The value of the pronucleus size of human single pronucleus zygotes as a predictor of fertility outcomes in conventional in vitro fertilization: a retrospective cohort study.

This study analyzed the outcomes of 1PN embryos derived from the first IVF cycles from May 2023 to April 2024. The overall incidence of 1PN zygotes was 5.38%. The baseline characteristics of the patients are presented in Table  1 , including 629 1PN embryos from 514 patients. The corresponding cycle characteristics of these patients are shown in Table  2 . The mean ± SD PN diameter in this study was 33.86 ± 6.05 μm. Table 1 Baseline characteristics of 514 patients resulting in 629 IVF–1PN zygotes Continuous variables Mean ± SD Median (IQR) Maternal age (y) 33.92 ± 4.88 34.00 (30.00–37.00) Paternal age (y) 34.71 ± 5.22 34.00 (31.00–37.50) Maternal BMI (kg/m 2 ) 23.07 ± 9.37 22.19 (20.17–24.61) Duration of infertility (y) 3.03 ± 2.68 2.00 (1.00–4.00) Gravidity ( n ) 1.14 ± 1.42 1.00 (0.00–2.00) Parity ( n ) 0.25 ± 0.58 0.00 (0.00–0.00) AFC 11.70 ± 7.03 11.00 (6.00–16.00) AMH (ng/ml) 4.38 ± 4.15 3.26 (1.51–5.77) Basal FSH (mIU/ml) 6.07 ± 2.36 5.56 (4.78–6.78) Basal LH (mIU/ml) 3.66 ± 2.70 3.06 (2.10–4.31) Basal E2 (pg/ml) 35.73 ± 28.85 30.00 (23.00–39.25) Basal P4 (ng/ml) 0.30 ± 0.53 0.20 (0.20–0.30) Categorical variables n Rate (%) Infertility causes Tubal factor 206 40.08 Ovulatory dysfunction (DOR) 55 10.70 Ovulatory dysfunction (others) 41 7.98 Unexplained factor 118 22.96 Uterine factor 21 4.09 Endometriosis 15 2.92 Mixed factor 58 11.28 The data are presented as the mean ± SD and median (interquartile range‌, IQR). The overall incidence of 1PN zygotes was 5.38% (629/11690 = 5.38%, a total of 11690 IVF zygotes from 1708 IVF cycles.) a A total of 629 IVF-1PN zygotes from 514 IVF cycles were included in this study Table 2 Characteristics of the 629 IVF–1PN embryos and their corresponding controlled ovarian stimulation cycles Variables Mean ± SD Median (IQR) PN diameter (μm) 33.86 ± 6.05 35.00 (30.00–35.00) Duration of stimulation(d) 11.06 ± 1.94 11.00 (10.00–12.00) Total gonadotropin dosage (IU) 2058.25 ± 668.42 2025 (1725–2400) E2 level on trigger day (pg/ml) 2896.82 ± 1703.19 2975.0 (1228.0–4868.3) Average E2 of follicles > 10 mm on trigger day (pg/ml) 227.86 ± 126.05 224.79 (144.64–294.13) Average E2 of follicles > 14 mm on trigger day (pg/ml) 331.51 ± 196.81 301.56 (201.20–420.19) Follicles > 10 mm ( n ) 14.48 ± 8.93 14.00 (8.00–19.50) Follicles > 14 mm ( n ) 10.48 ± 7.38 9.00 (5.00–15.00) Punctured follicles ( n ) 16.68 ± 11.48 15.00 (5.00–22.00) Retrieved oocytes ( n ) 14.48 ± 10.20 13.00 (6.00–19.00) Oocyte retrieval rate (%) 86.48 ± 15.60 90.90 (77.80–100.00) Interval between trigger and oocyte retrieval (h) 39.86 ± 3.49 39.83 (37.00–42.71) Sperm density after processing (10 6 /ml) 40.93 ± 141.82 35.00 (31.00–40.00) Sperm motility after processing (%) 81.34 ± 8.62 83.00 (80.00–86.00) Categorical variables n Rate (%) COH protocols PPOS 445 70.74 GnRH-ant 137 21.78 Mild stimulation 26 4.13 GnRH-a 21 3.33 The data are presented as the mean ± SD and median (interquartile range‌, IQR) Baseline characteristics of 514 patients resulting in 629 IVF–1PN zygotes The data are presented as the mean ± SD and median (interquartile range‌, IQR). The overall incidence of 1PN zygotes was 5.38% (629/11690 = 5.38%, a total of 11690 IVF zygotes from 1708 IVF cycles.) a A total of 629 IVF-1PN zygotes from 514 IVF cycles were included in this study Characteristics of the 629 IVF–1PN embryos and their corresponding controlled ovarian stimulation cycles The data are presented as the mean ± SD and median (interquartile range‌, IQR) The IVF-derived 1PN zygotes were divided into the following six subgroups on the basis of their PN diameter: ≤ 20 μm, 25 μm, 30 μm, 35 μm, 40 μm, and ≥ 45 μm. We found that the PN diameter of IVF-derived 1PN zygotes was significantly correlated with embryological outcomes (Figure S1 and Table  3 ). The data revealed that the developmental potential of IVF-derived 1PN embryos increased with increasing PN diameter. The number of embryos in the different PN diameter groups is shown in Figure S1a. Figure S1b and Table  3 show that although the cleavage rate was 100% for PN diameters ≤ 20 μm, the cleavage rate basically presented a steadily increasing trend as the PN diameter increased (25, 81.16%; 30, 85.16%; 35, 93.30%; 40, 91.11%; and ≥ 45, 96.92%; P = 0.003). Furthermore, the proportion of D3 6–8 cells increased significantly with increasing PN diameter (≤ 20, 35.71%; 25, 34.78%; 30, 38.46%; 35, 43.54%; 40, 47.78%; and ≥ 45, 63.08%; P = 0.001). When 1PN zygotes were cultured into blastocysts, the rate of blastocyst formation in the PN ≥ 45 μm group (24.62%) was significantly greater than that in the smaller PN diameter group (0–22.22%; P  = 0.023). For the good-quality blastocyst rate, with increasing PN diameter, the ratio tended to increase, although there was no significant difference (≤ 20, 0%; 25, 1.45%; 30, 5.49%; 35, 6.70%; 40, 7.78%; and ≥ 45, 9.23%; P  = 0.373) (Figure S1c and Table  3 ). There was no significant difference in the blastocyst grade ( P  = 0.984) or D5 blastocyst formation rate ( P  = 0.634) among