Pronuclear Research Articles

Development of Germ Cell Isolation and Optimal Cryopreservation Method for Lissachatina fulica (L. fulica)

This study aims to develop an optimized method for cryopreserving the germ cells of Lissachatina fulica (L. fulica) using vitrification, as an alternative approach for conserving endangered snail species. First, we isolated several key reproductive organs, including the sperm oviduct, albumen gland, hermaphrodite gland (ovotestis), and hermaphrodite duct from L. fulica. When the ovotestis was finely chopped, numerous sperm with long tails and distinct heads were observed. The staining of sperm nuclei was confirmed using Hoechst 33342 dye. Since the hermaphrodite gland, referred to as the ovotestis, contains both male and female germ cells, we performed tissue staining on the ovotestis using hematoxylin and eosin (H&E) dye. H&E staining of the ovotestis revealed numerous oval-shaped acini containing sperm and early germ cells. Spermatocytes and spermatids were observed within distinct boundaries, with mature sperm appearing following spermatogenesis. To preserve the species of the L. fulica, we introduced vitrification technology to cryopreserve its reproductive organs. The non-vitrification group showed an average cell viability of 96.6%, while the vitrification group had 86.8% after thawing. This study presents a reliable cryopreservation protocol for L. fulica, with potential applications for other endangered snails, supporting conservation efforts to preserve genetic resources and biodiversity.

17α-Ethynylestradiol alters testicular epigenetic profiles and histone-to-protamine exchange in mice

Spermatogenesis starts with the onset of puberty within the seminiferous epithelium of the testes. It is a complex process under intricate control of the endocrine system. Physiological regulations by steroid hormones in general and by estrogens in particular are due to their chemical nature prone to be disrupted by exogenous factors acting as endocrine disruptors (EDs). 17α-Ethynylestradiol (EE2) is an environmental pollutant with a confirmed ED activity and a well-known effect on spermatogenesis and chromatin remodeling in haploid germ cells. The aim of our study was to assess possible effects of two doses (2.5ng/ml; 2.5 μg/ml) of EE2 on both histone-to-protamine exchange and epigenetic profiles during spermatogenesis performing a multi/transgenerational study in mice. Our results demonstrated an impaired histone-to-protamine exchange with a significantly higher histone retention in sperm nuclei of exposed animals, when this process was accompanied by the changes of histone post-translational modifications (PTMs) abundancies with a prominent effect on H3K9Ac and partial changes in protamine 1 promoter methylation status. Furthermore, individual changes in molecular phenotypes were partially transmitted to subsequent generations, when no direct trans-generational effect was observed. Finally, the uncovered specific localization of the histone retention in sperm nuclei and their specific PTMs profile after EE2 exposure may indicate an estrogenic effect on sperm motility and early embryonic development via epigenetic mechanisms.

Toxicological effects and potential reproductive risk of microplastic-induced molecular changes in protamine-like proteins and their DNA binding

Today, plastic pollution is a widespread problem in all ecosystems and has a particularly severe impact on marine ecosystems and external fertilisers such as the mussel Mytilus galloprovincialis. The present study aims to assess the toxicological reproductive health effects in this organism following exposure to two concentrations of polystyrene microplastics (PS-MPs) (0.5 and 1 μg/L), representative of conditions in the Mediterranean Sea. After exposure, the electrophoretic pattern of protamine-like (PL) proteins, the major basic protein component of Mytilus galloprovincialis sperm chromatin, was analysed. Compared to the unexposed condition, differences were observed by SDS-PAGE and an increased ability of PL to bind and protect DNA from oxidative damage was then measured, particularly for PL from mussels exposed to 1 μg/L PS-MPs. At this dose of PS-MPs, a reduced release of all PLs from the sperm nuclei was also observed, whereas the digestion by micrococcal nuclease did not show any significant differences between the exposed and the unexposed conditions. Finally, the possibility of poly(ADP)-ribosylation of the PLs was investigated. PL-II showed an increase in poly(ADP)-ribosylation after PS-MPs exposure, which may account for the difference in the ability of the PLs to bind DNA. In conclusion, while all the results might suggest a molecular mechanism of gametic plasticity occurring upon exposure of mussels to PS-MPs 1 μg/L, they also indicate that this dose of exposure could be extremely detrimental to the reproductive health of Mytilus galloprovincialis because it could prevent the release of basic nuclear proteins from the sperm DNA at fertilisation.

