.014 (− 2.38 to 4.409) | 1.728 | 0.59 | 0.5575 |
| Day 5 | Reference | – | – | – | |
| Day 6 | 3.638 (1.326–5.950) | 1.177 | 3.09 | 0.0021 | |
| Day 7 | − 7.174 (− 13.102 to − 1.247) | 3.017 | − 2.38 | 0.0178 | |
| Time interval from tpPNa to tpPNBD | Day 4 | − 2.220 (− 3.128 to − 1.313) | 0.462 | − 4.80 | < 0.0001 |
| Day 5 | Reference | – | – | – | |
| Day 6 | 0.893 (0.359–1.428) | 0.272 | 3.28 | 0.0011 | |
| Day 7 | 1.465 (0.277–2.652) | 0.605 | 2.42 | 0.0157 | |
| Time interval from tmPNa to tPNj | Day 4 | − 1.188 (− 2.310 to − 0.066) | 0.571 | − 2.08 | 0.0379 |
| Day 5 | Reference | – | – | – | |
| Day 6 | − 0.569 (− 1.230 to 0.091) | 0.337 | − 1.69 | 0.0913 | |
| Day 7 | 2.732 (1.264–4.200) | 0.748 | 3.65 | 0.0003 | |
| Time interval from tmPNa to tmPNBD | Day 4 | − 2.282 (− 3.229 to − 1.336) | 0.482 | − 4.73 | < 0.0001 |
| Day 5 | Reference | – | – | – | |
| Day 6 | 0.970 (0.412–1.527) | 0.284 | 3.42 | 0.0007 | |
| Day 7 | 1.455 (0.216–2.694) | 0.631 | 2.30 | 0.0214 |
Confounding factors: maternal age, paternal age, serum anti-Müllerian hormone level, and previous oocyte retrievals
tPB2 time of second polar body extrusion, tCW time of cytoplasmic wave, tpPNa time of appearance of paternal pronucleus (PN), tHr time of halo redistribution, tHd time of halo disappearance, tHa time of halo appearance, tPNj time of PN juxtaposition, tpNPB time of NPB alignment in paternal PN, tpPNBD time of paternal PN breakdown, tmPNa time of appearance of maternal PN, tmPNBD time of maternal PN breakdown
Blastocyst quality
The morphological quality of the ICM and TE progressively worsened with the increasing duration of blastocyst development (Online Resource 1A). Additionally, the blastocysts were categorised by the day of formation, and the impact of maternal age on blastocyst quality was examined (Online Resource 1B–E). In this cohort, the quality was not associated with maternal age.
Pregnancy outcomes
In the subset of transferred blastocysts (Table 7), the day of blastocyst development was positively associated with maternal and paternal age but not with the number of previous cycles or body mass index. The morphological quality of the ICM and TE worsened progressively with the increasing time of blastocyst development in the same subset. Pregnancy outcomes, when not controlled for confounders, worsened progressively with the increasing days of blastocyst development. When adjusting for female age, male age, and the morphological grade of ICM and TE, the probabilities of implantation, clinical and ongoing pregnancies, and live births were significantly reduced in day 6 blastocysts compared with day 5 blastocysts (Table 8). Furthermore, the live birth rate was stratified by the day of blastocyst development and the median female age (Table 9). The adverse impacts of maternal ageing on the live birth rate were observed after transferring day 4 and 5 blastocysts but not day 6 blastocysts.
Table 7.