the different groups (Figure S1d and Table  3 ). Table 3 Embryological outcomes of the 629 IVF–1PN embryos in total and grouped by PN diameter Group PN diameter (μm) Total    = 45 P No. of 1PN zygotes 629 14 69 182 209 90 65 Cleavage rate (%) 89.83 (565/629) 100 (14/14) 81.16 (56/69) 85.16 (155/182) 93.30 (195/209) 91.11 (82/90) 96.92 (63/65) 0.003* 6–10 cells on D3 (%) 43.56 35.71 34.78 38.46 43.54 47.78 63.08 0.001* (274/629) (5/14) (24/69) (70/182) (91/209) (43/90) (41/65) Blastocyte rate (%) 16.69 0 8.70 13.19 18.66 22.22 24.62 0.023* (105/629) (0/14) (6/69) (24/182) (39/209) (20/90) (16/65) Good-quality blastocyst rate a (%) 6.04 (38/629) 0 (0/14) 1.45 (1/69) 5.49 (10/182) 6.70 (14/209) 7.78 (7/90) 9.23 (6/65) 0.373 Blastocyst grade b 0.984  Grade 1 ( n , %) 4 (3.81) / 0 (0) 1 (4.17) 1 (2.56) 1 (5.00) 1 (6.25%)  Grade 2 ( n , %) 34 (32.38) / 1 (16.67) 9 (37.50) 13 (33.33) 6 (30.00) 5 (31.25%)  Grade 3 ( n , %) 67 (63.81) / 5 (83.33) 14 (58.34) 25 (64.10) 13 (65.00) 10 (62.50) Blastocyst formation on D5 ( n , %) 53 (50.48) / 4 (66.67) 9 (37.50) 20 (51.28) 10 (50.00) 10 (62.50) 0.634 a A good-quality blastocyst refers to a 4–6 AA/AB/BA/BB/BC/CB blastocyst that formed on D5/D6 b Grade 1 included blastocysts graded with 4–6 AA/AB/BA; Grade 2 included blastocysts graded with 4–6 BB/AC/CA; Grade 3 included blastocysts graded with 4–6 BC/CB/CC * P  < 0.05 Embryological outcomes of the 629 IVF–1PN embryos in total and grouped by PN diameter a A good-quality blastocyst refers to a 4–6 AA/AB/BA/BB/BC/CB blastocyst that formed on D5/D6 b Grade 1 included blastocysts graded with 4–6 AA/AB/BA; Grade 2 included blastocysts graded with 4–6 BB/AC/CA; Grade 3 included blastocysts graded with 4–6 BC/CB/CC * P  < 0.05 A total of 36 IVF-derived 1PN single vitrified-warmed blastocysts were subsequently transferred. Table 4 shows the basic characteristics of these individuals during their transfer cycles. Furthermore, we analyzed pregnancy outcomes following 1PN blastocyst transfer throughout IVF cycles. As demonstrated in Table  4 , 1 PN blastocyst transfer during IVF cycles resulted in 23 biochemical pregnancies (63.89%) and 21 embryo implantations (58.33%). Twenty of these patients progressed to clinical pregnancy (55.56%), and one progressed to ectopic pregnancy (2.78%). In addition, three cases of miscarriage (8.33%) were reported. Among the 20 clinical pregnancies, 17 were ongoing (47.22%), followed by live births (47.22%). Table 4 Characteristics and pregnancy outcomes of the 36 frozen–thawed embryo transfer cycles with a single IVF–1PN blastocyst Variables Mean ± SD Median (IQR) Maternal age (y) 33.06 ± 4.35 34.50 (30.00–36.00) Paternal age (y) 33.94 ± 4.50 35.00 (31.25–36.00) Maternal BMI (kg/m 2 ) 21.75 ± 2.80 21.25 (20.06–23.40) Duration of infertility (y) 2.19 ± 2.54 1.50 (0.00–4.00) Gravidity ( n ) 1.36 ± 1.64 1.00 (0.00–2.00) Parity ( n ) 0.42 ± 0.77 0.00 (0.00–0.75) Endometrial thickness (mm) 11.09 ± 2.21 11.20 (9.10–12.80) E2 level on transfer day (pg/ml) 234.08 ± 158.46 171.50 (108.50–272.00) P2 level on transfer day (pg/ml) 27.48 ± 45.63 18.45 (13.25–24.08) PN diameter (μm) 36.94 ± 6.01 35.00 (30.00–43.75) Categorical variables n Rate (%) Infertility causes  Tubal factor 17 47.22  Ovulatory dysfunction (DOR) 5 13.89  Ovulatory dysfunction (others) 2 5.56  Unexplained factor 4 11.11  Uterine factor 4 11.11  Endometriosis 1 2.78  Mixed factor 3 8.33 Endometrium preparation protocols  NC/mNC 25 69.44  HRT 11 30.56 Blastocyst formation date  Day 5 22 61.11  Day 6 14 38.89 Blastocyst grade  Grade 1 2 5.56  Grade 2 14 38.89  Grade 3 20 55.56 Pregnancy outcomes n Rate (%)  Biochemical pregnancy 23 63.89  Clinical pregnancy 20 55.56  Ongoing pregnancy 17 47.22  Live birth 17 47.22  Ectopic pregnancy 1 2.78  Miscarriage 3 8.33  Implantation 21 58.33 Grade 1 included blastocysts graded with 4–6 AA/AB/BA Grade 2 included blastocysts graded with 4–6 BB/AC/CA Grade 3 included blastocysts graded with 4–6 BC/CB/CC * P  < 0.05 Characteristics and pregnancy outcomes of the 36 frozen–thawed embryo transfer cycles with a single IVF–1PN blastocyst Grade 1 included blastocysts graded with 4–6 AA/AB/BA Grade 2 included blastocysts graded with 4–6 BB/AC/CA Grade 3 included blastocysts graded with 4–6 BC/CB/CC * P  < 0.05 We further analyzed the effects of the PN diameter of IVF-1PN embryos on embryological outcomes and pregnancy outcomes (Table  5 ). The results revealed that the numbers of D3 ( r  = 0.161, P  < 0.001), blastocyst formation ( r  = 0.140, P  < 0.001) and good-quality blastocyst ( r  = 0.085, P  = 0.034) cells were positively correlated with PN diameter. After the interference of other variables was excluded, the results still revealed that the number of D3 cells ( r  = 0.165, P  < 0.001), degree of blastocyst formation ( r  = 0.145, P  < 0.001), and number of good-quality blastocysts ( r  = 0.086, P  = 0.032) were correlated with PN diameter. The results indicated that embryo quality clearly improved with increasing PN diameter. Furthermore, no significant correlation was observed between PN diameter and biochemical pregnancy ( r  = 0.092, P  = 0.594), clinical pregnancy ( r  = 0.047, P  = 0.785), ongoing pregnancy ( r  = 0.011, P  = 0.949) or live birth ( r  = 0.011, P  = 0.949). The results were consistent after controlling for other confounding variables. Table 5 Correlations between the PN diameter of IVF–1PN embryos and IVF outcome indicators Outcome indicators r a P r b P Cell number on D3 c 0.161  < 0.001* 0.165  < 0.001* Blastocyst formation d 0.140  < 0.001* 0.145  < 0.001* Good-quality blastocyst d 0.085 0.034* 0.086 0.032* Biochemical pregnancy d 0.092 0.594 − 0.162 0.273 Clinical pregnancy d 0.047 0.785 − 0.350 0.563 Ongoing pregnancy d 0.011 0.949 − 0.351 0.563 Live birth d 0.011 0.949 − 0.351 0.563 a Spearman rank correlation coefficients (via simple correlation analysis) b Spearman rank correlation coefficients. The associations between PN diameter and embryological outcomes were examined through partial correlation analysis after controlling for maternal age, paternal age, maternal BMI, duration of infertility, gravidity, infertility causes, AFC and ovarian stimulation protocols. The associations between PN diameter and pregnancy outcomes were examined through partial correlation analysis after controlling for maternal age, paternal age, maternal BMI, duration of infertility, gravity, infertility cause, endometrial preparation protocol, endometrial thickness on the transfer day, E2 level on the transfer day, P4 level on the transfer day, blastocyst formation date (D5/D6), and blastocyst grade c The indicator was considered a grade variable. Grade 0: no cleavage; Grade 1: 1–5 cells on D3; Grade 2: 6–10 cells on D3 d These indicators were considered grade variables, with grade 1 defined as occurrence (+), and grade 0 defined as no occurrence (–) * P  < 0.05 Correlations between the PN diameter of IVF–1PN embryos and IVF outcome indicators a Spearman rank correlation coefficients (via simple correlation analysis) b Spearman rank correlation coefficients. The associations between PN diameter and embryological outcomes were examined through partial correlation analysis after controlling for maternal age, paternal age, maternal BMI, duration of infertility, gravidity, infertility causes, AFC and ovarian stimulation protocols. The associations between PN diameter and pregnancy outcomes were examined through partial correlation analysis after controlling for maternal age, paternal age, maternal BMI, duration of infertility, gravity, infertility cause, endometrial preparation protocol, endometrial thickness on the transfer day, E2 level on the transfer day, P4 level on the transfer day, blastocyst formation date (D5/D6), and blastocyst grade c The indicator was considered a grade variable. Grade 0: no cleavage; Grade 1: 1–5 cells on D3; Grade 2: 6–10 cells on D3 d These indicators were considered grade variables, with grade 1 defined as occurrence (+), and grade 0 defined as no occurrence (–) * P  < 0.05 We then performed a binary regression analysis together with GEE for adjusting the inclusion of several 1PN zygotes from the same patient to analyze the aOR for each embryo outcome and pregnancy outcome (Table  6 ). After adjusting for confounding factors, there were statistically significant differences in embryo cleavage, blastocyst formation and good-quality blastocyst formation among the different PN diameter groups (embryo cleavage: aOR 1.093, 95% CI 1.041–1.148, P  < 0.001; blastocyst formation: aOR 1.080, 95% CI 1.037–1.125, P  < 0.001; good-quality blastocyst formation: aOR 1.079, 95% CI 1.009–1.154, P  = 0.026). Moreover, there were no statistically significant differences in biochemical or clinical pregnancy rates between the groups (biochemical pregnancy: aOR 1.041, 95% CI 0.906–1.198, P  = 0.569; clinical pregnancy: aOR 1.003, 95% CI 0.842–1.194, P  = 0.976). The aOR for live births was 0.968 (0.800–1.172), which was not statistically significant ( P  = 0.739). Table 6 Binary logistic regression analysis a of the influencing factors for embryological and pregnancy outcomes of embryos derived from 1PN in conventional IVF ( n  = 629) Outcome indicators aOR 95% CI P Cleavage 1.093 1.041–1.148  < 0.001* Blastocyst formation b 1.080 1.037–1.125  < 0.001* Good-quality blastocyst formation 1.079 1.009–1.154 0.026* Biochemical pregnancy 1.041 0.906–1.198 0.569 Clinical pregnancy 1.003 0.842–1.194 0.976 Live birth c 0.968 0.800–1.172 0.739 a The influence of PN diameter on embryological outcomes was examined through binary logistic analysis with the following variables introduced into the analysis: PN diameter, maternal age, paternal age, maternal BMI, duration of infertility, gravity, parity, infertility causes, AFC ovarian stimulation protocols, total gonadotropin dosage, duration of stimulation, and interval between trigger and oocyte retrieval. The influence of PN diameter on pregnancy outcomes was examined through binary logistic analysis with the following variables introduced into the analysis: PN diameter, maternal age, paternal age, maternal BMI, duration of infertility, gravity, endometrial thickness on transfer day, blastocyst formation date (D5/D6), and blastocyst grade. In both binary regression analyses, generalized estimating equations (GEEs) were used to adjust the inclusion of several 1PN