Protamine 2 deficiency results in Septin 12 abnormalities.

There is a well-established link between abnormal sperm chromatin states and poor motility, however, how these two processes are interdependent is unknown. Here, we identified a possible mechanistic insight by showing that Protamine 2, a nuclear DNA packaging protein in sperm, directly interacts with cytoskeletal protein Septin 12, which is associated with sperm motility. Septin 12 has several isoforms, and we show, that in the Prm2 -/- sperm, the short one (Mw 36kDa) is mis-localized, while two long isoforms (Mw 40 and 41kDa) are unexpectedly lost in Prm2 -/- sperm chromatin-bound protein fractions. Septin 12 co-immunoprecipitated with Protamine 2 in the testicular cell lysate of WT mice and with Lamin B1/2/3 in co-transfected HEK cells despite we did not observe changes in Lamin B2/B3 proteins or SUN4 expression in Prm2 -/- testes. Furthermore, the Prm2 -/- sperm have on average a smaller sperm nucleus and aberrant acrosome biogenesis. In humans, patients with low sperm motility (asthenozoospermia) have imbalanced histone-protamine 1/2 ratio, modified levels of cytoskeletal proteins and we detected retained Septin 12 isoforms (Mw 40 and 41kDa) in the sperm membrane, chromatin-bound and tubulin/mitochondria protein fractions. In conclusion, our findings present potential interaction between Septin 12 and Protamine 2 or Lamin B2/3 and describe a new connection between their expression and localization, contributing likely to low sperm motility and morphological abnormalities.

Incidence of haploidy and triploidy in trophectoderm biopsies of blastocysts derived from normally and abnormally fertilized oocytes.

We aimed to identify the correlation between morphological pronuclear (PN) status and the genetically determined ploidy configuration in preimplantation embryos. A retrospective observational study was conducted on 1982 embryos displaying normal fertilization and 380 embryos showing an atypical PN pattern, tested for aneuploidies and ploidy status via preimplantation genetic testing (PGT) between May 2019 and May 2024. Ploidy prediction was performed using a validated targeted-NGS approach and a proprietary bioinformatic pipeline analyzing SNPs B-allele frequency information. Ploidy results were obtained in relation to the morphological PN pattern and further stratified by mode of PN observation, maternal age, and embryo quality parameters. Abnormal ploidy results in 2PN-derived embryos were 1% (n = 20/1982): 0.8% showed triploidy and 0.2% haploidy. Ploidy results in relation to PN number in atypical fertilization were as follows: 0PN (n = 150/380) associated with 87.3% of diploidy, 8.7% of haploidy, and 4.0% of triploidy; 1PN-derived blastocysts (n = 73/153) were haploid in 47.7% of cases, 6.5% were triploid, and 45.7% diploid; 2.1PN (n = 23/280) and 3PN patterns (n = 54/280) predicted a triploid result in 34.8% and 74.1% of cases, respectively. PN observation with time-lapse increased ploidy status predictivity from 28.3% to 80.4% (p < 0.01) and reduced expected diploid rates to 19.6% (p < 0.01). Diploidy rate was higher for maternal age ≤ 35years and for morphologically high-grade embryos. Morphological PN check can be improved by incorporating ploidy analysis within the conventional PGT workflow. Euploid 2PN-derived embryos can be further selected removing haploids and triploids, and some atypical PN pattern can be better classified.

Kdm4d mutant mice show impaired sperm motility and subfertility.

Regulation of gene expression through histone modifications underlies cell homeostasis and differentiation. Kdm4d and Kdm4dl exhibit a high degree of similarity and demethylate H3K9me3. However, the physiological functions of these proteins remain unclear. In this study, we generated Kdm4dl mutant mice and found that Kdm4dl was dispensable for mouse development. However, through the generation of Kdm4d mutant mice, we unexpectedly found that Kdm4d mutant male mice were subfertile because of impaired sperm motility. The absence of Kdm4d was associated with an altered distribution of H3K9me3 in round spermatids, suggesting that the Kdm4d-mediated adjustment of H3K9me3 levels is required to generate motile sperm. Further analysis revealed that the absence of Kdm4d did not affect the functionality of sperm nuclei in generating offspring. As KDM4D is specifically expressed in the human testes, our results suggest that changes in KDM4D expression or its activity may be a risk factor for human infertility.