Pregnancy outcomes stratified by day 4–7 blastocysts
| Day 4 | Day 5 | Day 6 | Day 7 | P value | |
|---|---|---|---|---|---|
| ET cycles, n | 27 | 967 | 168 | 11 | |
| Female age (years) | 37.1 ± 0.8a | 38.0 ± 0.1a, b | 39.8 ± 0.3c | 40.1 ± 0.9b, c | < 0.0001 |
| Male age (years) | 39.9 ± 1.1a, b | 40.3 ± 0.2a | 41.9 ± 0.4b | 42.3 ± 1.7a,b | 0.0062 |
| Body mass index (kg/m2) | 21.6 ± 0.5 | 21.5 ± 0.1 | 21.5 ± 0.2 | 21.8 ± 0.7 | 0.9811 |
| Previous ET cycles | 1.7 ± 0.1 | 1.5 ± 0.0 | 1.4 ± 0.1 | 1.5 ± 0.2 | 0.1686 |
| Culture time (h) | 115.3 ± 0.2 | 121.8 ± 0.3 | 142.0 ± 0.3 | 163.7 ± 0.5 | < 0.0001 |
| Morphological grade of inner cell mass | |||||
| Grade A, n (%) | 22 (81.5)a | 550 (56.9)b | 20 (11.9)c | 1 (9.1)c | < 0.0001 |
| Grade B, n (%) | 4 (14.8)a, b | 297 (30.7)a | 43 (25.6)a, b | 0 (0)b | 0.0252 |
| Grade C, n (%) | 1 (3.7)a | 120 (12.4)b | 105 (62.5)c | 10 (90.9)c | < 0.0001 |
| Morphological grade of trophectoderm | |||||
| Grade A, n (%) | 23 (85.2)a | 497 (51.4)b | 7 (4.2)c | 0 (0)c | < 0.0001 |
| Grade B, n (%) | 4 (14.8)a, b | 242 (25.0)a | 19 (11.3)b | 1 (9.1)a, b | 0.0005 |
| Grade C, n (%) | 0 (0)a | 228 (23.6)b | 142 (84.5)c | 10 (90.9)c | < 0.0001 |
| Implantation, n (%) | 20 (74.1)a | 508 (52.5)b | 22 (13.1)c | 1 (9.1)c | < 0.0001 |
| Clinical pregnancies, n (%) | 19 (70.4)a | 442 (45.7)b | 19 (11.3)c | 1 (9.1)c | < 0.0001 |
| Ongoing pregnancies, n (%) | 17 (63.0)a | 382 (39.5)b | 15 (8.9)c | 1 (9.1)c | < 0.0001 |
| Live birth, n (%) | 15 (55.6)a | 324 (33.5)b | 11 (6.6)c | 1 (9.1)b, c | < 0.0001 |
| Early pregnancy loss, n (%) | 1 (5.0) | 66 (13.0) | 3 (13.6) | 0 (0) | 0.7360 |
| Miscarriages, n (%) | 4 (21.1) | 118 (26.7) | 8 (42.1) | 0 (0) | 0.4027 |
Values are presented as mean ± SEM or n (%). (a–b) Different letters indicate a significant difference at P < 0.05
SEM standard error of the mean, ET embryo transfer
Table 8.
Multivariate logistic regression analysis for pregnancy outcomes
| Outcome | Adjusted odds ratio* | 95% confidence intervals | P value | |
|---|---|---|---|---|
| Implantation | Day 4 | 1.694 | 0.683–4.203 | 0.2552 |
| Day 5 | Reference | – | – | |
| Day 6 | 0.365 | 0.218–0.614 | 0.0001 | |
| Day 7 | 0.333 | 0.041–2.717 | 0.3052 | |
| Clinical pregnancy | Day 4 | 2.001 | 0.838–4.775 | 0.1180 |
| Day 5 | Reference | – | – | |
| Day 6 | 0.398 | 0.230–0.689 | 0.0010 | |
| Day 7 | 0.449 | 0.055–3.672 | 0.4555 | |
| Ongoing pregnancy | Day 4 | 1.831 | 0.797–4.203 | 0.1536 |
| Day 5 | Reference | – | – | |
| Day 6 | 0.403 | 0.221–0.735 | 0.0030 | |
| Day 7 | 0.586 | 0.071–4.825 | 0.6201 | |
| Live birth | Day 4 | 1.726 | 0.759–3.928 | 0.1927 |
| Day 5 | Reference | – | – | |
| Day 6 | 0.393 | 0.198–0.779 | 0.0075 | |
| Day 7 | 0.819 | 0.098–6.820 | 0.8542 |
*Confounders: female age, male age, and morphological grade of inner cell mass and trophectoderm
Table 9.
