An expert opinion on rescuing atypically pronucleated human zygotes by molecular genetic fertilization checks in IVF.

IVF laboratories routinely adopt morphological pronuclear assessment at the zygote stage to identify abnormally fertilized embryos deemed unsuitable for clinical use. In essence, this is a pseudo-genetic test for ploidy motivated by the notion that biparental diploidy is required for normal human life and abnormal ploidy will lead to either failed implantation, miscarriage, or significant pregnancy complications, including molar pregnancy and chorionic carcinoma. Here, we review the literature associated with ploidy assessment of human embryos derived from zygotes displaying a pronuclear configuration other than the canonical two, and the related pregnancy outcome following transfer. We highlight that pronuclear assessment, although associated with aberrant ploidy outcomes, has a low specificity in the prediction of abnormal ploidy status in the developing embryo, while embryos deemed abnormally fertilized can yield healthy pregnancies. Therefore, this universal strategy of pronuclear assessment invariably leads to incorrect classification of over 50% of blastocysts derived from atypically pronucleated zygotes, and the systematic disposal of potentially viable embryos in IVF. To overcome this limitation of current practice, we discuss the new preimplantation genetic testing technologies that enable accurate identification of the ploidy status of preimplantation embryos and suggest a progress from morphology-based checks to molecular fertilization check as the new gold standard. This alternative molecular fertilization checking represents a possible non-incremental and controversy-free improvement to live birth rates in IVF as it adds to the pool of viable embryos available for transfer. This is especially important for the purposes of 'family building' or for poor-prognosis IVF patients where embryo numbers are often limited.

Diameter of Human Day Five Blastocysts and Birth Sex.

Background This study aimed to evaluate the offspring sex ratio, born through fresh and cryo-thawed single blastocyst (BL) transfers regarding a single morphological, static parameter, namely, BL diameter. Methodology This retrospective, observational study was conducted at an assisted reproductive technology (ART) center, Kinderwunschzentrum Niederrhein Germany. We conducted a statistical analysis of all births resulting from fresh and thawed in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI) cycles after a single embryo transfer (SET). The main outcome measure was the offspring sex ratio after SET of a day five BL in relation to the BL diameter measurement. Results There were more female than male babies born in our study. We observed a tendency for BL to have a higher diameter, resulting in female offspring, which was not statistically relevant. We also compared the BL diameter in the fresh embryo transfer (ET) group with that of the cryo-thawed ET group, showing a tendency toward a larger diameter in the fresh ET group. In the ICSI cycles, there was a higher tendency for a larger BL diameter when compared to IVF cycles. In the fresh ET cycles, BL leading to the male sex at birth had a tendency toward a larger diameter than the female BL. In the cryo-thaw ET cycles, BL leading to the female sex had a tendency toward a larger diameter than the male BL. Conclusions Our results showed a tendency in the sex of offspring toward the female sex and no significant difference in the BL diameter of BL leading to birth after ART and consecutive transfer of day five BL.

Could the presence and proportion of three/multiple pronuclei (3PN/MPN) zygotes indicate the cytoplasmic maturation state of oocyte cohort in conventional IVF patients?

Although abnormally fertilized zygotes with three or multiple pronuclei (3 PN/MPN) are commonly believed to be associated with improper maturation of the oocyte cytoplasm in conventional IVF cycles, no studies investigated the association between the proportion of MPN zygotes and the maturation state of the oocyte cohort. We compared the cytoplasmic maturity of oocytes from conventional IVF cycles with different proportions of 3 PN/MPN zygotes. A total of 1428 conventional IVF patients with ≥6 oocytes retrieved and fresh embryos transferred were divided into 4 groups according to the proportions of 3 PN/MPN zygotes. The pregnancy outcomes and the proportion of nuclear immature oocytes were analyzed to suggest the cytoplasmic maturation state of the oocyte cohort. Our results showed that the group with a low proportion of 3 PN/MPN zygotes had a higher clinical pregnancy rate (CPR) than those without 3 PN/MPN zygotes (P < 0.05). However, the live birth rate (LBR) was not significantly different between the two groups. The implantation rate (IR), CPR, and LBR did not differ between the low-proportion and high-proportion 3 PN/MPN groups. The proportion of nuclear immature oocytes on day 1 was highest in the group without 3 PN/MPN zygotes (23.8 %) and gradually decreased with an increased proportion of 3 PN/MPN zygotes (P < 0.001). Therefore, the presence of 3 PN/MPN zygotes after conventional IVF may indicate a more mature cytoplasmic state of the oocyte cohort, and the increased proportion of 3 PN/MPN zygotes is associated with an increased maturation state of the whole oocyte cohort. The occurrence and proportion of 3 PN/MPN zygotes may serve as an indicator for the cytoplasmic maturity of the oocyte cohort and help clinicians evaluate the efficiency of ovarian stimulation and optimize the stimulation protocols in subsequent cycles.

Healthy live birth following embryo transfer of a blastocyst of tetrapronuclear (4PN) origin: a case report.