The mechanism of INO80D involved in chromatin remodeling regulating spermatogenesis in Chinese mitten crab (Eriocheir sinensis).

The INO80D protein, a component of the INO80 chromatin remodeling complex, plays a pivotal role in chromatin remodeling, gene expression, and DNA repair within mammalian sperm. In contrast to the condensed nuclear structure of mammalian sperm, Chinese mitten crab, Eriocheir sinensis, exhibits a distinctively decondensed sperm nucleus. The distribution and function of INO80D during the E. sinensis spermatogenesis were previously enigmatic. Our research endeavored to elucidate the distribution and function of INO80D, thereby enhancing our comprehension of sperm decondensation and the process of spermatogenesis in this species. Employing transcriptome sequencing, RT-qPCR, western blot analysis, and immunofluorescence techniques, we observed a pronounced upregulation of INO80D in the adult E. sinensis in comparison to the juvenile. The protein predominantly resides in the cellular nucleus, with high levels in spermatogonia and spermatocytes, less in stage I and III spermatids, and lowest in mature sperm. The results indicated that INO80D is initially instrumental in chromatin decondensation to facilitate gene accessibility and DNA repair during the early phases of spermatogenesis. Its role subsequently shifts to maintaining decondensed chromatin stability and genetic integrity during spermiogenesis. The sustained presence of INO80D during spermiogenesis is essential for the ultimate maturation of the decondensed sperm nucleus, imperative for preserving the unique decondensed state and the protection of genetic material in E. sinensis. Our study concludes that INO80D exerts a multifaceted influence on the spermatogenesis of E. sinensis, impacting chromatin decondensation, genetic integrity, and the regulation of early gene expression. This understanding could potentially improve crab breeding in aquaculture.

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.

Experimental Varicocele Impairs DNA Methylation and Demethylation in Germ Cells, TESE, and Epididymal Spermatozoa, Impacting Active DNA Demethylation in Zygotes.

Varicocele causes infertility. The current study has investigated the impact of experimental varicocele on DNA methylation, demethylation, and damage in the germ cells, TESE-derived and epididymal spermatozoa. Moreover, the results were compared between epidydimal and TESE-derived spermatozoa. Finally, the varicocele-induced effect on active DNA demethylation (ADD) of male pronucleus and pre-implantation embryo development was assessed. The mature male rats were divided into control, control-sham (undergone simple laparotomy), and experimental varicocele-induced groups (n = 6/each group). The left renal vein semi-ligation was considered to induce varicocele. The expression levels of DNA methyltransferase 1 (DNMT1) and ten-eleven-translocation proteins (TET1, 2, 3), and global DNA methylation in testicular tissue, TESE, and epididymis-derived spermatozoa, and the ADD in zygotes male pronucleus as well as pre-implantation embryo development were assessed. The expression levels of DNMT1 and TET1, 2, 3 in testicles, TESE, and epididymis-derived spermatozoa were decreased in the varicocele group compared to the control and control-sham groups. The TESE-derived spermatozoa exhibited higher DNMT1, higher DNMT1, and TET 1, 2, and no change in TET3 expression compared to epididymis-derived spermatozoa. The varicocele group represented lower DNA methylation in the testicles, TESE-derived and epididymal spermatozoa, higher 5mC+ signal in male pronucleus, and a lower pre-implantation embryo development compared to control and control-sham rats. The TESE-derived spermatozoa exhibited higher 5mC protein expression compared to epididymal spermatozoa. In conclusion, varicocele can negatively impact the DNA methylation/demethylation processes impairing spermatogenesis and leading to fertilization failure, which may ultimately result in a decrease in embryo development by increasing susceptibility to DNA damage.

In Vitro Gene Conservation Status and the Quality of the Genetic Resources of Native Hungarian Sheep Breeds.