zygotes from the same patient b The following two variables were also demonstrated to influence the outcome indicator of blastocyst formation: 1) maternal BMI (aOR 0.882, 95% CI 0.813–0.956, P  = 0.002) and 2) AFC (aOR 1.042, 95% CI 1.003–1.083, P  = 0.033) c The following two variables were demonstrated to influence the outcome indicator of live birth: 1) blastocyst formation on day 5 (aOR 14.64, 95% CI 1.019–210.452, P  = 0.048, when day 6 was set as a reference) and 2) maternal BMI (aOR 0.598, 95% CI 0.353–0.915, P  = 0.049) * P  < 0.05 Binary logistic regression analysis a of the influencing factors for embryological and pregnancy outcomes of embryos derived from 1PN in conventional IVF ( n  = 629) a The influence of PN diameter on embryological outcomes was examined through binary logistic analysis with the following variables introduced into the analysis: PN diameter, maternal age, paternal age, maternal BMI, duration of infertility, gravity, parity, infertility causes, AFC ovarian stimulation protocols, total gonadotropin dosage, duration of stimulation, and interval between trigger and oocyte retrieval. The influence of PN diameter on pregnancy outcomes was examined through binary logistic analysis with the following variables introduced into the analysis: PN diameter, maternal age, paternal age, maternal BMI, duration of infertility, gravity, endometrial thickness on transfer day, blastocyst formation date (D5/D6), and blastocyst grade. In both binary regression analyses, generalized estimating equations (GEEs) were used to adjust the inclusion of several 1PN zygotes from the same patient b The following two variables were also demonstrated to influence the outcome indicator of blastocyst formation: 1) maternal BMI (aOR 0.882, 95% CI 0.813–0.956, P  = 0.002) and 2) AFC (aOR 1.042, 95% CI 1.003–1.083, P  = 0.033) c The following two variables were demonstrated to influence the outcome indicator of live birth: 1) blastocyst formation on day 5 (aOR 14.64, 95% CI 1.019–210.452, P  = 0.048, when day 6 was set as a reference) and 2) maternal BMI (aOR 0.598, 95% CI 0.353–0.915, P  = 0.049) * P  < 0.05 To further analyze the accuracy of using the diameter of 1PN zygotes to predict embryological outcomes, ROC curve analysis was performed, as shown in Fig. 2 results revealed that the diameter of the 1PN zygotes was a valuable marker for identifying blastocyst formation, with an AUC (area under the receiver operating curve) of 0.605. Therefore, a diameter of 1PN zygotes > 32.5 μm is a useful predictor of blastocyst formation. Fig. 2 Receiver operating characteristic (ROC) curve analysis of the PN diameter. The cutoff value was 32.5 μm for the PN diameter Receiver operating characteristic (ROC) curve analysis of the PN diameter. The cutoff value was 32.5 μm for the PN diameter

This retrospective study was conducted in the Department of Reproductive Medicine of the Ninth People's Hospital Affiliated with the Shanghai Jiao Tong University School of Medicine. All the 1PN zygotes ( n  = 629) from the first IVF cycle ( n  = 514) at our center between May 2023 and April 2024 were included for potential analysis. The embryonic outcomes of embryos obtained from IVF–1PN zygotes were compared. The pregnancy outcomes were compared following the initial transfer of single vitrified–warmed blastocysts ( n  = 36) obtained from IVF–1PN zygotes within the aforementioned time period. There all included 36 cycles were confirmed to have no available 2PN-derived blastocysts for transfer. A flow chart of the study design and cohort selection for this study is shown in Fig.  1 . This study adheres to the Declaration of Helsinki on Medical Research involving Human. Fig. 1 Flow diagram of cohort screening in the study Flow diagram of cohort screening in the study Subjects and has been approved by the Ethics Committee of the Ninth People's Hospital Affiliated to Shanghai Jiao Tong University School of Medicine (20161202). All enrolled patients were fully informed of the potential risks associated with transferring 1PN-derived blastocysts and wrote informed consents before the treatment cycles. Previously, the protocols for ovarian stimulation, fertilization and embryo culture have been described in detail [ 26 ]. In brief, oocytes were retrieved approximately 36 h after human chorionic gonadotrophin (hCG) administration. All aspirated oocytes were transferred to G-IVF PLUS culture medium (Vitrolife, Sweden) and fertilized via conventional IVF 4 h later. In the case of IVF, spermatozoa were collected via density gradient and swim-up procedures, with 50,000 motile sperm cells/mL in the insemination dish. Fertilization was examined 16–18 h after insemination, and zygotes derived from 1PN were transferred to dishes containing pre-equilibrated G-1 ™ cleavage culture medium (Vitrolife, Sweden), which was then transferred to G-2™ blastocyst culture medium (Vitrolife, Sweden) on day 3 and continuously cultured to the blastocyst stage. Regarding the pronuclear status assessment, conventional IVF was performed with overnight insemination in this study. Pronuclear status was assessed within 1 h after fertilization check on the morning of