Are tri‐pronuclear embryos that show two normal‐sized pronuclei and additional smaller pronuclei useful for embryo transfer?

Abstract Purpose This study aimed to analyze whether tripronuclear (3PN) zygotes, with two normal‐sized PNs and an additional smaller PN (2.1PN), can be used for embryo transfer. Method(s) A retrospective embryo cohort study was conducted on 695 patients who underwent intracytoplasmic sperm injection treatment. Blastocyst formation rates were compared between 2.1PN and 2PN zygotes and PGT‐A analysis was performed on 15 blastocysts derived from 2.1PN zygotes. Result(s) Blastocyst formation rates were comparable between 2.1PN (43.8%) and 2PN zygotes (54.8%; p = 0.212). The rates of blastocysts with good morphology derived from 2.1 PN and 2PN zygotes were 18.8% and 25.5%, respectively. No significant differences were detected (p = 0.383). All of the analyzed blastocysts were diploid; however, 13 of these were found to be aneuploid, with a further two being mosaic. Conclusion Our results suggest that 2.1PN embryos can reach blastocyst stage. These blastocysts were diploid, however, predominantly aneuploid, and therefore could not be used for embryo transfer.


| INTRODUC TI ON
In assisted reproductive technology (ART), fertilization is confirmed 16-18 h after insemination or intracytoplasmic sperm injection (ICSI).
The formation of two pronuclei (PN) is considered normal fertilization. The two pronuclei of a normally fertilized zygote are generally equal in size and centrally located. 1 If they are not of equal size, the resulting zygotes have little developmental potential 2,3 and a high degree of aneuploidy. [4][5][6] Zygotes with one or more than three PNs are considered abnormally fertilized. In addition, zygotes that show three or more pronuclei (3PN and ≥4PN) are generally discarded regardless of their size. 7 This is because embryos arising from zygotes with three PNs are considered to harbor a polyploid chromosomal constitution, and the transfer of these embryos is considered to result in a higher risk of miscarriage and/or molar pregnancy. [7][8][9] It has been reported that blastocysts derived from 3PN zygotes, showing two normal-sized PNs and additional smaller PN (2.1PN), are predominantly diploid and some of these have resulted in live births. 10 According to their study, the 2.1PN-derived blastocysts under study were also largely diploid (n = 12/14; 85.7%), while the remainder were triploid.
Various studies have shown that these tripronuclear human oocytes do not always develop into triploid embryos. 11,12 Although births derived from triploid zygotes have been reported, such infants generally do not live long after birth and only few have been reported as surviving for an unusual duration. 13 Thus, it is important to analyze the chromosomal abnormalities of embryos to determine whether they are suitable for embryo transfer. Therefore, in this study, we aimed to investigate the developmental potential of 2.1PN embryos and analyze their ploidy and chromosomal abnormalities by performed preimplantation genetic testing for aneuploidy (PGT-A).

| MATERIAL S AND ME THODS
This retrospective study included 1001 cycles involving 695 patients who underwent ICSI treatment between November 2020 and July 2021. Thirty-two 2.1PN zygotes were observed in 28 cycles from 26 patients. Written patient consent was obtained after providing a thorough explanation. This study was conducted with the approval of the ethics committee of Okayama Couple's Clinic (approval number 2020-04).

| Stimulation protocols
Ovarian stimulation, triggering final oocyte maturation, oocyte retrieval, fertilization, and embryo culture were conducted according to our standard protocols. 14 The ovarian stimulation protocols were chosen depending on each patient's age and serum anti-Müllerian hormone (AMH) level (Table 1). Patients in the long protocol group and the short protocol group were treated with a GnRH agonist (Buserelin acetate, Fuji Pharma). Treatment continued until the day on which 10 000 IU of human chorionic gonadotropins (hCG) (HCG, Fuji Pharma) was administered.
For the long protocol, administration of the GnRH agonist commenced a week following their most recent ovulation. For the short protocol, administration of the GnRH agonist commenced on the second day of the menstrual cycle.
For the GnRH antagonist protocol, the GnRH antagonist  When at least two follicles reached a diameter of 18 mm or larger, 10 000 IU hCG was administered to induce ovulation.

TA B L E 1 Ovarian stimulation protocols
Denuded oocytes were incubated in single-step medium (ONE STEP Medium; NAKA medical inc, Tokyo, Japan) covered with mineral oil.

| ICSI procedures and embryo culture
Intracytoplasmic sperm injection was performed on MII stage oocytes 39.5 h after hCG administration to induce ovulation.
Sperms were prepared using a density-gradient centrifugation technique with Isolate (Irvine, Cal., USA) and the swim-up method with insemination medium. The embryos were placed into the wellof-the-well (WOW) culture system (LinKID ® micro25; DNP, Tokyo, Japan) using a single-step medium and covered with mineral oil.
Fertilization was confirmed by the presence of two pronuclei 16-18 h after ICSI (Day 1). At the time of fertilization confirmation, the PNs diameter of 2PB3PN (a zygote with two polar bodies and three PNs) was measured, and zygotes that presented with two normal PNs and an additional smaller PN, not larger than one-third the size of normal, were defined as 2.1PN. 10 Cleavage stage embryos were evaluated based on Veeck's classification on the second day after oocyte retrieval. 15 Blastocyst development was evaluated based on Gardner's classification on the fifth and sixth days after oocyte retrieval. 16 Blastocyst formation with good morphology was defined as a grade of at least 3BB.

| Blastocyst vitrification
The

| Statistical analyses
We assessed continuous variables with a normal distribution and equal variances using Student's t-tests. The difference in blastocyst formation rates was compared between 2.1PN and 2PN embryos with a chi-squared test. Aneuploidy and ploidy analyses were evaluated by copy number variation (CNV) and single-nucleotide polymorphisms (SNP).
Preimplantation genetic testing for aneuploidy analysis was performed on 15 blastocysts derived from 2.1PN zygotes. All of the analyzed blastocysts were diploid; however, 13 of these were found to be aneuploid, with a further two being mosaic ( Table 4).
The comparison of PGT-A results between 2.1PN-derived and 2PN-derived embryos before and after propensity score matching are shown in Table S1. Note that the sample sizes for the comparison are smaller than the standard sizes. Nevertheless, in this study, patients' ages in the cycles with 2.1PN zygotes were higher than those without 2.1PN zygotes, and the two patients with mosaic embryos were relatively young (30 and 32 years). Although it has been reported that chromosomal aneuploidy is associated with age, 20-23 further research is required to gain a more complete understanding of whether the aneuploidy of 2.1PN embryos is associated with patient age.

Hiromi Takahashi, Rei Hirata, Junko Otsuki, Toshihiro Habara and
Nobuyoshi Hayashi declare that they have no conflicts of interest.

E TH I C A L A PPROVA L
Approval was obtained from the ethics committee of Okayama Couple's Clinic (approval number: 2020-04). The procedures used in this study adhere to the tenets of the declaration of Helsinki.

H U M A N R I G HT S S TATE M E NT S A N D I N FO R M E D CO N S E NT
All patients were well informed and written informed consent was obtained prior to the treatment period.

A N I M A L S TU D I E S
This article does not contain any experimental studies with animal subjects on the part of any of the authors.