The effect of follicle size and homogeneity of follicular development on the morphokinetics of human embryos

This prospective cohort study was registered at clinicaltrials.gov (NCT02230449) and obtained an ethical approval from the institutional review board (23/06/2014-19). It was conducted in a private IVF clinic between July 2014 and September 2015. The analysis was based on a total of 2526 cumulus oocyte complexes (COCs) belonging to 187 patients with culture until day 5 since 13 patients were excluded (one premature ovulation, one oocyte maturation defect, two low fertilizations, three fertilization failures, two cleavage-stage developmental arrests, four OHSS/freeze-all). In the cases of low fertilization, one patient had only one fertilized oocyte which was abnormal (three pronuclei (PN)) and in the other case, despite more than eight COCs being retrieved, there was only one MII oocyte which presented only one PN and did not cleave. In the two cases of cleavage-stage arrests at the two-cell stage, the blastomeres were significantly different in size, possibly indicating abnormality. The patients included in this study presented various infertility causes and the mean female age was 31.1 years. All protocols were approved by the institutional review board and all patients gave their informed consent prior to their inclusion in the study. Patients were selected based on inclusion criteria (age ≤39 years, body mass index (BMI) <30 kg/m², ≥8 COCs retrieved, <2 previous treatment cycles, hCG trigger) and exclusion criteria (recurrent pregnancy loss, severe endometriosis, PGD or PGS, COC > 24, embryo transfer (ET) < day 5, PCOS, uterine anomaly, severe sperm morphological abnormality such as dominantly macrocephalic or globozoospermic sample or cryptozoospermia, ≤1million motile sperm cells in total ejaculate). All embryos were obtained after fertilization by intracytoplasmic sperm injection (ICSI) and were part of our standard ART program. Embryo development was recorded using time-lapse technology (EmbryoScope™ time-lapse system, Vitrolife, Göteborg, Sweden).

The baseline estradiol, LH and progesterone levels were evaluated on cycle day 2, and baseline ultrasound scans were performed on the same day. Depending on the BMI, anti-mullerian hormone (AMH) level, basal antral follicle count and the history, if any, of a previous response to gonadotropins, recombinant FSH (rFSH; Gonal-F®; Merck Serono, Switzerland) was used for ovarian stimulation at a dosage of 150 to 225 IU. The standard dose was 150 IU, when necessary however, depending on body mass index, 225 IU was started. From the fourth or fifth day of rFSH therapy onwards, patients were monitored daily or every other day for hormone levels (estradiol, LH and progesterone whenever needed) and follicular measurements. A daily administration of 0.25 mg GnRH antagonist (Cetrotide®; Merck Serono, Switzerland) was administered when the size of the follicle was at least 12 mm, but never exceeding 13 mm. Follicular maturation was achieved by using 250 μg recombinant hCG (Ovitrelle®; Merck Serono, Switzerland) when at least three follicles reached a minimum mean diameter of at least 17 mm. Transvaginal ultrasound-guided oocyte retrieval was scheduled for 36 h later.

Each follicular aspiration was performed by the same doctor in order to reduce any possible inaccuracy of measurement to a minimum. Follicles <17 mm at the time of OPU were classified as small while those ≥17 mm were classified as large. Oocytes coming from large and small follicles were incubated separately.

The data was also evaluated according to the homogeneity of follicular development. Follicles being present in a homogenous spread of all sizes from large (>20 mm) to intermediate (17-20 mm) to small (<17 mm) was considered to be homogenous development, whereas a predominance of large (>20 mm) and small (<17 mm) follicles was considered to be heterogeneous. Each case met the criteria for trigger of a minimum of three follicles of at least 17 mm. Four subgroups have been defined according to the follicular size and cycle homogeneity: small follicles/heterogeneous follicular development (SHet), small follicles/homogenous follicular development (SHom), large follicles/heterogeneous follicular development (LHet) and large follicles/homogenous follicular development (LHom).

On the day of OPU, follicles were aspirated separately and COCs were washed in human tubal fluid medium (HTF; Life Global®, Brussels, Belgium). The gynaecologist who performed the pickup informed the embryology laboratory of the size of follicle (large ≥17 mm /small <17 mm) from which each oocyte was derived. One embryologist retrieved oocytes with a second embryologist assisting to ensure smooth operation and to document the process of isolation, identification and positioning of COCs in the culture dish.

The COCs were then incubated for 3.5 h at 6% CO₂, 5% O₂ and 37 °C before denudation, which was carried out by mechanical pipetting in ICSI Cumulase® (Origio, Måløv, Denmark). Each COC was denuded separately and the maturation status was then determined. Next, oocytes were allowed to incubate for an additional 30 min. ICSI was then performed in an HTF medium with HEPES (Life Global®, Brussels, Belgium) at ×400 magnification using Olympus IX70 and Olympus IX71 inverted microscopes.