D1. For oocytes observed to have 1PN, the PN diameter was measured manually using a visual scale (5 μm), and these zygotes were labeled and cultured separately from 2PN zygotes. Throughout the embryonic development period (D1 to D6), 1PN-derived embryos were tracked independently, with regular assessments of cleavage rate, blastocyst formation, and blastocyst quality. Embryo grading was performed at D3 (9:00–11:00 AM), D5 (9:00–11:00 AM), and D6 (9:00–11:00 AM). Insemination was performed between 1:00 and 3:00 PM on the day of oocyte retrieval. All the embryos were cultured in mineral oil balanced with N2 in an atmosphere containing 5% O2 and 6% CO2 at 37 °C. In this study, blastocysts with PN diameters were separated into six groups: ≤ 20, 25, 30, 35, 40, and ≥ 45 μm [ 23 – 25 ]. The blastocysts were evaluated morphologically according to the Gardner and Schoolcraft grading system [ 27 ], which was performed by experienced embryologists at our center. The score was determined by the degree of blastocyst expansion (full, expanded, hatched, and. hatched blastocyst), inner cell mass (ICM) quality, and trophectoderm (TE) (A, B and C). A good-quality blastocyst was defined as a 4–6 AA/AB/BA/BB/AC/CA blastocyst that formed on D5/D6. Grade 1 blastocysts were graded with 4–6 AA/AB/BA; Grade 2 blastocysts were graded with 4–6 BB/AC/CA; and Grade 3 blastocysts were graded with 4–6 BC/CB/CC. IVF–1PN-derived blastocysts were subsequently transferred during the vitrified–warmed cycle. Frozen–thawed embryo transfer (FET) was performed via either a natural or artificial cycle depending on the individual condition of the patient. Women with regular ovulation cycles were given a natural cycle of FET combined with the administration of hCG to induce ovulation. Patients with irregular cycles received artificial cycles to prepare their endometrium for FET. As previously described by Kuwayama et al. [ 28 ], the blastocysts were vitrified and warmed via a Cryotop carrier system combined with dimethylsulfoxe–glycol–sucrose as cryoprotectants. Thereafter, a single vitrified-warmed embryo was transferred to each patient. Progesterone supplements were used until 12 weeks of pregnancy. The cleavage rate was calculated as the number of cleaved embryos divided by the number of 1PN zygotes. The rate of blastocyst formation was determined as the ratio of the number of blastocysts to the number of 1PN zygotes. The proportion of good-quality blastocysts was calculated as the ratio of 4–6 AA/AB/BA/BB/AC/CA blastocysts formed on D5/D6 to total blastocysts. The biochemical pregnancy rate was the percentage of β-hCG-positive cycles after embryo transfer compared with the total number of transfer cycles. The clinical pregnancy rate was the number of cycles of pregnancy sacs with a heartbeat detected by ultrasound approximately 4 weeks following embryo transfer, given as a percentage of the total number of transfer cycles. The ongoing pregnancy rate was defined as the proportion of all transfer cycles with gestation cycles lasting 20 weeks or more following embryo transfer. The live birth rate was defined as the number of cycles in which a live baby was born after 28 weeks of gestation following embryo transfer, expressed as a percentage of the total number of cycles transferred. The implantation rate was defined as the number of gestational sacs observed via ultrasound following embryo transfer per number of cycles transferred. The miscarriage rate was defined as the number of clinical pregnancies lost prior to 20 gestational weeks following embryo transfer per number of cycles transferred. The ectopic pregnancy rate was defined as the number of gestational sacs outside the uterine cavity during ultrasound examination following embryo transfer per number of cycles transferred. Statistical analysis was performed with SPSS (version 26.0; SPSS Inc., USA). P values of < 0.05 were considered statistically significant. For continuous variables, normality was tested via histograms and Q‒Q plots as well as the Kolmogorov‒Smirnov test. The data are presented as the means ± standard deviations (SDs) for normally distributed data or medians (interquartile range, IQRs) for non-normally distributed data. Categorical variables are expressed as numbers and percentages and were compared via the chi-square test or Fisher’s exact test when appropriate. To determine the relationships between the PN diameter of IVF–1PN embryos and IVF outcome indicators (embryological outcomes and pregnancy outcomes), Spearman correlation analysis was performed. The associations between PN diameter and IVF outcomes were examined through partial correlation analysis after controlling for potential bias. Binary logistic regression analysis was subsequently performed to evaluate the influence of PN diameter on IVF outcomes using generalized estimating equations (GEEs) to adjust the inclusion of several 1PN zygotes from the same patient. The adjusted odds ratios (ORs) with corresponding 95% confidence intervals (CIs) were calculated. Receiver operating characteristic (ROC) curve analysis was used to evaluate whether the PN diameter could be used as a biomarker for predicting the possibility of blastocyst formation in IVF–1PN zygotes. The optimal cutoff value for PN diameter in 1PN zygotes was determined using the value that maximizes the measure of Youden's Index (sensitivity + specificity-1).