Studies revealed a global loss of genetic resources for local sheep breeds. Therefore, the current study aimed to introduce and highlight the progress made on Hungary's existing gene conservation program (small Gene Bank). Furthermore, we evaluated breed (Tsigai, Cikta, and Racka), season, and individual variabilities (n = 24) of the pre-freeze and post-thaw semen stored in the Gene Bank to enhance the gene conservation of the breeds. The samples were cryopreserved manually, and post-thaw spermatozoa were analyzed for motility (CASA), viability, chromatin structure, and morphometry of the sperm nuclei. Ejaculate volume, spermatozoa concentration, subjective motility and standard motility, kinematic parameters, and spermatozoa's head area standard deviation of the post-thaw samples differed significantly among breeds (p < 0.05). Season affected ejaculate volume, total spermatozoa number/ejaculate, STR, BCF, and ALH. We observed a significant (p < 0.001; 0.05) breed and season interaction on concentration, total spermatozoa number/ejaculate, VCL, LIN, WOB, spermatozoa's head average perimeter and nucleus length (Tsigai and Cikta differed but were statistically the same as Racka). Similarly, season significantly (p < 0.05) affected the proportion of ejaculate suitable for freezing. There was a significant (p < 0.05) difference in kinematic parameters and viability among the rams across the breeds. The spermatozoa's head morphometry of the Tsigai and Cikta breeds differed significantly (p < 0.05) among the rams. There were individual and breed differences in many spermatozoa quality parameters. The stored samples are of good quality, with more than 40% having intact membranes and low abnormal chromatin condensation.

Atypical structure of the nuclear membrane, distribution of nuclear pores and lamin B1 in spermatozoa of patients with complete and partial globozoospermia.

Globozoospermia is a form of male infertility characterized by spermatozoa with spherical heads lacking acrosomes. The aim of this study was to evaluate ultrastructural and molecular defects in different types of globozoospermia. Semen samples from 12 infertile patients (9 with complete globozoospermia and 3 with partial globozoospermia) and 10 normozoospermic men (control) were examined by transmission electron microscopy and immunocytochemistry with antibodies against lamin B1. The presence of lamin A and progerin was assessed by reverse transcription-PCR. Whole exome sequencing was performed in three patients. In semen samples with complete and partial globozoospermia, lamin B1 was observed at the periphery of sperm nuclei, whereas lamin A and progerin were absent. Nuclear envelope pores were found in spermatozoa from both patient groups, regardless of morphology and chromatin condensation, in contrast to the control group. Non-condensed chromatin was present in 51%-81% of cases of complete globozoospermia and in 36%-79% of cases of partial globozoospermia. Homozygous DPY19L2 and SPATA16 variants were identified in two patients with partial globozoospermia and one patient with complete globozoospermia. An atypical nuclear membrane with abnormal nuclear pore distribution and lamin B1 localization was observed in spermatozoa from patients with both complete and partial globozoospermia. The genetic defects in the DPY19L2 and SPATA16 genes detected in patients from both globozoospermic groups suggest a generalized disruption of nuclear structure in globozoospermia, highlighting the genetic and phenotypic similarities between complete and partial globozoospermia.

DSB profiles in human spermatozoa highlight the role of TMEJ in the male germline.

The male mammalian germline is characterized by substantial chromatin remodeling associated with the transition from histones to protamines during spermatogenesis, followed by the reversal to nucleohistones in the male pronucleus preceding the zygotic genome activation. Both transitions are associated with the extensive formation of DNA double-strand breaks (DSBs), requiring an estimated 5 to 10 million transient DSBs per spermatozoa. Additionally, the high transcription rate in early stages of spermatogenesis leads to transcription-coupled damage preceding meiotic homologous recombination, potentially further contributing to the DSB landscape in mature spermatozoa. Once meiosis is completed, spermatozoa remain haploid and therefore cannot rely on error-free homologous recombination, but instead depend on error-prone classical non-homologous end joining (cNHEJ). This DNA damage/repair-scenario is proposed to be one of the main causes of the observed paternal mutation propensity in human evolution. Recent studies have shown that DSBs in the male pronucleus are repaired by maternally provided Polθ in Caenorhabditis elegans through Polθ-mediated end joining (TMEJ). Additionally, population genetic datasets have revealed a preponderance of TMEJ signatures associated with human variation. Since these signatures are the result of the combined effect of TMEJ and DSB formation in spermatozoa and male pronuclei, we used a BLISS-based protocol to analyze recurrent DSBs in mature human sperm heads as a proxy of the male pronucleus before zygotic chromatin remodeling. The DSBs were found to be enriched in (YR)n short tandem repeats and in evolutionarily young SINEs, reminiscent to patterns observed in murine spermatids, indicating evolutionary hotspots of recurrent DSB formation in mammalian spermatozoa. Additionally, we detected a similar DSB pattern in diploid human IMR90 cells when cNHEJ was selectively inhibited, indicating the significant impact of absent cNHEJ on the sperm DSB landscape. Strikingly, regions associated with most retained histones, and therefore less condensed chromatin, were not strongly enriched with recurrent DSBs. In contrast, the fraction of retained H3K27me3 in the mature spermatozoa displayed a strong association with recurrent DSBs. DSBs in H3K27me3 are associated with a preference for TMEJ over cNHEJ during repair. We hypothesize that the retained H3K27me3 may trigger transgenerational DNA repair by priming maternal Polθ to these regions.

