2PN Zygotes Research Articles

High proportion of zygotes with multiple pronuclei increase the embryo multinucleation rate during conventional IVF

The incidence of multiple pronuclei (≥ 3PN) zygotes and blastomere multinucleation was found to be elevated in the presence of increased estradiol (E2) levels and a greater number of retrieved oocytes. This implies a potential link between the incidence of multinucleation at the two-cell stage (MN2) and a higher proportion of ≥ 3PN zygotes. We aimed to investigate the effect of high proportion of ≥ 3PN zygotes on MN2 incidence during conventional in vitro fertilization (C-IVF) by using time-lapse monitoring. This study included 1195 patients from January 2020 to December 2022. The patients were categorized into three groups: Group 1 comprised patients with no ≥ 3PN zygotes (n = 422), Group 2 included those with 0–25% ≥3PN zygotes (n = 617), and Group 3 consisted of patients with more than 25% ≥3PN zygotes (n = 156). The MN2 rate, types of MN2 and clinical outcomes were compared among the three groups. Our data indicated that the MN2 rate was significantly lower in groups 1 and 2 compared to group 3 (18.33 versus 25.62%; p < 0.001 and 19.45 versus 25.62%; p < 0.001). The MN2 embryos exhibited similar rates of high-quality embryos (42.27 versus 43.50 versus 40.67%; p = 0.401) and available embryos (84.96 versus 84.04 versus 83.21%; p = 0.460) rates among the three groups. There were no significant differences in the proportion of MN2 with different types among the three groups (p > 0.05). The embryos displaying binucleated at the two-cell stage in one blastomere (2BI1) and true multinucleated at the two-cell stage in one blastomere (2MULTI1) showed significantly higher blastocyst formation rates compared to embryos exhibiting true multinucleated at the two-cell stage in both blastomeres (2MULTI2) (59.50 versus 45.40%; p < 0.001 and 59.40 versus 45.40%; p < 0.001). In conclusion, the occurrence of MN2 events might be associated with high proportion of ≥ 3PN zygotes incidence. The types of MN2 had significant reference value when selecting embryos for transfer during the cleavage stage.

Birth of Thirty-Two Healthy Babies Following Transfer of Fresh and Frozen-Thawed Embryos Derived from Monopronuclear Zygotes: A Retrospective Study.

Background and Objectives: Fertilized zygotes normally display two pronuclei (PN), but abnormal fertilization patterns (0, 1 or >2PN) are observed daily in IVF labs. Multiple PN zygotes (>2) are generally discarded due to an increased risk of aneuploidy. However, the decision to transfer or not transfer 1PN-derived embryos remains controversial. The aims of our study were to analyze the neonatal outcomes of fresh or frozen-thawed embryos derived from 1PN zygotes, and to evaluate the influence of the fertilization method. Materials and Methods: Data were retrospectively collected from cycles performed between January 2018 and December 2022. Fresh cycles were analyzed for the comparative fate of 1PN zygotes (n = 1234) following conventional in vitro fertilization (cIVF; n = 648) or intracytoplasmic sperm injection (ICSI; n = 586), as well as the results of the 64 transfers of 1PN-derived embryos (pregnancy rate (PR) and neonatal outcomes). This pregnancy follow-up was also applied to 167 transfers of frozen-thawed 1PN-derived embryos. Results: In fresh cycles, 46% of the 1PN zygotes in the cIVF group developed into embryos of sufficient quality to be transferred or frozen (day 3 or 5/6). This rate was lower in the fresh ICSI cycles (33%). Blastulation rate was also significantly higher in the cIVF group (44%) in comparison to the ICSI group (20%). The fresh single embryo transfers (32 per group) allowed seven pregnancies in the cIVF group (PR = 21.9%) as compared to four pregnancies in the ICSI group (PR = 12.5%). In the cIVF group, five deliveries of healthy newborns were achieved, but only one in the ICSI group. In frozen/thawed cycles, 36 pregnancies were obtained out of the 167 transfers. A non-significant difference was observed between embryos derived from cIVF cycles (PR = 26%) and ICSI cycles (PR = 16%) with 18 and 8 healthy babies born, respectively. Conclusions: We observed better outcomes for 1PN zygotes in cIVF cycles in comparison to ICSI cycles. Our center policy to transfer good-quality 1PN-derived embryos allowed the birth of 32 healthy babies.

Associations between neonicotinoid insecticide levels in follicular fluid and serum and reproductive outcomes among women undergoing assisted reproductive technology: An observational study

Neonicotinoid insecticides (NEOs) are a widely used type of insecticide found globally, leading to broad human exposure. However, there is limited research on how internal exposure levels of NEOs and their metabolites impact in vitro fertilization/intracytoplasmic sperm injection (IVF/ICSI) outcomes. A study was conducted at the Sixth Affiliated Hospital of Sun Yat-sen University between 2017 and 2020 involving 436 women undergoing IVF/ICSI treatment. Data on demographics and clinical history were collected from medical records. The concentrations of 11 NEOs and 4 NEO metabolites in follicular fluid and serum were measured using a salting-out assisted liquid–liquid extraction method and liquid chromatography–tandem mass spectrometry. Our findings indicated that NEOs were prevalent in women with infertility. One NEO metabolite, N-dm-ACE, was detected in all samples with median concentrations of 0.221 ng/mL in follicular fluid and 0.228 ng/mL in serum. The study showed a decrease in the number of retrieved oocytes, mature oocytes, 2 PN zygotes, and high-quality embryos as the number of exposed NEOs in follicular fluid increased. Women in the highest tertile of N-dm-ACE exposure had fewer mature oocytes, 2 PN zygotes, and lower oocyte maturity rates compared to those in the lowest tertile. The findings suggest that exposure to NEOs may negatively impact reproductive outcomes in IVF/ICSI pregnancies, particularly affecting oocyte retrieval and embryo quality. This study highlights the potential adverse effects of environmental NEO exposure on IVF/ICSI outcomes, emphasizing the importance of considering such exposures in preconception care.