Recent research has shown that 1PN zygotes with larger PN diameters have higher blastocyst formation rates [ 23 – 25 ]. However, owing to the small sample size, the ability to reliably forecast the development of 1PN-derived blastocysts on the basis of PN diameter remains inadequate. To the best of our knowledge, this study is the largest retrospective cohort study to date to investigate the relationship between the diameter of 1PN zygotes and embryological outcomes as well as pregnancy outcomes. In this study, we found that the 1PN zygote diameter is an important marker for predicting blastocyst formation, further confirming that 1PN zygotes with larger PN diameters have better embryological outcomes. Once a blastocyst is formed, the PN diameter has no effect on pregnancy outcomes. For the past two decades, the use of 1PN zygotes in IVF has been controversial. Transfer of 1PN-derived blastocysts carries potential clinical risks that require careful consideration. 1PN zygotes exhibit poor developmental potential, analysis via SNP array has revealed that uniparental diploid 1PN zygotes exhibit whole-genome loss of heterozygosity while maintaining a normal chromosome number, contrasting sharply with normally fertilized 2PN embryos [ 8 , 29 ]. True parthenogenetic 1PN zygotes completely lack paternal genomic and nearly 100% present with genetic abnormalities [ 30 ]. Uniparental diploid 1PN embryos (including androgenetic and gynogenetic diploids) significantly increase the risk of adverse pregnancy outcomes: androgenetic diploid embryos fail to develop an embryoblast, whereas the trophoblast proliferates excessively, resulting in a hydatidiform mole, which may trigger complications, such as gestational trophoblastic disease [ 31 , 32 ]. In contrast, gynogenetic diploid embryos typically exhibit impaired or arrested development: they fail to form functional extraembryonic structures (e.g., placenta and fetal membranes) essential for embryonic nourishment and support, and their embryoblast undergoes high risk of abnormal differentiation into a teratoma [ 33 , 34 ]. Furthermore, true 1PN embryos generated via parthenogenetic activation exhibit impaired development, implantation failure and early miscarriage. The recently published Istanbul Consensus recommends evaluating the clinical application of 1PN zygotes by combining blastocyst culture with PGT-A technology suitable for biparental diploid assessment to mitigate associated risks [ 35 ]. To date, the mechanism of 1PN development, apart from those yet to be discovered, could be attributed to improper timing of PN formation and/or disappearance, paternal and maternal PN fusion, or parthenogenetic activation [ 4 , 36 ]. 1PN zygotes are classified into two distinct phenotypes based on their formation mechanisms: true 1PN and missed 2PN. True 1PN zygotes arise from intrinsic biological abnormalities, including pronuclear fusion (where male and female pronuclei fuse prematurely before observation) and parthenogenetic activation (spontaneous activation of the oocyte without sperm penetration, resulting in uniparental genome). Missed 2PN zygotes are a result of technical limitations in pronuclear observation: asynchrony in pronuclear appearance (male and female pronuclei form at different timepoints, leading to only one pronucleus being visible during the standard observation window) or premature pronuclear breakdown (pronuclei disintegrate before the observation time). Notably, missed 2PN zygotes can be effectively identified using short incubation time IVF combined with time-lapse monitoring technology, which enables continuous tracking of pronuclear dynamics throughout fertilization [ 3 – 6 , 36 ]. However, numerous cytogenetic analyses have shown that a large proportion of IVF-derived 1PN zygotes are diploid and have developmental capacity [ 7 , 37 ]. According to some reports, embryos originating from IVF-1PN zygotes successfully reach full-term development following transfer, resulting in healthy live births [ 19 , 38 ]. Moreover, the diploid rate of 1PN zygotes in IVF cycles is higher than that in ICSI cycles [ 14 , 39 ], which could be explained by the different mechanisms of 1PN formation between IVF and ICSI cycles [ 4 , 40 ]. A recent study on 1PN zygotes from conventional IVF cycles revealed that 78% are diploid and can be detected noninvasively by measuring PN diameter and monitoring PN loss [ 24 ]. Therefore, PN diameter measurement might be a promising noninvasive method to predict the developmental potential of IVF-1PN zygotes. To date, three studies have explored the relationship between the PN diameter of 1PN zygotes and embryo development. Otsu et al. examined the PN diameter of 1PN zygotes and reported that blastocyst formation rates were higher in large 1PN zygotes than in small 1PN zygotes, and FISH revealed that blastocysts produced by large 1PN zygotes were more likely to be diploid [ 23 ]. Later, Yoshiteru et al. reported that the