CRISPR/Cas9-mediated Nap knockout affects female reproduction and egg shape in Bombyx mori.

Insect reproductive capacity can affect effective pest control and infertility studies and has become an important focus in recent molecular genetic research. Nucleosome assembly protein (Nap) is highly conserved across multiple species and is involved in forming the sperm nucleus in many species. We used clustered regularly interspaced palindromic repeats/Cas9 technology to knockout BmNap in Bombyx mori and observed that the mutations caused female infertility, whereas male fertility was not affected. BmNap mutants grew and mated normally; however, female mutants laid smaller eggs that could not be fertilised and did not hatch. In addition, female sterility produced by the mutation could be inherited stably via male mutants; therefore, Nap could be used as a potential target for lepidopteran pest control through population regulation. In the current study, we elucidated a new function of BmNap, increased the understanding of the oogenesis regulation network in Lepidoptera and promoted the development of insect sterility technologies.

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-505 Clinical rescue rate of abnormally fertilized oocytes and the relationship with patient, embryo and laboratory features

Abstract Study question Which are the main patient, embryonic and laboratory variables associated with a diploid result in abnormally fertilized embryos? Summary answer Embryo diploid constitution is associated with maternal age, trophectoderm and inner cell mass quality as well as pronuclear (PN) number predicted by time-lapse incubator. What is known already In conventional in vitro fertilization cycles, morphological PN evaluation can identify proper fertilization and predict embryo ploidy constitution. Atypical PN patterns like 0PN, 1PN or more than 2PN are associated with ploidy abnormalities. However, we have previously shown that PN number is insufficient to predict embryo ploidy composition, and advanced single nucleotide polymorphism (SNP)-based methodologies for preimplantation genetic testing (PGT) detect a significant number of genetically diploid embryos from presumed abnormally fertilized oocytes. In this study, we characterized the diploid rescue rate associated with abnormal PN configurations, uncovering the link between ploidy results and relevant patient, embryonic and clinical/laboratory parameters. Study design, size, duration Retrospective observational study evaluating the association between diploid rescue rate of abnormally fertilized embryos and different patient, embryonic and laboratory features. Embryos were cultured in conventional or EmbryoScope™ time-lapse incubator (Vitrolife) until blastocyst stage and classified according to Gardner embryo grading system. Ploidy assessment was performed on 343 euploid trophectoderm (TE) biopsies collected between January 2021 and November 2023. Results were stratified according to patients age, embryo quality parameters and different laboratory procedures. Participants/materials, setting, methods Multi-center international study involving consenting patients using autologous gametes and undergoing PGT for aneuploidies (PGT-A) combined with ploidy analysis. Ploidy protocol was performed using a validated targeted NGS approach composed of a custom panel of 357 SNPs and a proprietary pipeline of analysis. Reliable ploidy classification was obtained using a combined algorithm strategy based on SNPs allele frequencies. Categorical variables were expressed in percentages. Nominal variables were analysed using the Cochran-Armitage test to detect trends. Main results and the role of chance Among 343 euploid embryos, diploidy rates obtained for 0PN, 1PN, 2.1PN and 3PN groups were 86.8% (n = 118/136;95%CI=79.89-91.96) 43.7% (n = 59/135;95%CI=35.19-52.50), 65.0% (n = 13/20;95%CI=40.78-84.61) and 26.9% (n = 14/52;95%CI=15.57-41.02) respectively. Regarding patient parameters, advanced maternal age, but not paternal age, was significantly associated with lower diploid rescue rates, with a decreasing trend from 72.5% (n = 79/109;95%CI=63.10-80.60) below age 35 to 42.9% (n = 30/70;95%CI=31.09-55.25) above age 40 (p &amp;lt; 0.01). When considering embryo morphological evaluations, Cochran Armitage test revealed a significant link between diploidy rates and TE quality with a decreasing trend going from 72.1% (n = 80/111;95%CI=62.76-80.17) to 52.7% (n = 79/150;95%CI=44.36-60.87) and 45.8% (n = 22/48;95%CI=31.37-60.83) for embryos classified with TE grade as A, B, and C, respectively (p-value&amp;lt;0.01). Similarly, a decreasing trend was reported in relation to inner cell mass (ICM) quality, when moving from grade A (67,1%, n = 94/140;95%CI=58.23-75.33) to grade C (54.8%, n = 17/31;95%CI=36.03-72.68) (p = 0.03). No clear association was reported for embryo expansion degree. Finally, among laboratory variables (oocyte freezing procedure, fertilization method, incubator type), only time-lapse significantly impacted expected diploid results. Overall, if abnormal PN number is confirmed by time-lapse (n = 42 samples), the accuracy in predicting ploidy abnormalities increased from 29.2% (n = 82/281;95%CI=23.93-34.87), in conventional incubators, to 83.3% (n = 35/42;95%CI=68.64-93.03) (p &amp;lt; 0.01). Consequently, expected diploid rescue rate decreased from 65.5% (n = 184/281;95%CI=59.60-71.03) to 16.7% (n = 7/42;95%CI=6.97-31.36) (p &amp;lt; 0.01). Limitations, reasons for caution Parental DNA analysis should be included to rule-out the residual risk of uniparental disomy in diploid embryos. The multi-center nature of the study didn’t support a uniform PN annotation, especially for centers lacking time-lapse observation. Future studies should focus on the clinical follow-up after the transfer of rescued euploid-diploid embryos. Wider implications of the findings We believe we reported the first evidence of maternal age effect on ploidy results in the largest dataset of abnormally fertilized embryos. We revealed a link between ploidy constitution and blastocyst morphological quality, providing important information for patient counselling. Time-lapse incubators offer a more accurate identification of abnormally fertilized oocytes. Trial registration number n/a

