Clinical outcomes of frozen-thawed blastocysts from zygotes with no or one pronucleus for in vitro fertilization and intracytoplasmic sperm injection cycles

doi:10.21203/rs.3.rs-2502145/v1

Purpose

To investigate the clinical outcomes of in vitro fertilization/intracytoplasmic sperm injection (IVF/ICSI) cycles using frozen-thawed blastocyst transfers derived from zygotes with no (0PN) or one pronucleus (1PN).

Methods

This retrospective study included 7084 0PN, 2238 1PN and 72266 two-pronuclear (2PN) embryos cultured into blastocyst stage from 19631 IVF and 12377 ICSI cycles between March 2018 and December 2021. Developmental potential and clinical outcomes of 0PN, 1PN and 2PN embryos were analyzed. A total of 290 0PN-derived, 92 1PN-derived and 1906 2PN-derived single frozen-thawed blastocyst transfers were performed. Chromosome analysis of 0PN-, 1PN- or 2PN-derived blastocysts used Infinium Asian Screening Array gene chip or next-generation sequencing.

Results

The available blastocyst rates of 0PN and 1PN embryos were significantly lower than those of 2PN embryos in both IVF and ICSI cycles. Single 0PN and 1PN blastocysts transferred in frozen-thawed cycles resulted in a similar clinical pregnancy rate, live birth rate and miscarriage rate compared with 2PN blastocysts, in both IVF and ICSI cycles. Genetic analysis showed that euploid rates of 0PN- and 1PN-derived blastocysts used for ICSI cycles were similar to that of 2PN-derived blastocysts.

Conclusion

Our study indicated that 0PN- and 1PN-derived blastocysts resulted in a similar CPR and LBR as 2PN-derived blastocysts. The 0PN- and 1PN-derived blastocysts from ICSI cycles can be transferred as well as those from IVF cycles when the number of 2PN-derived blastocysts is insufficient.

IVF

ICSI

blastocyst transfer

live birth rate

PGT-A

The assessment of in vitro fertilization (IVF) is generally performed 16–18 h after the fertilization procedure [1]. Typically, fertilized oocytes exhibiting two pronuclei (2PN) under light microscopy will be cultured further and selected for transfer. Mature oocytes without a pronucleus (0PN) or with one pronucleus (1PN) are generally considered to be a failed or abnormal fertilization. It has been reported that 11.3–20.0% and 1.6–7.7% of 0PN and 1PN zygotes, respectively, are present after IVF or ICSI [2–3]. Even though the embryos developing from 0PN and 1PN zygotes are capable of developing into blastocysts in vitro and may result in healthy babies, they are usually discarded because of a possible higher proportion of chromosomal abnormalities [4–7]. Some studies of the genetic characteristics of 0PN and 1PN embryos have suggested that a considerable number of 0PN and 1PN embryos may have a normal chromosomal composition, and thus may be considered for transfer when no embryos developing from 2PN zygotes are available [8–10]. Previous studies have reported that 0PN and 1PN cleavage stage embryos resulted in a lower implantation rate (IR) and live birth rate (LBR) when compared with 2PN cleavage stage embryos [11–12], while the IR and LBR were comparable between 0PN- and 1PN-derived blastocyst transfers and those of 2PN-derived blastocyst transfers [12–13]. However, some research suggested that 0PN- and 1PN-derived blastocyst transfers resulted in a lower IR and LBR than 2PN-derived blastocysts, especially in ICSI cycles [14–15]. Thus, the clinical outcomes of 0PN- and 1PN-derived blastocysts from different fertilization methods need to be further investigated.

In this study, a retrospective analysis of blastocyst formation of 0PN and 1PN embryos from IVF and ICSI cycles was conducted. The clinical outcomes of single 0PN- and 1PN-derived frozen–thawed blastocyst transfers were compared. The genetic results of 0PN- and 1PN-derived blastocysts of preimplantation genetic testing for aneuploidies (PGT-A) cycles were also analyzed. The aim of this study was to investigate whether 0PN- and 1PN-derived blastocysts from ICSI cycles were comparable to blastocyst transfers from IVF cycles.