Exposure of women undergoing in-vitro fertilization to per-and polyfluoroalkyl substances: Evidence on negative effects on fertilization and high-quality embryos

In April 2023, the World Health Organization (WHO) reported that 17.5% of the global adult population experience infertility. What may be the contribution of per-and polyfluoroalkyl (PFAS) to this global public health problem?This study explored the associations between in vitro fertilization (IVF) outcomes and plasma concentrations of individual PFAS and PFAS mixtures in women undergoing in vitro fertilization and embryo transfer (IVF-ET) and how these exposures might affect IVF outcomes. We analyzed 8 PFASs in plasma samples from women (N = 259) who underwent IVF treatment. In multivariable generalized linear mixed models, there were statistically significant associations of higher plasma concentrations of PFNA with reduced numbers of total retrieved oocytes [12.486 (95%CI: 0.446,25.418), p trend = 0.017], 2 PN zygotes [6.467(95%CI: 2.034,14.968), p trend = 0.007], and cleavage embryos [6.039(95%CI: 2.162,14.240), p trend = 0.008]. Similarly, there was a continuous decline in the numbers of retrieved 2 PN zygotes and cleavage embryos with increasing concentration of PFOS [6.467(95%CI: 2.034,14.968), p trend = 0.009 and 6.039(95%CI: 2.162,14.240), p trend = 0.031,respectively] and a negative association between PFHxS concentrations and clinical pregnancy during the initial cycles of frozen ET [0.525(95%CI:0.410,0.640), p trend = 0.021]. To investigate the joint effect of PFAS mixtures, a confounder-adjusted BKMR model analysis showed inverse relationship between PFAS mixtures and the number of high-quality embryos, 2 PN zygotes and cleavage embryos, to which the greatest contributors to the mixture effect are PFDeA and PFBS, respectively. It demonstrated that PFAS exposure might exert negative effects on oocyte yield, fertilization and high-quality embryo in women undergoing IVF. These findings suggest that exposure to PFAS may increase the risk of female infertility and further studies are needed to uncover the potential mechanisms underlying the reproductive effects associated with PFAS.

O-006 Oocyte factors as predictors of abnormal, mononucleated and trinucleated, zygotes and assessment of their morphokinetic differences

Abstract Study question Are there oocyte factors associated with the occurrence of mononucleated and trinucleated zygotes after ICSI? Does morphokinetics differ in embryos from normal and abnormal zygotes? Summary answer Formation of 1PN zygotes relates to vitrificated and donor oocytes. 3PN links to age and own oocytes. 1PN embryo morphokinetics are slower than 2PN, 3PN. What is known already In assisted reproduction, after monitoring fertilization, it is common to observe zygotes with one or more than 2 pronuclei. These abnormal zygotes are often discarded due to potential chromosomal abnormalities. The formation of these anomalous zygotes can be attributed to various causes, such as non-activation of the male pronucleus, non-extrusion of the second polar body, etc. Simultaneously, limited data exist on the morphokinetics of abnormal zygotes, explored in a few studies. The embryonic kinetics of 1PN zygotes seem slower than those of 2PN and 3PN zygotes, but further research is needed to confirm this aspect. Study design, size, duration Retrospective multicentre study involved time-lapse monitoring of 28,218 ICSI zygotes (26,252 2PN, 1,190 1PN, and 776 &amp;gt;3PN) generated between January 2019 and December 2023. The study includes donor and patient oocytes with various infertility diagnoses, whether fresh or vitrified. The comparison of embryonic morphokinetics from abnormal/normal zygotes was conducted on 254 embryos from a single centre, with 45 originating from 1PN zygotes, 198 from 2PN zygotes, and 11 from zygotes with 3 or more pronuclei. Participants/materials, setting, methods The study involved 1,053 couples treated in four different centres. Uniform culture media were used, and oocytes were cultivated in time-lapse technology-equipped incubators across all centres. A logistic regression analysis aimed to identify oocyte factors linked to an abnormal pronuclei count. Embryonic morphokinetics were analysed using the CHLOE artificial intelligence system (Fairtility, Israel). Evaluated parameters included: tPNa, tPNf, t2, t3, t4, t5, t6, t7, t8, t9+, tM, tSB, tB, and tEB. Main results and the role of chance Donor oocytes exhibited a 1.32 times higher risk of producing 1PN zygotes than 2PN or 3PN zygotes (p &amp;lt; 0.001). Similarly, vitrified oocytes had a 1.63-fold higher risk of generating 1PN zygotes than 2PN or 3PN zygotes (p &amp;lt; 0.001). Additionally, we found that both the use of own oocytes and advanced age were significantly associated with 3PN zygotes (p &amp;lt; 0.001). Regarding age, the risk increases by 10% for each additional year. The average durations for all morphokinetic parameters of 2PN and 3PN embryos were similar. In contrast, all events, except t3 (p = 0.2), occurred more slowly in embryos derived from 1PN zygotes compared to embryos from 2PN zygotes. Thus, the disappearance of pronuclei had a p-value &amp;lt;0.005, and from t4 to tEB, the p-value was &amp;lt;0.001. When comparing embryos from 1PN and 3PN zygotes, only tPNa, t4, t8, t9+, and tM were significantly different. If only zygotes that reached the blastocyst stage were used for analysis, the results were less conclusive due to a significantly reduced number of cases. Limitations, reasons for caution Embryos were cultured in an EmbryoScope incubator at three centres, while Geri was used in the fourth. Wider implications of the findings Using donor or vitrified oocytes may lead to an increased occurrence of zygotes with 1PN, as will occur with 3PN when patient or aged oocytes are used. The kinetics of embryos from 3PN oocytes resemble those from 2PN, while those from 1PN zygotes exhibit slower kinetics. Trial registration number not applicable