Each of the 12 individual wells of the EmbryoSlide® culture dish was filled with 25 μl of a single step culture medium (Life Global®, Brussels, Belgium), supplemented with 10% Plasmanate (Life Global®, Brussels, Belgium), and all wells were covered with an overlay of 1.5 mL paraffin oil (Life Global®, Brussels, Belgium). Following ICSI, injected oocytes were positioned in the wells of the slide, which was placed in a time-lapse incubator (EmbryoScope™) at 6% CO₂, 5% O₂ and 37 °C for 5 days until embryo transfer. The culture medium was refreshed on the afternoon of day 3 by replacing the incubated slide with a new pre-equilibrated slide prepared as described above. Image stacks were acquired at seven focal planes every 15 min, and data were continuously transferred to an external computer, EmbryoViewer® workstation (Vitrolife, Göteborg, Sweden). Embryo development was annotated by one investigator and cross-checked by two other assessors.

Morphokinetic variables for all cleavage events up to the expanded blastocyst stage were annotated. All relevant events (fertilization, cleavages, morula and blastocyst formation) were checked on a daily basis, and time of cleavage to two-cell embryo (t2) and subsequent divisions t3, t4, t5, t6, t7, t8 and t9+ were recorded in the EmbryoViewer® workstation. The time of all mitotic events was expressed as hours post-ICSI. In order to minimize the variation of ICSI time within oocytes of one patient, ICSI was split between two embryologists above ten oocytes. Therefore, the maximum ICSI duration did not exceed 15 min, which is below the default time interval of each picture taken by the camera of the EmbryoScope™ system. tM was annotated at the end of the compaction process, when compaction was observed to be full with no apparent cell contours. tSB marks the initiation or start of blastulation, the first frame when initiation of a cavity formation is observed. tB indicates a blastocyst, where the ICM and the cavity are formed. tEB shows an expanded blastocyst with 50% thinning of the zona pellucida. Blastocysts were scored according to Gardner’s classification (114–120 h post-ICSI) and selected for transfer based on the final morphology and the score obtained from the morphokinetic ratios published by Çetinkaya and colleagues (CS2–8 = ((t3-t2) + (t5-t4)) / (t8-t2); CS4–8 = (t8-t5) / (t8-t4)) [29].

After embryo transfer, for luteal phase support, patients received a twice daily dose of progesterone gel administered intravaginally (Crinone® 8%; Merck Serono, Switzerland). When pregnancy occurred, a daily dose was continued until the 10th week of gestation. Fourteen days after pickup, serum β-hCG was measured. At 7 weeks, a transvaginal ultrasound was performed to monitor early pregnancy. The implantation rate was calculated by dividing the number of implanted embryos by the total number of transferred embryos.

An earlier pilot study revealed that top and good quality blastocyst rates for large and small follicles were 45 and 32%, respectively. A power analysis indicated that, for an alpha level of 0.05 and a beta level of 0.20 (power = 0.80), 438 fertilized oocytes were sufficient to detect a significant difference between follicle size groups.

Demographics of patients were reported as minimum, maximum, mean ± sd and 95% confidence interval of the mean. Due to the dependent nature of the data, generalized linear mixed models with logit link function were conducted to analyse the differences of binary variables between four groups, namely LHom, LHet, SHom and SHet. Generalized linear mixed models with linear link function were conducted to analyse the differences of continuous variables between the four subgroups mentioned above.

Generalized linear mixed models with logit link function were conducted to analyse the possible effects of factors on the rate of top and good quality blastocysts. First, five models were conducted separately to test the effects of age, AMH, BMI, homogeneity and follicle size on the rate of top and good quality blastocysts. Second, a multivariable model was conducted, where variables with p < 0.20 significance level at univariable analysis were introduced as independent variables. A p value of <0.05 was considered statistically significant. All statistical analyses were performed using the MedCalc Statistical Software version 13.2.0 (MedCalc Software bvba, Ostend, Belgium) and R version 3.3.2.

A total of 187 patients were prospectively involved in the analysis, using strict inclusion criteria, which allowed the study of a homogeneous, young, infertile patient population with a low BMI and a good ovarian reserve (mean female age 31.1 ± 4.1, AMH 3.1 ± 2.0 ng/mL and BMI 23.6 ± 3.0 kg/m²). An average of 14 COCs was collected, 10.1 of which were at MII stage (maturation rate 73.6%). The fertilization rate after ICSI was 76.6%. The fertilized oocytes (71.7%) became grade 1 or 2 embryos on day 3 and 37.2% top or good quality blastocysts on day 5 (Table 1). The clinical and ongoing pregnancy rates achieved in the study cohort were 63.9 and 55.2%, respectively. The early clinical miscarriage rate was 13.7%. Two thirds of patients had surplus frozen blastocysts (66.3%). Finally, a live birth rate of 52% was reached (Supplementary Table 1).

Maturation and fertilization rates of oocytes deriving from large follicles were significantly higher than those deriving from small follicles. When subcategorizing the data, large follicles developed in homogenous cycles (LHom) had better outcomes than large follicles developed in heterogeneous cycles (LHet), small follicles in homogenous cycles (SHom) and finally small follicles in heterogeneous cycles (SHet). Also, rates of grade 1 and 2 embryos on day 3, blastocyst formation, top and good quality blastocysts were gradually higher in the same order mentioned above.