During IVF treatments, normal fertilization is generally evidenced by the appearance of two pronuclei, one arising from the oocyte and the other from the male gamete. Embryos derived from zygotes with a pronuclei number other than two are assumed to possess a ploidy abnormality and their transfer is usually avoided owing to increased risk of implantation failure, miscarriage, and molar pregnancies. Nonetheless, the inclusion of genotyping data in preimplantation genetic testing has revealed that a normal diploid configuration is possible in embryos deriving from zygotes with an abnormal pronuclei number such as tripronuclear and one pronucleus. Here, we present a one-of-a-kind transfer of a tetrapronuclear-derived embryo that was discovered to be diploid and negative for other whole chromosome or segmental aneuploidies during preimplantation genetic testing using a targeted next-generation sequencing approach. The transfer resulted in the live birth of a healthy infant who is now 4 years old and has no apparent health or developmental impairments.

The first mitotic division: a perilous bridge connecting the zygote and the early embryo.

Human embryos are very frequently affected by maternally inherited aneuploidies, which in the vast majority of cases determine developmental failure at pre- or post-implantation stages. However, recent evidence, generated by the alliance between diverse technologies now routinely employed in the IVF laboratory, has revealed a broader, more complex scenario. Aberrant patterns occurring at the cellular or molecular level can impact at multiple stages of the trajectory of development to blastocyst. In this context, fertilization is an extremely delicate phase, as it marks the transition between gametic and embryonic life. Centrosomes, essential for mitosis, are assembled ex novo from components of both parents. Very large and initially distant nuclei (the pronuclei) are brought together and positioned centrally. The overall cell arrangement is converted from being asymmetric to symmetric. The maternal and paternal chromosome sets, initially separate and scattered within their respective pronuclei, become clustered where the pronuclei juxtapose, to facilitate their assembly in the mitotic spindle. The meiotic spindle is replaced by a segregation machinery that may form as a transient or persistent dual mitotic spindle. Maternal proteins assist the decay of maternal mRNAs to allow the translation of newly synthesized zygotic transcripts. The diversity and complexity of these events, regulated in a precise temporal order and occurring in narrow time windows, make fertilization a highly error-prone process. As a consequence, at the first mitotic division, cellular or genomic integrity may be lost, with fatal consequences for embryonic development.

Variation of Female Pronucleus Reveals Oocyte or Embryo Chromosomal Copy Number Variations.

The characteristics of the human pronuclei (PNs), which exist 16-22 h after fertilization, appear to serve as good indicators to evaluate the quality of human oocyte and embryo, and may reflect the status of female and male chromosome composition. Here, a quantitative PN measurement method that is generated by applying expert experience combined with deep learning from large annotated datasets is reported. After mathematic reconstruction of PNs, significant differences are obtained in chromosome-normal rate and chromosomal small errors such as copy number variants by comparing the size of the reconstructive female PN. After integrating the whole procedure of PN dynamics and adjusting for errors that occur during PN identification, the results are robust. Notably, all positive prediction results are obtained from the female propositus population. Thus, the size of female PNs may mirror the internal quality of the chromosomal integrity of the oocyte. Embryos that develop from zygotes with larger female PNs may have a reduced risk of copy number variations.

Direct cleavage during the first mitosis is a sign of abnormal fertilization in cattle.

Direct cleavage, a type of abnormal cleavage in which one zygote divides into three or more blastomeres, has been reported in mammals. The incidence of direct cleavage increases in zygotes with three or more pronuclei (multi-PN) and those showing abnormal pronuclei migration. However, there are few reports on the relationship between pronuclei and direct cleavage, and the effects of these relationships on subsequent embryogenesis have not been clarified. It is difficult to observe pronuclei under visible light, especially in bovine zygotes, because of abundant dark lipid droplets in the cytoplasm. We visualized pronuclei by removing lipid droplets from bovine zygotes and analyzed the relationship between the number of pronuclei and direct cleavage using time-lapse cinematography. The direct cleavage rate of multi-PN zygotes was 78.6%, which was significantly higher than that of zygotes with one pronucleus (1 PN, 0.0%) and two pronuclei (2 PN, 8.2%). Observation of pronuclei migration in 2 PN zygotes showed that 3.1% of 2 PN zygotes had non-apposed pronuclei. The direct cleavage rate of zygotes with non-apposed pronuclei was 66.7%, which was significantly higher than that of zygotes with apposed pronuclei (6.4%). Among multi-PN zygotes, the proportions of zygotes with apposed pronuclei and non-apposed pronuclei were 37.5% and 64.3%, respectively. The direct cleavage rate of multi-PN zygotes with non-apposed pronuclei was 100.0%, which was significantly higher than that of zygotes with apposed pronuclei (40.0%). Three-dimensional live-cell imaging of bovine zygotes injected with the mRNA-encoding histone H2B-mCherry showed that the direct cleavage rates of 2 PN and multi-PN zygotes bypassing syngamy were 63.2% and 75.5%, respectively. These rates were significantly higher than that of 2 PN and multi-PN zygotes that underwent syngamy (5.6% and 20.0%, respectively). Regardless of the number of pronuclei, a high frequency of direct cleavage was observed in zygotes in which the pronuclei did not migrate inward the cytoplasm and bypassed syngamy. These results suggest that abnormal fertilization such as multi-PN and migration error of pronuclei in cattle is the primary reason for direct cleavage during the first mitosis. Assessment of direct cleavage during the first mitosis allows exclusion of embryos with abnormal fertilization and may contribute to in vitro produced embryo transfer success.