PN area and diameter of 1PN zygotes were significantly correlated with embryological outcomes [ 24 ]. Araki et al. also reported that the blastocyst formation rates and quality of 1PN zygotes with larger PN diameters were much greater than those with smaller PN diameters [ 25 ]. However, the sample sizes of the abovementioned studies are limited, and there are no studies on pregnancy outcomes. At present, the potential of accurately predicting 1PN-derived blastocyst development on the basis of PN diameter is still lacking; however, explorations of the application of PN diameter in evaluating pregnancy outcomes are needed. To address this issue further, we conducted a retrospective study with a large sample size to assess the relationship between the 1PN zygote diameter and embryological outcomes/pregnancy outcomes. In this study, we found that blastocyst development and high-quality blastocyst formation rates were significantly greater in 1PN zygotes with large PN diameters than in 1PN zygotes with small PN diameters, revealing a correlation between the PN size of 1PN zygotes and embryological outcomes, which is consistent with previous findings [ 23 – 25 ]. These results suggest that 1PN zygotes with large PN diameters are likely to contain both female and male genomes. Research has demonstrated that 1PN zygotes from IVF cycles are formed primarily by enclosing juxtaposed male and female nuclei in a common pronuclear envelope [ 4 ] and that male chromatin fuses near female chromatin before the nuclear envelope forms [ 41 ]. However, the PN diameter of these 1PN zygotes remains uncertain. The rate of embryonic development in 1PN zygotes with small PN diameters is thought to be due to parthenogenesis and failure to decondense the sperm head [ 36 ]. This study also revealed that once 1PN zygotes reach the blastocyst stage, the PN diameter is unrelated to pregnancy or the live birth rate. Previous studies have shown no significant difference in the rate of aneuploidy between 1 and 2PN blastocysts (39.3% and 36.5%, respectively) [ 7 ]. Research has also demonstrated that 1PN and 2PN-derived blastocyst transfer cycles result in equivalent pregnancy and neonatal outcomes [ 22 ]. Furthermore, this study used correlation and binary regression in combination with GEE analyses to rule out the influence of confounding factors. As a result, we recommend measuring the PN diameter of all 1PN zygotes (> 32.5 μm is a predictor of blastocyst formation) and culturing them until they reach the blastocyst stage. The advantage of this study is that with a larger sample size, 1PN zygotes with larger PN diameters are associated with better embryological outcomes, making the findings more trustworthy. In addition, owing to the large sample size, we were able to further divide the IVF-derived 1PN zygotes into several subgroups according to their PN diameters and report their respective embryological outcomes. We also determined a PN diameter cutoff value of 32.5 μm in predicting blastocyst formation to provide clinicians with more information when making decisions on IVF-derived 1PN zygotes (continuing to culture or abandon them). Moreover, we observed pregnancy outcomes and analyzed their relationship with PN diameter in this study for the first time, and the results again corroborated the adoption of blastocysts developed from 1PN zygotes in conventional IVF. In addition, we further confirmed our conclusions by controlling for as many important confounding factors as possible statistically. However, the study has certain limitations. Currently, the sample size is still too small, and retrospective cohort studies are prone to selection bias. Therefore, prospective studies with larger sample sizes are needed in the future to increase the strength of our studies. Furthermore, since we did not employ short-time IVF combined with time-lapse monitoring to dynamically observe the process of pronuclear formation, the exact mechanism underlying the 1PN observed in this study remains unclear. In addition, not all patients underwent a reassessment of PN quantity. Finally, this study lacked genetic analysis, preventing us from determining the euploidy status of these embryos. Consequently, we recommend conducting PGT-A on 1PN blastocysts, if feasible, and emphasize the need for further research to ascertain whether PN size is correlated with aneuploidy.

This is the largest cohort study and the first to investigate the relationship between the 1PN zygote diameter and embryological/pregnancy outcomes. We confirmed that the 1PN zygote diameter is an important marker for predicting blastocyst formation (cutoff value of 32.5 μm) and that 1PN zygotes with a larger PN diameter have better embryological outcomes. Once a blastocyst has formed, the PN diameter has no significant effect on pregnancy outcomes. We anticipate that our findings will contribute to the development of a screening tool for available 1PN zygotes for clinical use and improve the likelihood of conception in infertile patients by increasing the possibility of FET in 1PN zygotes. However, these findings still need to be validated by future prospective studies with large samples.