P-568 Correlation of ploidy analysis in two types of zygotes with 3 pronuclei (3PN) observed: same size 3PNs and one micronucleus 3PNs

Abstract Study question Do all zygotes with three observed pronuclei have an actual triploid chromosomal complement? Summary answer Not all zygotes with 3 pronuclei observed are triploid, most of embryos with two even pronuclei and one smaller micronucleus are diploid. What is known already In the in vitro fertilization (IVF) setting, normal oocyte fertilisation is commonly confirmed 16–19 hours after conventional insemination or intracytoplasmic sperm injection (ICSI) with the appearance of two pronuclei (2PN). The presence three pronuclei (3PN) shows an abnormal oocyte fertilization. The incidence of 3PN zygotes in IVF is between 5.0%- 8.1% in conventional insemination and between 2.0%- 6.2% in ICSI. 3PN fertilized embryos are commonly discarded due to the suspected increased risk of a triploid chromosomal complement. However, no definitive genetic evidence has been obtained to show that all 3PN zygotes are chromosomally abnormal. Study design, size, duration An Ethics Committee for Investigation accredited this prospective observational study performed in a private centre between June 2023 and January 2024. 120 3PN zygotes from 113 cycles were cultivated until blastocyst stage. 46 were biopsied to clarify ploidy and euploidy status. 3PNs were divided into 2 groups based on the size of their pronuclei: Micro Triploid (MT) (smallest pronucleus &amp;lt;70% of the diameter of the largest pronucleus) and Pure Triploid (PT) (smallest &amp;gt;70% of the largest). Participants/materials, setting, methods Patients with 1 or more 3PN embryo were offered to participate, culturing and assessing their 3PNs. If 3PNs reached blastocyst stage, they were biopsied and analysed using Next-Generation Sequencing strategy with Single Nucleotide Polymorphisms technology without the need for parental samples, to assess ploidy and aneuploidy status. Measures of the pronuclei size were taken to sort the 3PNs in both groups. Comparisons between these groups regarding blastocyst rates, ploidy status and aneuploidy rates were assessed. Main results and the role of chance 120 3PNs zygotes were identified in 1054 cycles, representing 1.9% of all fertilized embryos. The blastocyst rate (BR) for overall 3PNs was 38.3%, while the BR for 2PNs on those 113 cycles was 58.3%, showing significant differences (p &amp;lt; 0.001). 3PN zygotes were classified into the MT and PT groups described above. The MT group had 40.4% BR, and the PT group had 37.0% BR. No significant differences were found. Of the 46 biopsied embryos, 37 were informative (80.4%), 17 for the MT group and 20 for the PT group. The diploidy rate for the overall 3PNs was 37.8% (n = 14). The MT group had 82.4% (n = 14) of diploidy rate, while the PT group had 0% diploid rate (n = 0), showing significant differences between them. (p &amp;lt; 0.001). The overall euploidy rate for 3PN zygotes was 28.6% (n = 12) (36.2 mean age), which was similar to the euploidy rate (30.3%) for our overall 2PNs biopsied during 2023 (38.2 mean age). No differences found. Out of the 14 diploid 3PNs, 28.6% (n = 4) were euploid, showing no differences with our 2023 euploidy rate. Two diploid 3PNs were mosaic, resulting in a total of 6 embryos that could potentially be transferred. Limitations, reasons for caution This study is limited by the number of triploid embryos recruited. As blastocyst rate for 3PNs population is lower than observed in the 2PN population the number of biopsied embryos is reduced. Euploidy rates for 3PNs and 2PNs are very similar, but the mean age differs by 2 years. Wider implications of the findings It is very important to distinguish between both types of 3PNs observed: PT are not worthy to keep and analyse, while MT embryos are very likely to be diploid, and it is worthwhile to culture and analyse them. Patients with few embryos available in particular could benefit from this study. Trial registration number not applicable

O-065 PGT-A reveals triploid embryos among normally fertilized oocytes: Frequency and blastocyst features