Study design

This study was performed at the Department of Reproductive Medicine, Sir Run Run Shaw Hospital, affiliated with Zhejiang University School of Medicine. The available blastocyst rates of 7084 0PN, 2238 1PN and 72266 2PN embryos from 19631 IVF and 12377 ICSI cycles cultured to blastocyst stage from March 2018 to December 2021 were analyzed. The clinical pregnancy rate (CPR), LBR and miscarriage rate (MR) after transfer of 0PN-, 1PN- and 2PN-zygote derived single frozen-thawed blastocysts (hereafter denoted 0PN, 1PN and 2PN blastocysts) were compared. The chromosomal characteristics of 0PN, 1PN and 2PN blastocysts from PGT-A cycles were analyzed. All of the PGT-A cycles underwent ICSI treatment. This study was approved by the medical ethical committee of Sir Run Run Shaw Hospital.

Fertilization evaluation, embryo culture and handling

Patients underwent controlled ovarian stimulation as previously described [16]. Sequential culture media (Sydney IVF, Cook Medical, USA) were used for embryo culture. The 0PN, 1PN and 2PN zygotes were further cultured. Cleavage stage embryos on day 3 with more than four blastomeres graded 3 and over were cultured into day 5 and 6 blastocyst stage. The formed blastocysts graded as 3 to 6 with inner cell mass (ICM) greater than grade B or with ICM grade C, trophoblast cell (TE) greater than grade B, according to Gardner’s grading system [17], were selected for vitrification. The blastocysts graded as 4 to 6 with ICM and TE scores both equal to or better than grade B were considered to be high-quality blastocysts. Single blastocyst transfer was performed following warming of the vitrified blastocysts. In PGT-A cycles, TE biopsy was performed on day 5 and 6 blastocysts without pre-hatching, so embryos were undisturbed until the expanded blastocyst stage. Three to five TE cells from the side opposite of the ICM were dissected and separated from the blastocysts by laser combined with mechanical dissection.

Genetic analysis of blastocysts

Whole genome amplification (WGA) was performed according to the instructions of the Sample Processing Kit for Gene Sequencing (XK-028, Yikon Genomics, China) for biopsied TE cell samples. The WGA products were used for library construction with a library size of 200 to 500 bp, generating approximately 2 million raw reads for each TE sample [18]. The WGA products from 2PN blastocysts were sequenced using an Illumina Nextseq 550 platform. The WGA products from 0PN and 1PN blastocysts and genome of parental peripheral blood cells were collected and analyzed using Illumina Infinium Asian Screening Array-24 + v1.0 (ASA) gene chips. Whole genome copy number variations were analyzed to determine the euploidy or aneuploidy status of TE cell samples. Single nucleotide polymorphisms information of TE cell samples and parent cells were analyzed to exclude ploidy abnormalities, such as polyploids, haploid or uniparental disomy.

Follow-up

Serum human chorionic gonadotropin β-subunit levels were evaluated 10 days after blastocyst transfer. The presence of a gestation sac by ultrasound after 5 weeks was defined as clinical pregnancy. Live birth was defined as at least one live baby born after 28 weeks’ gestation, and implantation rate was calculated as the number of gestational sacs per number of embryos transferred. Miscarriage was defined as the loss of a previously visualized intrauterine gestational sac by ultrasound before 20 weeks. Data on pregnancy loss and delivery were acquired through telephone interviews.

Statistical analysis

All statistical analyses were performed using SPSS software version 19.0. For mean values, independent sample t-test and variance analysis were used for inter-group and intra-group differences, respectively. For proportional values, chi-square test and chi-square test of multiple comparisons were used for inter-group and intra-group differences. A P value of <0.05 was considered statistically significant.