Pregnancy outcomes in live birth cycles stratified by the female age and day of cryopreservation
| Female age (years) | Adjusted odds ratio* | 95% confidence intervals | P value | |||
|---|---|---|---|---|---|---|
| < 38 | ≥ 38 | (Reference: < 38) | ||||
| Day 4 | ET cycles, n | 15 | 12 | |||
| Live birth, n (%) | 11 (73.3) | 4 (33.3) | 0.122 | 0.013–0.762 | 0.0234 | |
| Day 5 | ET cycles, n | 425 | 542 | |||
| Live birth, n (%) | 186 (43.8) | 138 (25.5) | 0.434 | 0.327–0.575 | < 0.0001 | |
| Day 6 | ET cycles, n | 42 | 126 | |||
| Live birth, n (%) | 3 (7.1) | 8 (6.4) | 0.935 | 0.227–4.822 | 0.9293 | |
*Confounders: morphological grade of inner cell mass and trophectoderm
ET embryo transfer
Discussion
Our study demonstrated that embryos developing to the blastocyst stage between days 4 and 7 displayed different morphokinetic velocities throughout fertilisation in the cytoplasmic and pronuclear compartments (Fig. 2). This difference already emerged at the earliest morphokinetic marker of fertilisation: PB2 extrusion.
Fig. 2.
Morphokinetic differences during fertilisation between days 4 and 7 blastocysts. tPB2, time of second polar body extrusion; tCW, time of cytoplasmic wave; tpPNa, time of appearance of paternal pronucleus (PN); tmPNa, time of appearance of maternal PN; tHa, time of halo appearance; tPNj, time of PN juxtaposition; tmNPB, time of nucleolus precursor body (NPB) alignment in maternal PN; tHc, time of halo centring; tpNPB, time of NPB alignment in paternal PN; tHr, time of halo redistribution; tHd, time of halo disappearance; tmPNBD, time of maternal PN breakdown; tpPNBD, time of paternal PN breakdown
PB2 extrusion was earlier in day 4 blastocysts than in days 5 and 6 blastocysts. Paternal PN appeared earlier in day 4 blastocysts than in day 5 blastocysts but was delayed in day 6 blastocysts. Sperm-borne phospholipase C-zeta triggers activation by generating Ca2+ oscillations [35]. This activation is essential for establishing cortical polarization, PB2 extrusion, and PN formation [36–38]. Therefore, we hypothesized that the observed significantly early time differences might reflect the speed at which activation mechanisms are initiated after sperm microinjection. We also speculated that sperm quality might influence embryonic developmental velocity from the outset; however, we did not observe a correlation between semen parameters and early morphokinetic events. This may depend on the fact that the sperm selected for ICSI does not necessarily reflect the general quality of the semen sample of origin. However, maternal and paternal ages were the only patient characteristics negatively associated with the day of blastocyst development. Variations in the timing of male PN formation and downstream events may be due to the speed at which the paternal chromatin is rearranged after microinjection. Previous studies have reported that the sperm nuclear membrane breaks down following gamete fusion (or ICSI), and maternally derived histones replace protamines in male chromatin [39]. Protamines are completely replaced within the first 2–4 h post-insemination (hpi) [40], a time consistent with the tpPNa observed in the present study. Therefore, a difference in tpPNa among days 4–7 blastocysts may be due to a delay or prolongation of the time required for protamine replacement. Further molecular studies, including DNA damage assessments, are required to examine this hypothesis.
