The incidence of 3PN fertilization was 2.5–6.2% in ICSI cycles[1]. The mechanisms resulting in polyspermy fertilization formation is still unclear. Different mechanisms were suggested between c-IVF and ICSI cycles[1]. Immunofluorescent analysis shows that most of 3PN zygotes derived from c-IVF had a female PN (fPN)and two male PNs (mPN), suggesting polyspermy[9]. On the other hand, 3PN zygotes after ICSI showed two fPNs and a mPN, indicating failure of the extrusion of the second polar body[4, 9, 10, 11]. In the 3PN embryos after ICSI, equal ratios of XXX and XXY were observed and no XYY embryos were present[12]. However, Time-lapse technology (TLT) has revealed extrusion of the second polar body was observed in a vast majority of 3PN zygotes (92.1%) in ICSI cycles[13].
3PN incidence may serve as a prognostic indicator for IVF cycle outcome using embryos derived from normally fertilized oocytes and make a negative effect on pregnancy outcomes in eSBET[3, 4, 14]. However, Ye YH et al showed that polyspermic fertilization is correlated with improved oocyte receptivity to sperm and could be considered as an encouraging sign for the success of cIVF[15]. While some studies showed that 3PN incidence might be caused by oocyte quality or sperm abnormality in cIVF cycles[16], and the high cIVF 3PN incidence (3PN > 20%) may predict poor outcomes in blastocyst-stage embryo transfer cycles, but not influence for cleavage-stage pregnancy outcome[17].
The higher proportions of 3PN also affected embryos led to worse clinical outcomes in ICSI cycles[4]. When the 3PN rate was less than 20% or 25%, the ICSI outcomes were not impaired[5, 6]. Only when the 3PN zygote rate was > 20% or > 25%, the proportion of 3PN zygotes after ICSI serves as a negative prognostic indicator for ICSI cycle outcome[5, 6]. However, This study showed that the number of 3PN zygotes per cycle was only 1.31(106/81). The presence of 3PN can serve as a surrogate marker of PGT outcomes for the remaining cohort of zygotes. 3PN zygote rate > 20% or > 25% was fewer, so we did not analysis the influence of the difference 3PN zygote rate.
In couples with a normal semen analysis undergoing ICSI, the 3PN rate may serve as a surrogate marker of oocyte quality and the 3PN rate is a significant predictor of implantation rate for the remaining cohort of zygotes[4]. The starting and total dose of gonadotropins administered and the total days of stimulation are independent predictors of the 3PN rate[4]. High proportion of 3PN zygotes made a negative effect on clinical outcomes for IVF-ET cycles with long-term protocol[18]. 3PN formation may also be increased in women that are high responders to gonadotropins [4]. We also analysis the COH protocol, there were no significantly difference in COH-anti and COH-Long rate between the 3PN group and Non 3PN group.
However, more retrieved oocytes could result in 3PN incidence easily which is consistent with previous study [19]. The reason is that once more oocytes were retrieved, they display a more degenerated, immature, and aging phenotype[19]. In this study, we also observed more retrieved oocytes in 3PN group than Non 3PN group. However, number of blastocyst per cycle was no significantly difference between two groups (3.92 ± 3.55 vs.3.30 ± 2.70, P > 0.05). And lower MII oocyte rate, 2PN fertilizaion rate, D3 high embryo rate, blastocyst rate, per oocyte utilization and D5 blastocyst rate in 3PN group than Non 3PN group (P < 0.05).
Both maternal and paternal ages were higher in cases involving 3PN fertilization in ICSI[13]. However, in this study although female and male's ages were significantly older in PGT-A cycles than PGT-M/SR cycles. 3PN cycle rate was no significantly different in two groups(15.8% vs. 20.7%, P > 0.05). And the patients age was no significant difference between 3PN group and Non 3PN group. Similar as Sachs et al. result that no significant association of aging between women who had 3PN zygotes after ICSI[20]. Our study first comparing the 3PN rate between PGT-A cycles and PGT-M/SR cycles(15.2% vs.10.5%, P > 0.05).
No correlation was observed between the semen analysis abnormalities and the 3PN formation rates in ICSI[21]. Some studies suggest severe sperm abnormalities and oocyte aging can contribute to the process[4, 22]. Male factor infertility impacts the rate of mosaic blastocysts in cycles of PGT-A[23]. In this study, we did not observed correlation between the semen analysis abnormalities and the 3PN formation rates in PGT cycles.
Some studies researched relationship between Anti-Müllerian hormone (AMH), luteinizing hormone (LH) and 3PN. AMH significantly correlates with the presence of multiple pronuclei in the zygote. The presence of multiple pronuclei increased when AMH levels were higher[24]. Supplementation of exogenous LH activity to ovarian stimulation may be associated with lower prevalence of 3PN zygotes in IVF cycles, but not in ICSI cycles[1]. 3PN zygotes may occur less frequently in LH activity-added cycles[1].
Nowadays most reference laboratories require use of ICSI and blastocyst biopsy for PGT[25]. The results presented here suggest that 3PN rate do not affect the euploidy and aneuploid rate of the blastocyst. However, the mosaic rate was significantly higher in 3PN group than Non 3PN group (16.3 vs. 12.1; P < 0.05). There was a preliminary study revealed not all morphologically 3PN embryos are genetically abnormal[26]. Some 3PN zygotes, with two normal-sized PNs and an additional smaller PN (2.1PN) embryos were diploid, however, predominantly aneuploid, and therefore could not be used for embryo transfer[27]. The deficiency of this study was that lack of analyzing the 3PN blastocysts genetic testing.
In conclusion, No correlation was observed between 3PN formation rate and PGT-A or PGT-M/SR cycles. the presence of 3PN in PGT cycles was associated with the greater number of retrieved oocytes. The occurrence of 3PN seems to impair the developing blastocyst and interfere with good embryo formation rate and mosaic rate in PGT. But the occurrence of 3PN does not seem to impair the euploid rate and aneuploid rate.