The blastocyst formation from 0PN and 1PN embryos in IVF and ICSI cycles

In both IVF and ICSI cycles, the available blastocyst rate of 0PN embryos was lower than that of 2PN embryos (47.7% vs. 53.4% in IVF, P<0.05; 32.0% vs. 49.8% in ICSI, P<0.05; Table 1). However, in both IVF and ICSI cycles, the rates of day 5 blastocysts and high-quality blastocysts from 0PN embryos were higher than those from 2PN embryos (85.5% vs. 69.6% and 56.1% vs. 44.7%, respectively, in IVF, P<0.05; 75.5% vs. 61.6% and 57.1% vs. 42.9%, respectively, in ICSI, P<0.05).

The available blastocyst rate of 1PN embryos was also decreased in both IVF and ICSI cycles, when compared with 2PN embryos (28.9% vs. 53.4% in IVF, P<0.05; 16.1% vs. 49.8%, P<0.05, respectively). The rate of day 5 blastocysts from 1PN embryos was lower than those from 2PN embryos in ICSI cycles (47.4% vs 61.6%, P<0.05). The rates of day 5 blastocysts in IVF cycles and high-quality blastocysts in both IVF and ICSI cycles did not differ between 1PN and 2PN embryos.

The rates of available blastocysts and day 5 blastocysts from 0PN and 1PN embryos were much lower in ICSI cycles than IVF cycles (P<0.05). The fertilization methods significantly influenced blastocyst formation from 0PN and 1PN embryos.

Clinical outcomes of transfer single frozen-thawed 0PN and 1PN blastocysts

In this study, 290 0PN and 92 1PN single frozen-thawed blastocyst transfers were performed, resulting in 106 0PN- and 35 1PN-derived singletons, respectively, compared with 1906 2PN single blastocyst transfers and 815 2PN-derived singletons, respectively. We compared the clinical outcomes of 0PN and 1PN blastocyst transfers with 2PN blastocyst transfers in IVF and ICSI cycles. As shown in Table 2, the CPRs of 0PN, 1PN and 2PN blastocyst transfers in IVF cycles were 46.2%, 52.6% and 50.7%, respectively; the LBRs were 36.1%, 37.2% and 42.3%, respectively; and the MRs were 20.0%, 26.8% and 16.2%, respectively. The CPR, LBR and MR did not differ significantly among the three groups. Forty-one 0PN and 14 1PN blastocysts were transferred in ICSI cycles. The 0PN blastocyst transfers resulted in a CPR of 46.3%, a LBR of 39.0% and a MR of 16.7%. The 1PN blastocyst transfers resulted in a CPR of 42.9% and all clinical pregnancies achieved live birth. No significant differences in CPR, LBR and MR for 0PN and 1PN blastocyst transfers were observed when compared with 2PN blastocyst transfers.

Genetic analysis of 0PN and 1PN blastocysts derived from ICSI cycles

To investigate whether there was a decrease in euploid rates in 0PN and 1PN blastocysts derived from ICSI cycles, we evaluated the TE biopsy samples from 64 0PN, 20 1PN and 2009 2PN blastocysts undergoing PGT-A. ICSI was performed in all PGT-A cycles. The WGA rate was similar for the three groups (Table 3). The euploid rates of 0PN, 1PN and 2PN blastocysts were 62.5%, 60.0% and 54.4%, respectively. The euploid, aneuploid and mosaic rates of 0PN and 1PN blastocysts were comparable with those of 2PN blastocysts. No ploidy abnormalities were found in euploid 0PN and 1PN blastocysts by ASA gene chip analysis.

This study found that 0PN and 1PN embryos exhibited a lower available blastocyst rate than 2PN embryos, especially in ICSI cycles. Single 0PN and 1PN blastocysts transferred in frozen-thawed cycles resulted in a similar CPR, LBR and MR compared with 2PN blastocysts, in both IVF and ICSI cycles. No significant differences were observed in chromosomal euploid rates for 0PN, 1PN and 2PN blastocysts in ICSI cycles.