Male and female PN typically emerge almost simultaneously and are subsequently juxtaposed at 8–10 hpi; NPB alignment occurs with female PN earlier (8–13 hpi) than male PN (11–17 hpi), and the two PN break down synchronously [9]. We observed similar results in this study and did not observe large changes in male and female PN between days 5 and 7; however, PN juxtaposition was delayed in the day 7 blastocyst group. Cavazza et al. [41] reported that molecular players, such as microtubules, dynein, nuclear pore complexes, and centrosomes, are responsible for driving human PN migration and juxtaposition. Coticchio et al. [8] reported that the female PN approaches the male PN rectilinearly in almost all cases, guided by the microtubular network. A mammalian study revealed that the actin cytoskeleton indirectly diminishes microtubule network mobility and blocks further PN migration [42]. Therefore, the differences in tPNj between days 4 and 7 may be caused by a slower action of motor proteins, including dynein, or delays in female PN movement hindered by dysfunction of cytoplasmic components, such as actin. Consistent with this hypothesis, multivariate linear regression analysis revealed prolonged halo dynamics in slow-growing blastocysts, indicating slower movement of cytoplasmic granules and organelles. Strikingly, in cytoplasmic dynamics, our study found that even the duration of the significantly short interval intervening between tHr and tHd is longer in later-developing blastocysts. A previous study reported that the non-juxtaposed PN pattern was associated with reduced pre-implantation development [30]. Therefore, PNj occurrence and tPNj may be an effective predictor of embryo development velocity.
Finally, a detailed analysis of PN morphometry (size and volume) and NPB patterning (alignment) revealed no differences among the study groups. This suggests that the mechanisms of PN assembly and chromatin rearrangement within the PNs are not involved in causing developmental delays.
The strength of this study was that each patient’s protocol was standardised. However, this study has some limitations. First, only natural or mild stimulation cycles were used. Second, our unique blastocyst classification criteria for vitrification may have influenced the results; they are based on the blastocyst diameter on the day of development (days 4–6, 160 µm; day 7, 180 µm) [43]. Third, this study only analysed zygotes generated by ICSI. The analysis of conventional IVF cases may reveal different correlations between fertilisation patterns and embryo development. Finally, patients who used cryopreserved gametes were excluded; therefore, further analyses would be beneficial to evaluate the dynamics of this cohort.
In conclusion, embryos that developed to the blastocyst stage between days 4 and 7 exhibited different morphokinetic speeds throughout fertilisation, starting as early as PB2 extrusion and male PN formation. These initial differences might depend on activation mechanisms. Such differences progressively increased, especially until tPNj. Aberrant rearrangement of cytoplasmic granules, organelles, and PN may be involved in this second phase of changes in fertilisation dynamics. Therefore, the delay in fertilisation morphokinetics observed in late-forming blastocysts appears to have multifactorial origins attributable to activation mechanisms (signified by the times of PB2 extrusion and male PN formation) and cytoplasmic factors (suggested by prolonged times of PN repositioning).
Further molecular studies are required to reveal the biological mechanisms of fertilisation events that affect subsequent developmental competence and velocity. Coticchio et al. [44] focused on the cytoplasmic movements of early human embryos and explored a novel approach using artificial intelligence (AI) to predict blastocyst development. This study’s findings suggest that AI interpretation of sequential images subjected to particle image velocimetry analysis could be harnessed to improve the early prediction of embryo development ability. Further analysis of cytoplasmic movements throughout fertilisation events using these approaches will be crucial for future studies, contributing to the non-invasive prediction of blastocyst development and velocity as early as the zygote stage.
Supplementary information
Below is the link to the electronic supplementary material.
Online Resource 1: Blastocyst morphology stratified by the day of blastocyst formation. A) Blastocyst grades based on Gardner’s criteria: excellent (AA), good (AB, BA, and BB), and poor (AC, BC, CA, CB, and CC). A–C, a–c Different superscript letters indicate a significant difference at P < 0.05. Blastocyst grades are stratified by median maternal age: Days 4 (B), 5 (C), 6 (D), and 7 (E) blastocysts.