Abstract Study question Is there any embryo parameter that could differentiate 2 pronucleus (PN) triploid from 2 PN euploid embryos? Summary answer Accuracy in the fertilization check is still necessary as 2 PN but triploid embryos are due mostly to retention of the second polar body (PB). What is known already Since blastocyst culture has evolved and the development of genetic technologies enables to know the karyotipe of the embryos, the concept of normal fertilization is being revised. Commonly, correct fertilization is denoted by the appearance of 2 PN 16-18h after insemination. Deviation from 2PN is considered evidence of abnormal fertilization and ploidy anomalies. However, not all oocytes exhibiting 2 PN are euploids. After performing NGS in trophectoderm biopsies for preimplantation genetic testing for aneuploidy (PGT-A), a percentage of embryos are categorized as triploids. Study design, size, duration Retrospective study including 1862 cycles from 1605 patients aged 18-45 years old who underwent PGT-A with NGS of trophectoderm biopsies in our IVF center from 2019 to 2023. From 6526 analyzed blastocysts in an independent Genetics laboratory, 59 were categorized as triploids. These 59 blastocysts were obtained from 57 PGT-A cycles and belonged to 54 women. In total, these patients had 266 biopsied blastocysts. From the remaining 207 embryos, 42 were called as euploids. Participants/materials, setting, methods In embryos cultured in time-lapse, fertilization was re-evaluated to check the appearance of 2PN and the second PB extrusion. Genetic triploid embryos were analysed in relation to the day of biopsy (day 5 vs day 6), the trophectoderm quality and the timing of the first cell divisions (time of cleavage from 2 to 6 cells: t2, t3, t4, t5, t6). Categorical variables are shown as rate. T-student tests were performed. P-value&amp;lt;0.05 was considered statistically significant. Main results and the role of chance The re-evaluation of the fertilization of the 59 triploids categorized as 2 PN revealed 50 oocytes 2 PN, 8 embryos 3 PN and 1 embryo 2 PN + 1 micronuclei configuration. Therefore, the prevalence of real triploidy in 2PN embryos was 0,8% (n = 50/6526; CI95%:0,6-1,0%). These data showed that 2PN zygotes may give rise to embryos with altered ploidy. From 27 triploid 2PN embryos was possible to access to the whole development time-lapse images. Just 33,3% of triploid embryos exhibited the extrusion of the second PB, while 100% of euploid sibling embryos showed this extrusion (N = 9/27 vs N = 24/24,;p&amp;lt;0,005). Comparing 2 PN triploid and the rest of the embryos from the same cohorts, there were no differences regarding the day of biopsy (N = 31/176 day 5 biopsy, 17.6% vs N = 19/81 day 6 biopsy, 23.5%; p &amp;gt; 0.05). On the contrary, a worse blastocyst quality showed a significant relation with triploidy (N = 23/161,14,2% of good quality embryos are triploid vs N = 27/96, 38,6% of fair quality embryos are triploids; p:0.006).Embryos cleavage time points up to 6-cell stage did not show any significative difference between triploid and euploid embryos (t2: 27.36h vs 25.8h; t3: 38.5h vs 35.6h; t4 39.4h vs 37.2h; t5 52.8h vs 47.0h; t6 56.2h vs 52.1h; all comparisons p &amp;gt; 0.05). Limitations, reasons for caution The incidence of triplody from 2PN zygotes may vary among IVF laboratory settings. The small sample size may be responsible of no difference in morphokinetics. Wider implications of the findings Fertilization check should persist in IVF procedures. Special consideration should be given to the second PB extrusion as triploidy is the most common form of aneuploidy observed in the first trimester miscarriages. The role of diploid spermatozoa, the size of the pronucleus and location should be explored in further studies. Trial registration number NOT APPLICABLE

P-142 The developmental time to reach the blastocyst stage in the human is an indicator of diploid mono-pronuclear embryos

Abstract Study question Is it possible to determine diploid mono-pronuclear (1PN) human embryos by the speed of development to blastocyst formation? Summary answer The developmental time to reach the blastocyst stage is a discrimination indicator for diploid 1PN embryos. What is known already We have previously reported that 80.7% of 1PN zygotes had a biparental chromosome using a Live-Cell imaging technique, and some of these developed to the blastocyst stage (Tokoro et al. ASRM 2013). Furthermore, we have reported that these blastocysts can result in a healthy live birth (Tsuji et al. ASRM 2020), and there was no effect on growth up to 5 years of age (Tsuji et al. ASRM 2023). On the other hand, it was also shown that a few of the 1PN embryos that developed into blastocysts were haploid embryos. Study design, size, duration This was a retrospective study that included 28 blastocysts derived from a 1PN zygote. The time period was 72 months (January 2017 to December 2022). These embryos are used for research following obtaining informed consent. Participants/materials, setting, methods The frequency rate of diploid embryos was compared from time of PN fading to blastocyst formation (Gardner’s classification: ≥ expansion grade 3). Two development time range groups were examined and designated: fast-developing embryos (65.7-78.7h) and slow-developing embryos (116.8-139.7h). Ploidy status was determined using whole blastocyst cells and NGS-based SNP analysis. Statistical significance was determined using Fisher’s exact test (level of P&amp;lt;0.05). Main results and the role of chance There was no significant difference in the average age (years) of the two development time groups (fast-developing embryos group; 35.5 +/- 5.5, slow-developing embryos group; 34.1 +/- 2.8). The frequency of diploid status in 1PN embryos with fast developing embryos was 100% (14/14), and in 1PN embryos designated as having slow development, the frequency of diploid embryos was only 14.3% (2/14), haploid embryos 71.4% (10/14), and other was 14.3% (2/14). As a result, the diploid embryo rate was significantly higher in fast-developing embryos than in slow-developing embryos (P&amp;lt;0.001). Limitations, reasons for caution This study was limited to extremely fast or slow-developing embryos to see trends. It is important to study the threshold time between diploidy and haploidy in 1PN embryos. Wider implications of the findings These results confirmed that fast developing 1PN embryos have a high diploid rate and can be used as transferable embryos in addition to 2PN embryos. Conversely, slow developing 1PN embryos should be considered to have a lower priority for transfer because of the high rate of haploid embryos. Trial registration number not applicable

P-173 An exploratory clinical study in patients with recurrent ART-failure due to severe fragmentation during early-cleavage: the effects of artificial removal of zona-pellucida at the pronuclear-stage