The availability of 0PN and 1PN embryos for clinical use is currently disputed. They are generally selected to use only when 2PN embryos are not available. However, the clinical outcomes of 0PN and 1PN embryo transfers varies significantly between different IVF clinics, and the influence of different fertilization methods remains unclear.

It has been described that 0PN may result from the lack of formation of PN, disappearance of PN, or unfertilized oocytes [15]. Previous studies have reported that the cleavage rate of 0PN zygotes was lower than that of 2PN zygotes, indicating that 0PN zygotes had a higher possibility of fertilization failure. Disappearance of pronuclei in normally fertilized oocytes occurs approximately 23–25 h after fertilization, and the PN of fast-developing embryos may disappear earlier than the PN of normally developing embryos [19-20]. A recent study performed time-lapse monitoring for PN assessment and revealed that the PN in 7.59% of embryos had already disappeared at 20 h after fertilization, indicating that PN may not be observed in these fertilized zygotes at a fixed time when they were in an ordinary incubator [21]. This may also explain that some 0PN embryos have a normal 2PN origin and the potential to develop into blastocysts and implant as 2PN-like embryos.

Blastocyst culture is commonly recommended to select development potential for 0PN embryos [22]. Chen et al. reported that the available blastocyst rates of 0PN embryos were decreased when compared with those of 2PN embryos, and the cell number of 0PN embryos on day 3 affected subsequent blastocyst formation [23].The available blastocyst rate of 0PN embryos was higher for the ≥6 cells than <6 cells day 3 embryos. The current results demonstrated that 47.7% and 32.0% of 0PN embryos in IVF and ICSI cycles, respectively, developed to the blastocyst stage. This development was significantly lower than 2PN embryos, and the rate was especially lower in ICSI cycles compared with IVF cycles. However, in both IVF and ICSI cycles, 0PN embryos had higher day 5 and high-quality blastocyst rates than 2PN embryos. Moreover, the CPR, LBR and MR of single frozen-thawed 0PN blastocysts obtained from IVF and ICSI cycles were nearly the same as those of 2PN blastocysts.

Embryos with abnormal chromosomes may be reduced by prolonging culture into the blastocyst stage, when embryos complete the transformation from maternal to embryonic-derived genes [24-26]. In the present study, next generation sequencing (NGS) was used to assess chromosomal abnormalities of 2PN blastocysts undergoing PGT-A. However, NGS was unable to detect ploidy alterations, and as a result, haploid or polyploid cells may be present in blastocysts diagnosed as euploid by NGS. Therefore, the ASA gene chip was adopted to detect the chromosomal characteristics of 0PN and 1PN blastocysts. There were no significant differences in the euploid rates between 0PN and 2PN blastocysts from ICSI cycles, and no ploidy abnormalities were observed in euploid 0PN blastocysts. Our findings were consistent with previous results showing no significant differences in chromosomal abnormalities between 0PN and 2PN blastocysts [27-28]. The 0PN blastocysts derived from both IVF and ICSI cycles can be transferred and produce equivalent outcomes to the 2PN blastocysts.