Author contribution
Nanoha Fujiwara and Kenji Ezoe contributed to the study design, data collection, interpretation, and writing. Giovanni Coticchio contributed to the study design, interpretation, and manuscript writing. Danilo Cimadomo, Laura Rienzi, and Keiichi Kato contributed to the manuscript revision. All the authors have read and approved the final version of the manuscript.
Funding
The authors did not receive support from any organisation for the submitted work.
Data availability
The primary data for this study are available from the authors upon request.
Code availability
Not applicable.
Declarations
Ethics approval
The Institutional Review Board of Kato Ladies Clinic approved this study on 30 March 2022 (approval number: 21–21).
Consent to participate
Written informed consent for the retrospective analysis of de-identified data was obtained from patients undergoing in vitro fertilisation treatment at the centre.
Consent for publication
Not applicable.
Competing interests
The authors declare no competing interests.
Footnotes
Publisher's Note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Nanoha Fujiwara and Kenji Ezoe contributed equally to this manuscript.
Contributor Information
Kenji Ezoe, Email: k-ezoe@towako-kato.com.
Keiichi Kato, Email: k-kato@towako.net.
References
- 1.Velez MP, Soule A, Gaudet L, Pudwell J, Nguyen P, Ray JG. Multifetal pregnancy after implementation of a publicly funded fertility program. JAMA Netw Open. 2024;7:e248496. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 2.Li H, Zhang W, Chen J, Lu X, Li L, Sun X, Ng EH. Immediate versus delayed single blastocyst transfer following the first stimulated IVF cycle in the freeze-all strategy: a study protocol for a randomised controlled trial. BMJ Open. 2024;14:e081018. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 3.Gleicher N, Patrizio P, Brivanlou A. Preimplantation genetic testing for aneuploidy — a castle built on sand. Trends Mol Med. 2021;27:731–42. [DOI] [PubMed] [Google Scholar]
- 4.Nagy ZP, Janssenswillen C, Janssens R, De Vos A, Staessen C, Van de Velde H, Van Steirteghem AC. Timing of oocyte activation, pronucleus formation and cleavage in humans after intracytoplasmic sperm injection (ICSI) with testicular spermatozoa and after ICSI or in-vitro fertilization on sibling oocytes with ejaculated spermatozoa. Hum Reprod. 1998;13:1606–12. [DOI] [PubMed] [Google Scholar]
- 5.Nagy ZP, Liu J, Joris H, Devroey P, Van Steirteghem A. Time-course of oocyte activation, pronucleus formation and cleavage in human oocytes fertilized by intracytoplasmic sperm injection. Hum Reprod. 1994;9:1743–8. [DOI] [PubMed] [Google Scholar]
- 6.Ueno S, Berntsen J, Ito M, Uchiyama K, Okimura T, Yabuuchi A, Kato K. Pregnancy prediction performance of an annotation-free embryo scoring system on the basis of deep learning after single vitrified-warmed blastocyst transfer: a single-center large cohort retrospective study. Fertil Steril. 2021;116:1172–80. [DOI] [PubMed] [Google Scholar]
- 7.Coticchio G, Barrie A, Lagalla C, Borini A, Fishel S, Griffin D, Campbell A. Plasticity of the human preimplantation embryo: developmental dogmas, variations on themes and self-correction. Hum Reprod Update. 2021;27:848–65. [DOI] [PubMed] [Google Scholar]
- 8.Coticchio G, Mignini Renzini M, Novara PV, Lain M, De Ponti E, Turchi D, Fadini R, Dal Canto M. Focused time-lapse analysis reveals novel aspects of human fertilization and suggests new parameters of embryo viability. Hum Reprod. 2018;33:23–31. [DOI] [PubMed] [Google Scholar]