Abstract Study question Can the artificial removal of the zona pellucida (ZP) at the pronuclear (PN)-stage prevent severe fragmentation in early-cleavage embryos and improve good-quality blastocyst development rate? Summary answer The artificial removal of the ZP at the 2PN-stage could prevent significantly severe fragmentation in early-cleavage embryos and improved the good-quality blastocyst development rate. What is known already Recent analysis using time-lapse culture demonstrated that fragments during early-cleavage occur at the site of the perivitelline threads between the ZP and the embryo membrane (Reprod Biomed Online 2017;35:640-5,646-56). Based on these findings we conducted a pilot study on embryonic development after ZP-removal using abnormally-fertilized oocytes (J Assist Reprod Gene2020;37:1349-54). We showed that the inter-blastomere adhesion was maintained and fragmentation was significantly lower in the ZP-free embryos than in the ZP-intact embryos. Therefore, we attempted to apply this method to patients who have given full consent and had repeatedly failed to conceive due to severe fragmentation in early cleavage-stage embryos. Study design, size, duration This is a single-centre retrospective exploratory study of 34 patients that were scheduled to undergo ART-treatment in our clinic between February 2020 and January 2021. They had been previously treated in our facilities (January 2016 to January 2020) with less than 10% morphologically good-quality blastocysts due to severe fragmentation. None of these patients became pregnant during their initial treatments. Totally, 173 2PN-zygotes were obtained, with 101 allocated to ZP-free group and 72 to ZP-intact group. Participants/materials, setting, methods Normally-fertilized oocytes were placed in 0.125M sucrose-containing HEPES-medium (approximately 5min) and cultured under: 1) ZP-free, in which the ZP was completely removed from the ooplasm by laser-irradiation and a medium-blowing method with a biopsy-pipette, or 2) ZP-intact. ZP-free and ZP-intact zygotes were cultured in sequential medium. Embryos were either freshly transferred on Day-2(ZP-intact group only, according to patients’ wishes), or Day-5/6, or they were cryopreserved on Day-5/6/7 for future embryo transfer cycles for both groups. Main results and the role of chance Even allocation of the normally fertilised oocytes to the two groups was not possible, due to the patients’ request to include more zygotes in the ZP-free group. Therefore, no specific criteria were set other than the rule that all the normally fertilised oocytes of each patient must be allocated into both groups at the time of allocation. As a result, each patient had 2PN zygotes assigned to both ZP-free and ZP-intact groups. Rates of good-quality embryos, blastocyst development, morphologically good-quality blastocyst development and cryopreservation demonstrated significant improvement (all P &amp;lt; 0.01) in the ZP-free group compared to the ZP-intact group. Pregnancy rates in ZP-intact group were 5.9% per patient and 5.3% per embryo transferred, while in ZP-free group 37.5% per patient and 24.3% per embryo transferred. Our results demonstrate that in difficult-to-treat cases where embryos with good morphology cannot be obtained due to excessive fragmentation at the early-cleavage, embryonic development can be significantly improved by artificial removal of ZP at the 2PN-stage. We believe that the ZP-free method has opened a new door in assisted reproductive medicine and can provide the opportunity to obtain blastocysts with good-morphology from an embryo that would probably not have become a blastocyst otherwise. Limitations, reasons for caution A comparative analysis between the two groups was not possible due to the small number of patients, the inclusion of fresh embryos as well as frozen embryos, and the non-randomized data. The imbalance was attributed to the fact that patients wanted us to allocate more oocytes to the ZP-free group. Wider implications of the findings The development of treatment methods based on new ideas should always take into consideration the possibility of associated risks. In the future, we will examine the criteria for selecting patients for whom this method is useful, as well as verify its safety and effectiveness by conducting a prospective randomized analysis. Trial registration number not applicable

The Blastulation Rate Is Negatively Associated With Euploid Rate

ObjectivesTo study the association between the blastulation rate, the presence of 1 pronucleus (1PN) zygotes, and the ploidy of the cohort of blastocysts. MethodsA cross-sectional study using the existing databases of 2 university fertility centres in Canada. We included 345 cycles from 235 couples who underwent next-generation sequencing preimplantation genetic testing for the detection of aneuploidy in the study. ResultsA total of 1456 blastocysts were biopsied. In multivariate analysis, only female age and the number of 1PN/2PN embryos showed a negative association with euploid ratio. Surprisingly, when the analysis was limited to cycles with no delayed blastulation, the blastulation rate was also negatively associated with the euploid ratio. ConclusionsThis study sheds some light on the stages of early embryo development. Further study on the mechanisms governing embryo development and the different cell cycle checkpoints in embryo development is warranted.

High coverage of targeted lipidomics revealed lipid changes in the follicular fluid of patients with insulin-resistant polycystic ovary syndrome and a positive correlation between plasmalogens and oocyte quality.

Polycystic ovary syndrome with insulin resistance (PCOS-IR) is the most common endocrine and metabolic disease in women of reproductive age, and low fertility in PCOS patients may be associated with oocyte quality; however, the molecular mechanism through which PCOS-IR affects oocyte quality remains unknown. A total of 22 women with PCOS-IR and 23 women without polycystic ovary syndrome (control) who underwent in vitro fertilization and embryo transfer were recruited, and clinical information pertaining to oocyte quality was analyzed. Lipid components of follicular fluid (FF) were detected using high-coverage targeted lipidomics, which identified 344 lipid species belonging to 19 lipid classes. The exact lipid species associated with oocyte quality were identified. The number (rate) of two pronuclear (2PN) zygotes, the number (rate) of 2PN cleaved embryos, and the number of high-quality embryos were significantly lower in the PCOS-IR group. A total of 19 individual lipid classes and 344 lipid species were identified and quantified. The concentrations of the 19 lipid species in the normal follicular fluid (control) ranged between 10-3 mol/L and 10-9 mol/L. In addition, 39 lipid species were significantly reduced in the PCOS-IR group, among which plasmalogens were positively correlated with oocyte quality. This study measured the levels of various lipids in follicular fluid, identified a significantly altered lipid profile in the FF of PCOS-IR patients, and established a correlation between poor oocyte quality and plasmalogens in PCOS-IR patients. These findings have contributed to the development of plasmalogen replacement therapy to enhance oocyte quality and have improved culture medium formulations for oocyte in vitro maturation (IVM).