The 1PN zygote may result from inappropriate timing of PN formation and/or disappearance of pronuclei, fusion of paternal and maternal PN, or parthenogenetic activation [29-30]. Several studies have reported that 1PN embryos were found to have a reduced developmental potential compared with 2PN embryos [31-32]. The blastocyst formation rates of 1PN and 2PN embryos were 14.8% and 36.4%, respectively, in IVF cycles, and 6.6% and 34.0%, respectively, in ICSI cycles [32]. Thus, more 1PN embryos derived from ICSI cycles are discarded because of poor clinical application. The results from the present study were similar: 28.9% of 1PN embryos from IVF cycles developed into available blastocysts, while only 16.1% of 1PN embryos from ICSI cycles developed into available blastocysts. However, 1PN blastocysts from IVF and ICSI cycles resulted in a comparable CPR and LBR with 2PN blastocysts. Although the sample size was limited in the current study, it appeared that 1PN embryos reaching blastocyst stage from ICSI cycles could achieve similar clinical outcomes as blastocysts from IVF. Recently, Mateo et al. revealed that 1PN embryos from ICSI cycles presented differences in developmental capacity and morphokinetic behavior compared with 2PN embryos [33]. Blastocysts from 1PN embryos from ICSI cycles had a similar morphokinetic development to those from 2PN embryos. Blastocyst culture may effectively select normal 1PN embryos derived from ICSI cycles for transfer.

Previous studies have reported that the chromosomal euploid rates of 1PN zygotes from ICSI cycles were lower than those from IVF cycles [9-10]. However, other evidence indicates that 1PN blastocysts derived from ICSI cycles were mostly diploid. Bradley et al. analyzed 74 IVF and 32 ICSI 1PN blastocysts for PGT-A using comparative genomic hybridization or NGS [32]. Their results showed that the chromosomal euploid rates of 1PN blastocysts from IVF and ICSI did not differ significantly. The current study also found that the euploid rates of 1PN and 2PN blastocysts derived from ICSI cycles were not significantly different, consistent with past results [32, 34-35]. Importantly, all the euploid 1PN blastocysts were diploid. This observation further strengthens the potential use of 1PN blastocysts from ICSI cycles. The 1PN blastocysts from both IVF and ICSI cycles could be selected for transfer, similar to 2PN blastocysts.

This study has several limitations. First, this is a single-center study, and multiple center studies are needed to further investigate our findings. Second, the transfer of 0PN and 1PN embryos is uncommon, therefore, the total numbers of patients undergoing transfer of such embryos were limited and the safety of 0PN and 1PN blastocyst transfer still needs further exploration.

Overall, our results indicated that 0PN and 1PN blastocyst transfers can result in a similar CPR and LBR as 2PN blastocysts. This finding may increase future opportunities of obtaining pregnancy via assisted reproductive technology, because it suggests that it is possible to transfer 0PN and 1PN blastocysts for patients that do not produce or have difficulty providing 2PN embryos. The 0PN and 1PN blastocysts derived from ICSI cycles can be used the same as those from IVF cycles.

What does this study add to the clinical work

The 0PN and 1PN embryos reaching blastocyst stage from ICSI cycles could achieve similar clinical outcomes of frozen-thawed embryo transfers as blastocysts from IVF. This study further strengthens the potential use of 0PN and 1PN blastocysts from ICSI cycles.

Acknowledgments

We thank Liwen Bianji (Edanz) (www.liwenbianji.cn/) for editing the English text of a draft of this manuscript.

Funding

This work was supported by the "Pioneer" and "Leading Goose" R&D Program of Zhejiang (No. 2023C03034) and Medical health science and technology project in Zhejiang province (2022RC039).

Author contributions

All authors contributed to the study conception and design. Material preparation and data collection were performed by Xiaomei Tong, Jiamin Jin and Lingying Jiang. Data analysis was performed by Lu Fang. Procedures of oocyte pick-up, ICSI, embryo culture, blastocyst freezing and thawing and blastocyst biopsy were performed by Yamei Xue, Haiyan Zhu and Lingying Jiang. The first draft of the manuscript was written by Xiaomei Tong and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.

Ethics declarations

Conflict of Interest:

The authors declare that they have no conflict of interest.

Ethics approval

Ethical approval was obtained from the medical ethical committee of Sir Run Run Shaw Hospital, Zhejiang University School of Medicine.

Consent to participate

Informed consent was exempted as it was a retrospective study using already available data for clinical purposes

Consent to publish

Not applicable.