O-307 Decrease of human sperm hypercondensed chromatin volume after slow freezing revealed by three-dimensional (3D) nuclear morphometry

Abstract Study question Are the 3D morphometry parameters of the human sperm nucleus affected by slow freezing? Summary answer The slow freezing-thawing cycle induces significant alterations on hypercondensed nucleus zone by reducing its volume and splits it into several small zones. What is known already Sperm cryopreservation is an essential technique for infertility care, but it seems to highly affect the genetic and epigenetic integrity of sperm nuclei with poorly identified mechanisms. Moreover, it is known that sperm nuclear alterations may be involved in pregnancy failures. Therefore, sperm nuclear biomarkers are an essential issue to assess the outcome of ART procedures when frozen sperm is used. Thereby, 3D visualization of chromatin is a good way to understand modifications in the organization and composition of sperm chromatin after a slow freezing procedure. Study design, size, duration Ten semen of patients were analyzed (Germetheque biobank) after informed consent. Standard semen parameters, DNA fragmentation, chromatin decondensation, sperm telomere length (STL) and 3D nuclear morphometry were performed before and after slow freezing. Morphometric measurements were performed from a digital representation of the sperm nucleus in 3 dimensions that allow us to measure the volume, flatness and elongation of each sperm nucleus and characterize hypercondensed zones, with live/dead spermatozoa distinction. Participants/materials, setting, methods After addition of cryoprotective medium v/v (Cryosperm®), sperm was frozen with a programmable freezer (NanoDigitcool®). Standards sperm parameters were measured in accordance with WHO guidelines 2021, DNA fragmentation by flow cytometry TUNEL assay, chromatin decondensation by epifluorescent microscopy with Chromomycin A3 and STL using qPCR. 3D nuclear representation was performed by a z-axis image acquisition series of DAPI/PI stained slides with optigrid epifluorescent microscopy. Analyses were carried out using the NucleusJ2.0 software with NODeJ plugin. Main results and the role of chance Freezing induces an alteration of sperm vitality (p &amp;lt; 0.0001) and progressive and total motility (p &amp;lt; 0.001 and p &amp;lt; 0.0001 respectively) and an increase in the percentage of sperm with DNA fragmentation (18.30% fresh vs 31.6% frozen, p &amp;lt; 0.05). We did not observe a modification of the median volume of the sperm nucleus before and after freezing (37.15 [IC = 36.5;38.14] and 37.10 [33.27;37.78] µm3 respectively), as the same for flatness and elongation (2.48 vs. 2.50 a.u. and 2.04 vs. 2.04 a.u. respectively). Nevertheless, five samples showed significant alterations of nucleus volume (p &amp;lt; 0.0001), due to diminution of flatness and more or less elongated. A significant decrease of the total volume of the hypercondensed chromatin zone was observed after freezing (0.94 [0.84;0.99] before vs. 0.61 [0.53;0.69] µm3 after freezing-thawing, p &amp;lt; 0.0001), with an effect size of -1.48 [-2.10; -0.86]. Decrease of total volume of hypercondensed region was measured homogeny in all the samples tested, particularly on live (1.00 vs. 0.68 µm3 respectively, p &amp;lt; 0.0001) compared with dead spermatozoa (0.72 vs. 0.57 µm3, p &amp;lt; 0.05). We showed also a significant increase of the number of hypercondensed zones in each nucleus (p &amp;lt; 0.001), reflecting a split into several parts of the hypercondensed chromatin. Limitations, reasons for caution A 3D-FISH analysis would allow to characterize the hypercondensed chromatin portion, probably as heterochromatin and which we suspect to be the nucleus ring. Moreover, a chromosomes specific marking would allow for the identification of whether whole chromosomes or specific parts of chromosomes (centromeres or telomeres) are affected by this decompaction. Wider implications of the findings This study is the first to make exacts measurements of human sperm nucleus because of a non-decondensing method. We demonstrate that slow freezing affects hypercondensed chromatin probably through a decondensation and modification of the nucleus-specific organization. This new assessment will allow a better proficiency of men fertility preservation techniques. Trial registration number NCT04715828