The kinetics of embryos derived from small and large follicles was evaluated according to cycle homogeneity. Statistically significant differences were found beginning from the first cleavage (t2) until the expanded blastocyst time (tEB) except for t3 and t4. Embryos from small follicles developed faster than embryos originating from large follicles, for all cleavage timings. Embryos from SHet developed faster, except for t9 and tB (Table 2). tB time was significantly different between follicular size and homogeneity groups (p = 0.001). Bonferroni corrected post-hoc analysis revealed that tB time was shorter in the SHom group than LHet or LHom groups (p = 0.002; p = 0.027, respectively), whereas embryos developing from the SHet group had a tB shorter than embryos coming from the LHet group (p = 0.002). When median values were compared, the time to achieve a blastocyst for a small follicle was 1.4 h faster than for a large follicle (p = 0.0036) (Fig. 1).

Another difference observed, when embryos developing from small and large follicles in homogenous or heterogeneous cycles were compared, was the significantly different rate of direct cleavages (t3-t2 < 5 h) (p = 0.011) (Table 2). Bonferroni corrected post-hoc analysis revealed that the direct cleavage rate was higher in the SHet group (26.7%) than in the LHet (18.2%) (p = 0.016) and in the LHom groups (16.8%) (p = 0.006).

Also, when time intervals and time ratios were evaluated, cc2 (t3-t2), s2 (t4-t3), cc3 (t5-t3), t5-t4, t8-t2, CS2–8 ([(t3-t2) + (t5-t4)] / (t8-t2)) and CS4–8 ((t8-t5) / (t8-t4)) were significantly different between follicular size and homogeneity of follicular development subgroups (Table 2). cc2 and cc3 time intervals were significantly different between all subgroups (p < 0.001 and p = 0.016, respectively). Bonferroni corrected post-hoc analyses revealed that cc2 and cc3 time intervals were shorter in embryos developing from the SHet group than in embryos developing from LHet or LHom groups (for cc2, p = 0.003; p = 0.001; and for cc3, p = 0.020; p = 0.039, respectively). When looking at homogenous cycles, cc2 and cc3 were shorter in embryos developing from small follicles than in those from large follicles (p = 0.004; p = 0.026; p = 0.002, respectively). Also, CS2–8 time interval showed a similar pattern in the aforementioned four categories (p = 0.045; p = 0.002; p = 0.002, respectively).

Embryos originating from small follicles had a higher arrest rate than embryos originating from large follicles when analysing those having a first cleavage t2 (Fig. 2).

Cumulative developmental arrest rates of embryos originating from small and large follicles were calculated by adding all arrested embryos up to that time point for each category divided by embryos having achieved the two-cell stage. When looking at the time to achieve the eight-cell stage (t8), 91.4% of embryos developing from large follicles reached t8, compared to only 86.9% of embryos developing from small follicles with a 4.5% lower arrest rate in the former group (p = 0.0032) (Fig. 2). No significant differences were found between the developmental arrest rates of embryos from t8 to expanded blastocyst (tEB).

Although not statistically significant, embryos developing from the LHom group had a higher implantation rate when compared first to SHom, second to LHet and finally to SHet groups (58.1, 47.5, 36.7, 35.2%, respectively; p = 0.237) (Table 2).

Univariable analyses revealed that AMH, homogeneity of follicular development and follicular size had a significant effect on blastocyst quality (p = 0.010; p = 0.018; p < 0.001, respectively). One unit increase in AMH level resulted in a 1.086-fold increase in top or good quality blastocysts development [OR (95% CI) 1.086 (1.020, 1.157); p = 0.010]. For follicles derived from homogenous cycles, the odds of resulting in top or good quality blastocysts were 1.370-fold larger than the odds for heterogeneous follicles becoming top or good quality blastocysts [OR (95% CI) 1.370 (1.055, 1.780); p = 0.010]. For large follicles, the odds of developing into top or good quality blastocysts were 1.803-fold larger than the odds for small follicles resulting in top or good quality blastocysts [OR (95% CI) 1.803 (1.494, 2.177); p < 0.001] (Table 3).

Multivariable analysis revealed that AMH, homogeneity of follicular development and follicular size had a significant effect on blastocyst quality (p = 0.010; p = 0.018; p < 0.001, respectively). One unit increase in AMH level resulted in a 1.075-fold increase in top or good quality blastocysts development [OR (95% CI) 1.075 (1.009, 1.146); p = 0.026]. For homogenous cycles, the odds of becoming top or good quality blastocysts were 1.302-fold larger than the odds for heterogeneous follicles developing into top or good quality blastocysts [OR (95% CI) 1.302 (1.001, 1.693); p = 0.049]. For large follicles, the odds of resulting in top or good quality blastocysts were 1.797-fold larger than the odds for small follicles becoming top or good quality blastocysts [OR (95% CI) 1.797 (1.488, 2.169); p < 0.001] (Table 3).