Nonpronuclear- and monopronuclear-derived blastocysts do not impair subsequent perinatal and maternal outcomes.

The routine clinical practice is to prioritize the transfer of blastocysts derived from 2PN embryos if they are available. For women who only have blastocysts resulting from 0PN and 1PN embryos, whether to transfer these embryos or discard them has been an ongoing debate over the years. To investigate the perinatal and obstetric outcomes following the transfer of vitrified-warmed single blastocysts derived from 0PN and 1PN zygotes. Retrospective cohort study. University-affiliated IVF center. This study included singletons born to women who had undergone 0PN and 1PN vitrified-warmed single blastocyst transfers, compared to those resulting from 2PN vitrified-warmed single blastocyst transfers from 2012 to 2020. None. Perinatal and obstetric outcomes. A total of 7,284 women were included in the final analysis. Of these, 386, 316, and 6582 cycles resulted from 0PN-, 1PN-, and 2PN-derived blastocysts transfer, respectively. The rates of clinical pregnancy, miscarriage, and live birth were similar across the study cohorts in both unadjusted and adjusted analyses. When comparing the 0PN and 2PN groups, no differences were found in birth outcomes after adjusting for confounders. Similarly, maternal complications and mode of delivery were comparable between these two study cohorts. Birth parameters were also similar between the 1PN and 2PN blastocyst groups, except for more male births in the 1PN cohort. Furthermore, a comparison between the 1PN and 2PN groups did not reveal any significant differences in maternal outcomes. The current study showed that the transfer of 0PN and 1PN blastocysts did not compromise reproductive outcomes or increase maternal and perinatal complications. This information is valuable for clinicians to counsel couples effectively and guide them in making informed decisions.

Outcomes and the effect of PGT-M in women with hormone-related hereditary tumor syndrome.

To review the outcome of PGT-M in hormone-related hereditary tumor syndrome and evaluate the effect of ovarian induction on tumor growth in those patients. Medical records of PGT-M were retrospectively analyzed in patients with hormone-related heritage tumors in our reproductive center. A total of eleven women with hereditary breast and ovarian cancer (HBOC) (including BRCA1/2 mutation carriers), and Lynch syndrome (including MMR gene mutation carriers) were included. Thirteen IVF/PGT-M cycles were performed. Eleven for PGT-M and two for fertility preservation. The ovulation protocol, numbers of oocytes retrieved and two pronuclei (2PN) zygotes, PGT-M results, and clinical outcomes were analyzed. Tumor progression was also estimated by comparing transvaginal ultrasound (TVS), MR, CT, or colonoscopy according to the follow-up requirements of different tumors. Eleven IVF/PGT-M cycles were performed with an antagonist protocol; Two cycles were performed with a mild stimulation protocol. The total dose of gonadotropin (Gn) was 1827 IU per patient (range from 1200 to 2625 IU). The median number of oocytes retrieved was 13 (range from 4 to 30), and the median number of 2PN zygotes was 8 (range from 2 to 16). A total of 32 embryos underwent PGT-M, and 9 (28.1%) embryos were suitable for transfer. Six transfer cycles were performed, and 5 cycles got clinical pregnancy (83%) with five newborns (83%). The follow-up examinations conducted 10-18 months after PGT-M/delivery revealed no new lesions or tumor progression. PGT-M results can provide important information for improving the consultation of hormone-related heritage tumor patients regarding their fertility preservation and reproductive options. Ovarian induction for women with hormone-related hereditary tumor syndrome is not associated with tumor progression.

Evaluating the developmental potential of 2.1PN-derived embryos and associated chromosomal analysis.

Zygotes with 2.1 pronuclei (2.1PN) present with two normal-sized pronuclei, and an additional smaller pronucleus, that is approximately smaller thantwo thirds the size of a normal pronucleus. It remains unclear whether the additional pronucleus causes embryonic chromosome abnormalities. In the majority of cases, in vitro fertilization (IVF) clinics discarded 2.1PN zygotes. Thus, the present study aimed to evaluate the developmental potential and value of 2.1PN zygotes. 2.1PN-derived embryos from 164 patients who underwent IVF or intracytoplasmic sperm injection (ICSI) treatment between January 2021 and December 2022 were included in the present study. All embryos were monitored using a time-lapse system, and blastocyst formation was used to assess 2.1PN-derived embryo developmental potential. The blastocyst formation was quantified using generalized estimating equations, and chromosome euploidy was analyzed using next-generation sequencing (NGS). In addition, the potential association between age and occurrence of 2.1PN zygotes was determined. The present study demonstrated that numerous 2.1PN zygotes developed into blastocysts. Early cleavage patterns and embryo quality on Day 3 were the independent predictors for the blastocyst formation of 2.1PN-derived embryos. The 2.1PN zygotes displayed a comparable developmental potential compared to 2PN zygotes in advanced age patients (≥ 38). Moreover, there was a tendency that 2.1PN-derived blastocysts showed a similar euploidy rate compared to 2PN-derived blastocysts. Clinicians should consider using 2.1PN-derived euploid embryos for transfer after preimplantation genetic testing in the absence of available 2PN embryo cycles. 2.1PN-derived embryos could be a candidate, particularly beneficial for patients at advanced age.