P-154 Structural changes of the human sperm tail during the early post-fertilization

Abstract Study question What happens to the sperm tails that are no longer needed after the goal of fertilization has been achieved? Summary answer The sperm tail lost its fibrous sheath and changed to a coiled morphology with a whisker root-like structure at the tip during the early post-fertilization. What is known already The sperm tail has been thought to play only a role in allowing the head and neck to reach the oocyte. The flagellum is found in the mammalian sperm tail and consists of a middle, main, and terminal part. It also contains an axon composed of two central microtubules surrounded by nine peripheral microtubule doublets (9d + 2s microtubule pattern) surrounded by an accessory structure called the fibrous sheath (FS), but few studies have been conducted on the post-fertilization fate of the mammalian sperm tail. Study design, size, duration To investigate sperm tail structure from the pronuclear stage to the second cleavage, we used abnormally fertilized oocytes derived from either conventional in vitro fertilization (cIVF, n = 41) or intracytoplasmic sperm injection (ICSI, n = 11). In this study, abnormally fertilized oocytes were collected between March 2022 and November 2023 and subjected to immunofluorescence analysis and field emission scanning electron microscopy (FE-SEM) analysis. Participants/materials, setting, methods Under ethical approval, abnormally fertilized oocytes were donated by infertile couples undergoing cIVF or ICSI cycles at the Yamashita Shonan Yume Clinic in Japan. Informed consent was obtained from all participants. Immunofluorescence assay was performed to analyze sperm tail morphology using antibodies against H3K27me3, α-tubulin, and AKAP4. FE-SEM analysis was performed to detect the 9d + 2s structure characteristic of the sperm tail axon. Main results and the role of chance Tri-pronuclear zygotes derived from conventional in vitro fertilization (cIVF-derived 3PN) had two male pronuclei, whereas those derived from intracytoplasmic sperm injection (ICSI-derived 3PN) had one. Each zygote had the same number of coiled microtubule structures as the male pronucleus, and the 9d + 2s structure characteristic of the axon was identified, suggesting a sperm tail. No difference in sperm tail shape was observed between ICSI-derived 3PN and cIVF-derived 3PN. AKAP4, which is specifically localized in the FS associated with the principal piece of the flagellum, was observed in sperm tails attached to the zona pellucida but not in those in the oocyte, suggesting that the FS of the flagellum is lost before the pronuclear stage, leaving the microtubules of the axonemes exposed. The sperm tail was attached to one of the spindle poles during the first cleavage of abnormally fertilized oocytes, but no differences in the structure of the spindle pole with or without the sperm tail were observed. The tip of the sperm tail was branched like a “whisker root”, suggesting that the sperm tail was degraded from the end, but the degree of tip branching did not differ from the pronuclear stage to the second cleavage. Limitations, reasons for caution Normal fertilized oocytes were not used in this study because they are used for clinical treatment. The cell biological significance of sperm tail branching is currently unknown and requires further experimentation. Wider implications of the findings These findings shed light on the processes that occur during human fertilization, suggest new roles for the sperm tail, and may have implications for assisted fertilization approaches in the future. 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