- 9.Ezoe K, Takahashi T, Miki T, Kato K. Developmental perturbation in human embryos: clinical and biological significance learned from time-lapse images. Reprod Med Biol. 2024;23:e12593. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 10.Meseguer M, Herrero J, Tejera A, Hilligsoe KM, Ramsing NB, Remohi J. The use of morphokinetics as a predictor of embryo implantation. Hum Reprod. 2011;26:2658–71. [DOI] [PubMed] [Google Scholar]
- 11.technology EWgoT-l, Apter S, Ebner T, Freour T, Guns Y, Kovacic B, Le Clef N, Marques M, Meseguer M, Montjean D, Sfontouris I, Sturmey R, Coticchio G. Good practice recommendations for the use of time-lapse technology(dagger). Hum Reprod Open. 2020;2020:hoaa008. [DOI] [PMC free article] [PubMed]
- 12.Hernandez-Nieto C, Lee JA, Slifkin R, Sandler B, Copperman AB, Flisser E. What is the reproductive potential of day 7 euploid embryos? Hum Reprod. 2019;34:1697–706. [DOI] [PubMed] [Google Scholar]
- 13.Michaeli J, Ge N, Huszti E, Greenblatt EM. Is a day 7 blastocyst predictive of the reproductive potential of sibling day 5 and day 6 blastocysts? J Assist Reprod Genet. 2024;41:1835–42. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 14.Ozgur K, Berkkanoglu M, Bulut H, Donmez L, Isikli A, Coetzee K. Blastocyst age, expansion, trophectoderm morphology, and number cryopreserved are variables predicting clinical implantation in single blastocyst frozen embryo transfers in freeze-only-IVF. J Assist Reprod Genet. 2021;38:1077–87. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 15.Tiegs AW, Sun L, Patounakis G, Scott RT. Worth the wait? Day 7 blastocysts have lower euploidy rates but similar sustained implantation rates as Day 5 and Day 6 blastocysts. Hum Reprod. 2019;34:1632–9. [DOI] [PubMed] [Google Scholar]
- 16.Fu J, Wang XJ, Wang YW, Sun J, Gemzell-Danielsson K, Sun XX. The influence of early cleavage on embryo developmental potential and IVF/ICSI outcome. J Assist Reprod Genet. 2009;26:437–41. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 17.Iwata K, Mio Y. Observation of human embryonic behavior in vitro by high-resolution time-lapse cinematography. Reprod Med Biol. 2016;15:145–54. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 18.Kato K, Ueno S, Yabuuchi A, Uchiyama K, Okuno T, Kobayashi T, Segawa T, Teramoto S. Women’s age and embryo developmental speed accurately predict clinical pregnancy after single vitrified-warmed blastocyst transfer. Reprod Biomed Online. 2014;29:411–6. [DOI] [PubMed] [Google Scholar]
- 19.Coticchio G, Ezoe K, Lagalla C, Zaca C, Borini A, Kato K. The destinies of human embryos reaching blastocyst stage between Day 4 and Day 7 diverge as early as fertilization. Hum Reprod. 2023;38:1690–9. [DOI] [PubMed] [Google Scholar]
- 20.Coticchio G, Marchio L, Bartolacci A, Cimadomo D, Zaca C, Lagalla C, Tarozzi N, Borini A, Rienzi L. Delays in the final stages of fertilization are strongly associated with trichotomous cytokinesis and cleavage arrest. J Assist Reprod Genet. 2025;42:107–14. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 21.Chamayou S, Romano S, Alecci C, Storaci G, Ragolia C, Palagiano A, Guglielmino A. Oocyte vitrification modifies nucleolar remodeling and zygote kinetics-a sibling study. J Assist Reprod Genet. 2015;32:581–6. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 22.van Marion ES, Speksnijder JP, Hoek J, Boellaard WPA, Dinkelman-Smit M, Chavli EA, Steegers-Theunissen RPM, Laven JSE, Baart EB. Time-lapse imaging of human embryos fertilized with testicular sperm reveals an impact on the first embryonic cell cycle. Biol Reprod. 