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Table 1 Available blastocyst formation from embryos with no (0PN), one (1PN) and two pronucleus (2PN) for IVF and ICSI cycles. Data are presented as n (%). For inter-group comparison of IVF and ICSI cycles, different superscripts in the same row indicate significant differences (P<0.05), and the same superscripts indicate no significant difference (P> 0.05). N/A, not applicable; D5+D6, day 5 and day 6.

	IVF			P value	ICSI			P value	P value (IVF vs. ICSI)
	0PN	1PN	2PN		0PN	1PN	2PN		0PN	1PN	2PN
No. of embryos cultured to blastocyst stage (n)	5708	1767	42955	N/A	1376	471	29311	N/A	N/A	N/A	N/A
Available blastocyst rate (n, %)	2725 (47.7%)^a	510 (28.9%)^b	22952 (53.4%)^c	0.000	441 (32.0%)^a	76 (16.1%)^b	14594 (49.8%)^c	0.000	0.000	0.000	0.000
Day 5 blastocysts (n, %)	2331 (85.5%)^a	359 (70.4%)^b	15980 (69.6%)^b	0.000	333 (75.5%)^a	36 (47.4%)^b	8987 (61.6%)^c	0.000	0.000	0.000	0.000
High-quality blastocysts (n, %) （D5+D6）	1528 (56.1%)^a	254 (49.8%)^b	10250 (44.7%)^b	0.000	252 (57.1%)^a	29 (38.2%)^b	6264 (42.9%)^b	0.000	0.674	0.058	0.001

Table 2 Table 2 Clinical outcomes of no (0PN), one (1PN) and two pronucleus (2PN) embryo-derived single frozen-thawed blastocyst transfers in IVF and ICSI cycles. Data are presented as mean ± standard deviation or n (%). N/A, not applicable.

	IVF			P value	ICSI			P value	P value (IVF vs. ICSI)
	0PN	1PN	2PN		0PN	1PN	2PN		0PN	1PN	2PN
No. Of transferred cycles	249	78	1366	N/A	41	14	540	N/A	N/A	N/A	N/A
Endometrial thickness (mm)	9.41±1.57	9.31±1.78	9.32±1.51	0.700	9.65±1.75	9.38±2.54	9.30±1.56	0.415	0.384	0.902	0.792
Clinical pregnancy (n, %)	115 (46.2%)	41 (52.6%)	692 (50.7%)	0.389	19 (46.3%)	6 (42.9%)	266 (49.2%)	0.844	0.985	0.503	0.582
Miscarriage (n, %)	23 (20.0%)	11 (26.8%)	112 (16.2%)	0.150	3 (15.8%)	0 (0)	27 (10.2%)	0.518	0.667	0.147	0.018
Live birth (n, %)	90 (36.1%)	29 (37.2%)	578 (42.3%)	0.146	16/41 (39%)	6/14 (42.9%)	237/540 (43.9%)	0.831	0.723	0.687	0.531

Table 3 The chromosomal aneuploidies of no pronucleus (0PN), one pronucleus (1PN) and two pronuclear (2PN) embryo-derived blastocysts from ICSI cycles. Data are presented as n (%). N/A, not applicable.

	0PN	1PN	2PN	P value
No. of blastocysts for PGT-A	64	20	2009	N/A
Whole-genome amplification rate (%)	100%	100%	99.2%	0.714
Euploid blastocysts (n, %)	40(62.5%)	12(60%)	1093(54.4%)	0.393
Aneuploid blastocysts (n, %)	23(35.9%)	8(40.0%)	749(37.3%)	0.945
Mosaic blastocysts (n, %)	1(1.6%)	0	151(7.5%)	0.089

Clinical outcomes of frozen-thawed blastocysts from zygotes with no or one pronucleus for in vitro fertilization and intracytoplasmic sperm injection cycles

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Abstract

Introduction

Methods

Results

Discussion

Declarations

References

Tables

Status:

Journal Publication

Version 1