O-069 The first mitotic spindle instability impacts nucleation, fragmentation, and development in human embryos

Abstract Study question How multinucleation and fragmentation occur in the first mitosis in human embryos? Summary answer The first mitotic spindles had heterogeneity, and the shapes and movements of the spindles were strongly associated with multinucleation and fragmentation. What is known already Studies have shown that many problems occur during the first mitosis, such as fragmentation and multinucleation. However, the cell biologic mechanisms whereby these errors occur during the first mitosis in human embryos remain unknown. To clarify this aspect, it is necessary to analysis the dynamics of DNA and cytoskeleton using live human embryos. Although microinjections of DNA or mRNA into mammalian embryos were widely used for live imaging during early mitosis, the method into human poses the risk of ethical issues. Therefore, we established a method used for real-time imaging in human embryos using chemical fluorescent labeling. Study design, size, duration Frozen human two-pronuclear stage embryos were donated for research by couples who had completed fertility treatment. We obtained written informed consent from patients who donated human embryos. The mean age of patients at embryo freezing was 32.00 ± 3.03 (SD) years. We imaged thirty-one living human embryos using chemical fluorescent labeling and confocal microscopy to visualize DNA and tubulin. Participants/materials, setting, methods Thirty-one human embryos were cultured in medium containing the live-cell stain probes SPY555-DNA and SPY650-Tubulin. The Embryos were imaged using an LSM980 laser scanning confocal microscope every 10–20 min for a period of eight hours. Three-dimensional (3D) visualizations of human embryos were performed using the Imaris software. The first spindle shapes were quantified by image analysis and calculated the aspect ratio of the spindles using ImageJ. Main results and the role of chance We discovered that over 81% of two-pronuclear zygotes became multinucleated 2-cell stage embryos. We demonstrated that there was variation in the first mitotic spindles. There was a significant difference between the aspect ratio (AR) of the spindles leading to the mononuclear and multinuclear types at the 2-cell stage (p &amp;lt; 0.005). In addition, the low-AR spindles were unstable and often had more defocused poles than the high-AR spindles, which lead to multinucleation at the 2-cell stage (55 % vs. 0%, p &amp;lt; 0.05). More embryo having high-aspect ratio spindles tend to develop blastocysts than those having low aspect-ratio spindles (p &amp;lt; 0.05). Moreover, the movement of the central spindles varied after chromosome segregation. The embryos with more fragments at two cell stage had a significantly longer period for the central spindles to transform into midbodies compared to those with fewer fragments. The central spindles of embryos with more fragments moved a longer distance from the beginning of furrow ingression to the completion of the first division (p &amp;lt; 0.05). These results suggest that the first mitotic spindle instability and heterogeneity cause many errors such as multinucleation, fragmentation, and embryonic development. Limitations, reasons for caution Imaged human embryos were donated from a fertility clinic, and detailed patient information, such as underlying disease and method of ovarian stimulation, was not available. It is possible that the human zygotes used in this study had a bias in patient characteristics. Wider implications of the findings Our study showed that there were instability and heterogeneity in the first mitotic spindle, which was strongly associated with multinucleation, fragmentation, and embryonic development. Revealing the mechanism underlying spindle formation in cleavage-stage embryos will contribute to improving the success rate of assisted reproduction technologies in humans. Trial registration number not applicable

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