2021;104:1218–27. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 23.Karakida S, Ezoe K, Fukuda J, Yabuuchi A, Kobayashi T, Kato K. Effects of gonadotropin administration on clinical outcomes in clomiphene citrate-based minimal stimulation cycle IVF. Reprod Med Biol. 2020;19:128–34. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 24.Kato K, Ezoe K, Yabuuchi A, Fukuda J, Kuroda T, Ueno S, Fujita H, Kobayashi T. Comparison of pregnancy outcomes following fresh and electively frozen single blastocyst transfer in natural cycle and clomiphene-stimulated IVF cycles. Hum Reprod Open. 2018;2018:hoy006. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 25.Ezoe K, Hickman C, Miki T, Okimura T, Uchiyama K, Yabuuchi A, Kobayashi T, Coticchio G, Kato K. Cytoplasmic halo characteristics during fertilization and their implications for human preimplantation embryo development and pregnancy outcome. Reprod Biomed Online. 2020;41:191–202. [DOI] [PubMed] [Google Scholar]
- 26.World Health Organization. WHO laboratory manual for the examination and processing of human semen, 6th ed. 2021. https://www.who.int/publications/i/item/9789240030787. Accessed 8/3/2024.
- 27.Ezoe K, Takahashi T, Shimazaki K, Miki T, Tanimura Y, Amagai A, Sawado A, Akaike H, Mogi M, Kaneko S, Kato M, Kato K, Tarozzi N, Borini A, Coticchio G. Human 1PN and 3PN zygotes recapitulate all morphokinetic events of normal fertilization but reveal novel developmental errors. Hum Reprod. 2022;37:2307–19. [DOI] [PubMed] [Google Scholar]
- 28.Ezoe K, Miki T, Akaike H, Shimazaki K, Takahashi T, Tanimura Y, Amagai A, Sawado A, Mogi M, Kaneko S, Ueno S, Coticchio G, Cimadomo D, Borini A, Rienzi L, Kato K. Maternal age affects pronuclear and chromatin dynamics, morula compaction and cell polarity, and blastulation of human embryos. Hum Reprod. 2023;38:387–99. [DOI] [PubMed] [Google Scholar]
- 29.Ezoe K, Miki T, Okimura T, Uchiyama K, Yabuuchi A, Kobayashi T, Kato K. Characteristics of the cytoplasmic halo during fertilisation correlate with the live birth rate after fresh cleaved embryo transfer on day 2 in minimal ovarian stimulation cycles: a retrospective observational study. Reprod Biol Endocrinol. 2021;19:172. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 30.Ezoe K, Coticchio G, Takenouchi H, Taoda S, Namerikawa S, Honda K, Miki T, Okimura T, Kobayashi T, Borini A, Kato K. Spatiotemporal perturbations of pronuclear breakdown preceding syngamy affect early human embryo development: a retrospective observational study. J Assist Reprod Genet. 2022;39:75–84. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 31.Sawado A, Ezoe K, Miki T, Ohata K, Amagai A, Shimazaki K, Okimura T, Kato K. Fatty acid supplementation during warming improves pregnancy outcomes after frozen blastocyst transfers: a propensity score-matched study. Sci Rep. 2024;14:9343. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 32.Onogi S, Ezoe K, Nishihara S, Fukuda J, Kobayashi T, Kato K. Endometrial thickness on the day of the LH surge: an effective predictor of pregnancy outcomes after modified natural cycle-frozen blastocyst transfer. Hum Reprod Open. 2020;2020:hoaa060. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 33.Ueno S, Ezoe K, Abe T, Yabuuchi A, Uchiyama K, Okuno T, Kobayashi T, Kato K. Maternal age and initial beta-hCG levels predict pregnancy outcome after single vitrified-warmed blastocyst transfer. J Assist Reprod Genet. 2014;31:1175–81. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 34.Ezoe K, Fukuda J, Takeshima K, Shinohara K, Kato K. Letrozole-induced endometrial preparation improved the pregnancy outcomes after frozen blastocyst transfer compared to the natural cycle: a retrospective cohort study. BMC Pregnancy Childbirth. 2022;22:824. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 35.Saleh A, Thanassoulas A, Aliyev E, Swann K, Naija A, Yalcin HC, Lai FA, Nomikos M. Development of recombinant PLC-zeta protein as a therapeutic intervention for the clinical treatment of oocyte activation failure. Biomedicines. 2024;12:12. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 36.Ajduk A, Zernicka-Goetz M. Polarity and cell division orientation in the cleavage embryo: from worm to human. Mol Hum Reprod. 2016;22:691–703. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 37.Dehapiot B, Carriere V, Carroll J, Halet G. Polarized Cdc42 activation promotes polar body protrusion and asymmetric division in mouse oocytes. Dev Biol. 2013;377:202–12. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 38.Palermo GD, Avrech OM, Colombero LT, Wu H, Wolny YM, Fissore RA, Rosenwaks Z. Human sperm cytosolic factor triggers Ca2+ oscillations and overcomes activation failure of mammalian oocytes. Mol Hum Reprod. 1997;3:367–74. [DOI] [PubMed] [Google Scholar]
- 39.Tarozzi N, Nadalini M, Coticchio G, Zaca C, Lagalla C, Borini A. The paternal toolbox for embryo development and health. Mol Hum Reprod. 2021;27(7):gaab042. [DOI] [PubMed]
- 40.Ward WS. Function of sperm chromatin structural elements in fertilization and development. Mol Hum Reprod. 2010;16:30–6. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 41.Cavazza T, Takeda Y, Politi AZ, Aushev M, Aldag P, Baker C, Choudhary M, Bucevicius J, Lukinavicius G, Elder K, Blayney M, Lucas-Hahn A, Niemann H, Herbert M, Schuh M. Parental genome unification is highly error-prone in mammalian embryos. Cell. 2021;184(2860–77): e22. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 42.Dunkley S, Scheffler K, Mogessie B. Cytoskeletal form and function in mammalian oocytes and zygotes. Curr Opin Cell Biol. 2022;75:102073. [DOI] [PubMed] [Google Scholar]
- 43.Ueno S, Uchiyama K, Kuroda T, Okimura T, Yabuuchi A, Kobayashi T, Kato K. Establishment of day 7 blastocyst freezing criteria using blastocyst diameter for single vitrified-warmed blastocyst transfer from live birth outcomes: a single-center, large cohort, retrospectively matched study. J Assist Reprod Genet. 2020;37:2327–35. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 44.Coticchio G, Fiorentino G, Nicora G, Sciajno R, Cavalera F, Bellazzi R, Garagna S, Borini A, Zuccotti M. Cytoplasmic movements of the early human embryo: imaging and artificial intelligence to predict blastocyst development. Reprod Biomed Online. 2021;42:521–8. [DOI] [PubMed] [Google Scholar]
Associated Data
This section collects any data citations, data availability statements, or supplementary materials included in this article.
Supplementary Materials
Online Resource 1: Blastocyst morphology stratified by the day of blastocyst formation. A) Blastocyst grades based on Gardner’s criteria: excellent (AA), good (AB, BA, and BB), and poor (AC, BC, CA, CB, and CC). A–C, a–c Different superscript letters indicate a significant difference at P < 0.05. Blastocyst grades are stratified by median maternal age: Days 4 (B), 5 (C), 6 (D), and 7 (E) blastocysts.
Data Availability Statement
The primary data for this study are available from the authors upon request.
Not applicable.