The utility of human two plus one small pronucleated zygotes (2.1PN) based on clinical outcomes and the focused ploidy analysis

PurposeAre human embryos arising from two plus one small pronucleated zygotes, called 2.1 pronuclei (PN), clinically useful?MethodsIn a retrospective embryo cohort study and prospective experimental study, a total of 287 cycles in which at least one 2.1PN was identified in the fertilization check were included. Embryonic development and clinical outcome were compared for the 1395 2PN zygotes and 304 2.1PN zygotes that were siblings. All embryos were individually cultured in time-lapse systems. Twenty-five 2.1PN-derived blastocysts, donated for research, were used in focused single-nucleotide variant ploidy analysis to identify the distribution pattern of heterozygosity.ResultsThe average diameter of PN was 24.9 ± 2.4 µm for large PN and 10.2 ± 2.4 µm for small PN; 79.9% of small PN was derived from female pronuclei. Blastocyst formation rate and good-quality blastocyst rate were significantly lower with 2.1PN embryos than with 2PN embryos (40.0% vs. 57.7%, 21.4% vs. 33.5%, respectively). A total of 13 embryos derived from 2.1PN were transferred, and three healthy babies were born. In ploidy constitutions of trophectoderm (TE), 2.1PN-derived blastocyst TE was shown to be mostly diploid (95.8%, 23/24), and only one blastocyst showed triploid.ConclusionsIt was suggested that 2.1PN embryos have lower embryonic developmental potential than 2PN embryos, but most of the 2.1PN were diploid, indicating that they are likely to be clinically usable. It is recommended to perform embryo transfer following a combination of PGT-A and ploidy analysis.

Developmental potential of non- and mono-pronuclear zygotes and associated clinical outcomes in IVF cycles.

This study aims to evaluate the developmental potential of 0PN, 1PN, and 2PN zygotes in IVF cycles and compare their clinical outcomes. We conducted a retrospective cohort study involving IVF patients. Blastocyst formation rates were assessed with 0PN, 1PN, and 2PN zygotes. Subsequently, we collected clinical outcome data following the transfer of these zygotes. The overall blastulation rate was similar between 0PN (29.6%) and 2PN (32.1%) zygotes, but 1PN zygotes exhibited a significantly lower blastulation rate (17.0%) compared to both 0PN and 2PN zygotes. Similarly, the overall rate of good-quality blastulation was comparable between 0PN (15.3%) and 2PN (17.5%) zygotes, while 1PN zygotes showed a significantly lower rate (7.0%) compared to both 0PN and 2PN. Clinical pregnancy, ectopic pregnancy, implantation, and live birth rates were similar among single blastocyst frozen embryo transfers (FET) of 0PN, 1PN, and 2PN. Additionally, no significant differences were observed between single- and double-blastocyst FET of 0PN and 2PN. Our findings suggest that 0PN and 2PN zygotes have comparable developmental potential, while 1PN embryos exhibit lower developmental potential. Blastocyst FET outcomes appear similar among 0PN, 1PN, and 2PN zygotes.

Associations between Phthalate Metabolite Concentrations in Follicular Fluid and Reproductive Outcomes among Women Undergoing in Vitro Fertilization/Intracytoplasmic Sperm Injection Treatment.

Phthalates have been reported to impair fertility in various studies. However, evidence exploring the associations between phthalate metabolites in follicular fluid (FF) and reproductive outcomes is lacking. To investigate the associations between phthalate metabolite concentrations in FF and in vitro fertilization/intracytoplasmic sperm injection (IVF/ICSI) outcomes among women recruited from a fertility clinic. We included 641 women undergoing IVF/ICSI treatment from December 2018 to January 2020. The levels of eight phthalate metabolites, including monoethyl phthalate (MEP), mono-isobutyl phthalate (MiBP), mono--butyl phthalate (MBP), monobenzyl phthalate (MBzP), mono(2-ethylhexyl) phthalate (MEHP), mono(2-ethyl-5-hydroxyhexyl) phthalate (MEHHP), mono(2-ethyl-5-oxohexyl) phthalate (MEOHP), and mono(2-ethyl-5-carboxypentyl) phthalate (MECPP), were quantified in FF collected on the oocyte retrieval day. Associations between quartiles of individual phthalate metabolite concentrations and nine IVF/ICSI outcomes, including oocyte yield, mature oocyte number, two distinct pronuclei (2PN) zygote number, fertilization rate, blastocyst formation rate, implantation, clinical pregnancy, miscarriage, and live birth, were estimated with generalized linear models. The effects of phthalate mixtures on IVF/ICSI outcomes were assessed using Bayesian kernel machine regression (BKMR) models. After adjusting for relevant confounders, elevated quartiles of MBzP, MEHHP, and MEHP in FF were inversely associated with the numbers of retrieved oocytes, mature oocytes, and 2PN zygotes (all for trends ). In comparison with the lowest quartile, the highest quartile of molar sum of di(2-ethylhexyl) phthalate metabolites () was associated with a reduction of 9.1% [95% confidence interval (CI): , ] and 10.3% (95% CI: , ) in yielded oocyte and mature oocyte numbers, respectively. Furthermore, the BKMR models revealed inverse associations between phthalate mixtures and the numbers of retrieved oocytes and mature oocytes. We generally found null results for implantation, clinical pregnancy, miscarriage, and live birth. Certain phthalate metabolites in FF are inversely associated with the numbers of retrieved oocytes, mature oocytes, and 2PN zygotes among women undergoing IVF/ICSI treatment. https://doi.org/10.1289/EHP11998.

Original delayed-start ovarian stimulation protocol with a gonadotropin-releasing hormone antagonist, medroxyprogesterone acetate, and high-dose gonadotropin for poor responders and patients with poor-quality embryos.