In assisted reproductive technologies, normal fertilization is confirmed by the presence of two pronuclei (2PNs) and two polar bodies (PBs) 16–18 h after insemination. However, zygotes with a single pronucleus (1PN), which are usually classified as failed or abnormal fertilization, are also commonly observed. The incidence of 1PN ranges from 4 to 8% after conventional in vitro fertilization (IVF) and 2% to 5% after intracytoplasmic sperm injection (ICSI) [ 1 , 2 ]. Nowadays, 1PN zygotes result from biological anomalies or observational limits. They can be separated into true 1PN (such as PN fusion or parthenogenetic activation) and missed 2PN (caused by asynchrony in pronuclear appearance or premature pronuclear breakdown) [ 3 – 6 ]. While parthenogenesis results in abnormal embryos, other patterns might involve normal fertilization and have the potential to produce normal blastocysts. Previous studies have demonstrated that 1PN zygotes are less likely to cleave and develop into blastocysts [ 2 , 7 , 8 ]. In both IVF and ICSI, the blastocyst formation rate of 1PN zygotes was lower than that of 2PN zygotes, at 14.8% and 6.6%, respectively [ 7 ]. Moreover, as revealed by fluorescence in situ hybridization (FISH), the diploid rate of 1PN embryos increased from 48.7% in IVF (27.9% in ICSI) in the cleavage stage [ 2 ] to 74.6% in blastocysts (from IVF or ICSI) [ 9 ]. Recently, with the use of comparative genomic hybridization (CGH), some studies reported that 50.0 [ 10 ]–69.2% [ 11 ] of 1PN-derived blastocysts were diploid. The diploid rate of 1PN embryos tends to increase significantly from cleavage to blastocyst, even if the diploid rate of 1PN-derived blastocysts varies slightly in different publications because of the various experimental methods used for the assessment of chromosome normality. Therefore, the blastocyst culture of 1PN zygotes could be a reliable tool for screening 1PN embryos with abnormal chromosomes and preliminarily identifying viable 1PN embryos [ 9 , 12 ]. In addition, since the rate of diploid chromosomal constitution among 1PN embryos was significantly higher in IVF than in ICSI, for those with only 1PN embryos available, 1PN-derived blastocysts from conventional IVF might be an alternative option to increase the chances of pregnancy [ 2 , 13 – 15 ]. Although 1PN blastocysts are often not recommended for transfer [ 16 ], live births involving these embryos have been reported by several researchers [ 17 – 21 ]. They reported that once the 1PN zygotes reached the blastocyst stage, the probability of live birth was similar to that of 2PN-derived blastocysts [ 9 , 19 , 20 ]. In addition, in our previous study, we analyzed 316 and 6582 cycles with 1PN- and 2PN-derived blastocyst transfer (which resulted in 115 and 2402 live births), in which the pregnancy and neonatal outcomes were comparable between the two cohorts [ 22 ]. Furthermore, to avoid the risk of adverse outcomes, preimplantation genetic testing for aneuploidies (PGT-A) and time-lapse embryo monitoring could be applied to support the diagnosis of a diploid genome and assess the implantation potential of 1PN-derived embryos. Nonetheless, a prompt, economic and noninvasive method to predict the developmental potential of 1PN zygotes in IVF is highly important. In 2004, pronuclear (PN) diameter measurement in 1PN zygotes was first proposed by Otsu et al., who reported that whereas the rate of blastocyst formation among large 1PN zygotes (29–34 μm) was 18%, small 1PN zygotes (23–26 μm) did not develop into blastocysts [ 23 ]. In 2018, Yoshiteru et al. reported that a PN diameter ≥ 32.2 μm just before PN breakdown might be a useful criterion for selecting 1PN zygotes capable of further cleavage and with a relatively high blastocyst formation rate (80%, 4/5) [ 24 ]. Another study performed by Araki et al. in 2018 revealed that the rates of blastocyst formation and good-quality blastocysts from 1PN zygotes with large PN diameters (≥ 31 μm) were significantly greater than those with smaller PN diameters (≤ 24, 25–27, and 28–30 μm) [ 25 ]. However, these three studies cannot draw clear conclusions because of the limited sample size, and accurate predictions of the developmental potential of 1PN-derived blastocysts on the basis of PN diameter are still lacking. In the present study, we aimed to explore the relationship between the PN diameter of 1PN zygotes in conventional IVF and their developmental potential (embryological outcomes and pregnancy outcomes) to establish an effective method to predict the clinical outcomes of 1PN embryos.

Supplementary Material 1: Comparison of embryological outcomes among different PN diameter groups.The number of embryos in different PN diameter groups; the n values are shown above.The proportion of D3 cellsin different PN diameter groups; the percentage is shown in the bar chart.The proportion of blastocyst qualityin different PN diameter groups; the percentage is shown in the bar chart.The proportion of blastocyst gradein different PN diameter groups; the percentage is shown in the bar chart. Supplementary Material 1: Comparison of embryological outcomes among different PN diameter groups.The number of embryos in different PN diameter groups; the n values are shown above.The proportion of D3 cellsin different PN diameter groups; the percentage is shown in the bar chart.The proportion of blastocyst qualityin different PN diameter groups; the percentage is shown in the bar chart.The proportion of blastocyst gradein different PN diameter groups; the percentage is shown in the bar chart.