The delayed-start gonadotropin-releasing hormone antagonist protocol seems effective for patients who are poor ovarian responders, but there are insufficient data on whether it is also effective for patients with poor-quality embryos and low rates of good blastocyst formation. Specifically, the effectiveness of delayed-start gonadotropin-releasing hormone antagonists with progesterone has not been adequately investigated. Therefore, we compared the efficacy of the original delayed-start gonadotropin-releasing hormone antagonist protocol using medroxyprogesterone acetate (MPA) and high-dose gonadotropin in patients with poor ovarian response. Overall, 156 patients with recurrent assisted reproductive technology failure who underwent the original protocol were included. They received cetrorelix acetate (3 mg) and MPA (10 mg) on cycle day 3, and high-dose gonadotropin was initiated on day 11. When the leading follicle reached 14mm, ganirelix acetate (0.25 mg) was administered until the trigger day. The number of oocytes retrieved, metaphase II (MII) oocytes, two pronuclear (2PN) zygotes, and good blastocysts and live birth rates were compared between the previous (Cycle A) and original (Cycle B) cycles in three groups (Group A, all patients; Group B, poor responders; and Group C, patients with poor-quality embryos). In Group A (n=156), the number of MII oocytes (3.6 ± 3.3 versus 4.5 ± 3.6), 2PN zygotes (2.8 ± 2.9 versus 3.8 ± 3.1), good blastocysts (0.5 ± 0.9 versus 1.2 ± 1.6), and live birth rates (0.6 versus 24.4) significantly increased in Cycle B. Similar results were obtained in Group B (n=83; 2PN zygotes [1.7 ± 1.7 versus 2.3 ± 1.8], good blastocysts [0.4 ± 0.7 versus 0.9 ± 1.3], live birth rates [0 versus 18.1]) and Group C (n=73; MII oocytes [5.1 ± 3.8 versus 6.6 ± 4.0], 2PN zygotes [4.0 ± 3.4 versus 5.4 ± 3.4], good blastocysts [0.7 ± 1.1 versus 1.6 ± 1.9], and live birth rates [1.4 versus 31.5]). This original protocol increased the number of MII oocytes retrieved, 2PN zygotes, good blastocysts, and live birth rates in both poor responders and in patients with poor-quality embryos.

Effect of ejaculatory abstinence period on fertilization and clinical outcomes in ICSI cycles: a retrospective analysis

Does ejaculatory abstinence impact fertilization outcomes in intracytoplasmic sperm injection (ICSI) cycles in infertile couples? This single-centre retrospective observational study included 6919 ICSI cycles from 2013 to 2022. The primary outcome was the assessment of oocyte fertilization, measured in terms of the rate of formation of two-pronuclear (2PN), 3PN and 1PN zygotes. Secondary outcomes were blastulation, cumulative positive β-human chorionic gonadotrophin test and clinical pregnancy rates. Relationships between ejaculatory abstinence and fertilization outcomes, and ejaculatory abstinence and clinical outcomes were evaluated with multivariable analysis, including possible confounders. A positive association was observed between ejaculatory abstinence and semen sample volume (P < 0.001), sperm concentration (P < 0.001) and total motile sperm count (P < 0.001). No association was found between the 1PN zygote rate and ejaculatory abstinence (P = 0.97). Conversely, for each additional day of ejaculatory abstinence, the likelihood of obtaining 2PN zygotes from all inseminated oocytes decreased by 3% [adjusted odds ratio (aOR) 0.97, 95% CI 0.94-0.99], whilst the likelihood of obtaining 3PN zygotes from all inseminated oocytes increased significantly by 14% (aOR 1.14, 95% CI 1.07-1.22). No significant associations were found between ejaculatory abstinence and blastulation, cumulative pregnancy or miscarriage rates. A longer ejaculatory abstinence period significantly decreases the rate of 2PN zygotes, and increases the rate of 3PN zygotes without directly affect blastulation and pregnancy rates.

Developmental competence and neonatal outcomes of nonpronuclear zygotes following single vitrified-warmed blastocyst transfers using propensity score matching analysis.

To investigate developmental competence and neonatal outcomes of nonpronuclear (0PN) zygotes following single vitrified-warmed blastocyst transfers (VBT). The clinical, laboratorial and neonatal data of 996 patients with ≤ 38years who underwent blastocyst culture and single VBT were retrospectively analyzed. The pregnancy and neonatal outcomes of VBT were compared between 0PN and 2PN blastocysts using propensity score matching (PSM). Moreover, Day 3 (D3) embryo development and blastocyst formation were compared between 0PN and 2PN zygotes. There were no significant differences in clinical pregnancy rate (CPR), live birth rate (LBR) and neonatal outcomes of VBT between the 0PN and 2PN blastocysts irrespectively of whether PSM was used. However, early abortion rate (EAR) was higher in blastocysts from 0PN D3 embryos > 10 cells (p < 0.05) before PSM. Moreover, the early developmental competence of 0PN zygotes was different from that of 2PN zygotes presenting higher percentages of D3 embryos ≤ 6 cells (p < 0.01) and > 10 cells (p < 0.01), lower available blastocyst formation rate (ABFR) (p < 0.01) and good-quality blastocyst formation rate (GBFR) (p < 0.01) in D3 embryos with 4-6 cells. ABFR and GBFR increased with cell number when compared among embryos with 4-6 cells, 7-10 cells and > 10 cells, irrespectively of 0PN or 2PN embryos. The early developmental competence of 0PN zygotes was different from that of 2PN zygotes, but did not influence pregnancy and neonatal outcomes following VBT. ABFR and GBFR increased with cell number, irrespectively of 0PN or 2PN embryos.