The migration speed of nucleolar precursor bodies in pronuclei affects in vitro fertilization-derived human embryo ploidy status and live birth.

Purpose: To study the relationship between clinical outcomes after assisted reproduction and the migration speed of nucleolus precursor bodies (NPBs) in male and female pronuclei (mPN; fPN). Methods: NPB migration speed, embryo ploidy status, and live birth (LB) were retrospectively analyzed in IVF-derived zygotes. The central coordinates of the mPN, fPN, and NPBs were noted at multiple timepoints. The migration distance of NPBs between two sequential images was measured to calculate NPB migration speed. Results: The NPB migration speeds in mPN and fPN were significantly faster in euploid zygotes than in aneuploid zygotes. In multivariate logistic analysis, NPB migration speed in mPN and the female age were associated with euploidy. The NPB migration speeds in mPN and fPN were also significantly faster in zygotes that led to LB than in zygotes that led to no pregnancy. In a receiver operating characteristic curve analysis of LB by NPB migration speed in mPN, the cut-off value was 3.74 µm/h (AUC: 0.825, 95%CI: 0.688-0.963). When the zygotes were categorized by this cut-off value, there were significantly more LBs in zygotes with migration speed ≥ the cut-off (78.9% vs. 21.1%). Conclusions: Zygotes with quickly migrating NPBs demonstrated the developmental potential to become a baby.

Snf2 Proteins Are Required to Generate Gamete Pronuclei in Tetrahymena thermophila.

During sexual reproduction/conjugation of the ciliate Tetrahymena thermophila, the germinal micronucleus undergoes meiosis resulting in four haploid micronuclei (hMICs). All hMICs undergo post-meiotic DNA double-strand break (PM-DSB) formation, cleaving their genome. DNA lesions are subsequently repaired in only one 'selected' hMIC, which eventually produces gametic pronuclei. DNA repair in the selected hMIC involves chromatin remodeling by switching from the heterochromatic to the euchromatic state of its genome. Here, we demonstrate that, among the 15 Tetrahymena Snf2 family proteins, a core of the ATP-dependent chromatin remodeling complex in Tetrahymena, the germline nucleus specific Iswi in Tetrahymena IswiGTt and Rad5Tt is crucial for the generation of gametic pronuclei. In either gene knockout, the selected hMIC which shows euchromatin markers such as lysine-acetylated histone H3 does not appear, but all hMICs in which markers for DNA lesions persist are degraded, indicating that both IswiGTt and Rad5Tt have important roles in repairing PM-DSB DNA lesions and remodeling chromatin for the euchromatic state in the selected hMIC.

Microinjection of Zygotes for CRISPR/Cas9-Mediated Insertion of Transgenes into the Murine Rosa26 Safe Harbor.

Genetically modified (GM) mice are widely used in biomedical research because they can address complex questions in an in-vivo setting that could not otherwise be addressed in-vitro. Microinjection of zygotes remains the most common technique to generate GM animals to date. Here, we describe the targeted insertion (knock-in) of transgenes by microinjection of 1-cell or 2-cell stage embryos into the murine Rosa26 safe harbor.

State of the art of nuclear transfer technologies for assisting mammalian reproduction.

The transfer of nuclear genomic DNA from a cell to a previously enucleated oocyte or zygote constitutes one of the main tools for studying epigenetic reprogramming, nucleus-cytoplasm compatibility, pluripotency state, and for genetic preservation or edition in animals. More than 50 years ago, the first experiences in nuclear transfer began to reveal that factors stored in the cytoplasm of oocytes could reprogram the nucleus of another cell and support the development of an embryo with new genetic information. Furthermore, when the nuclear donor cell is an oocyte, egg, or a zygote, the implementation of these technologies acquires clinical relevance for patients with repeated failures in ART associated with poor oocyte quality or mitochondrial dysfunctions. This review describes the current state, scope, and future perspectives of nuclear transfer techniques currently available for assisting mammal reproduction.

Conjoined twins after single blastocyst transfer: a case report including detailed time-lapse recording of the earliest embryogenesis, from zygote to expanded blastocyst.

Conjoined twins are estimated to occur in 1:50 000 pregnancies. Eighteen cases of pregnancies achieved by ART have been published of which three were achieved after single embryo transfer, allowing discussion of embryo characteristics. We report, to the best of our knowledge, the first case of parapagus conjoined twins after ART. Furthermore, this is the first report of conjoined twins with detailed morphokinetics of the earliest embryogenesis from zygote to expanded and hatched blastocyst stage. The case zygote had three refractile bodies, which were all allocated to one blastomere at first cleavage following an asynchronous pronuclei fading. Within 2 h, this blastomere cleaved to four and fragmented. The remaining blastomere cleaved symmetrically and regularly and a blastocyst (score: 4AB) was vitrified 120 h after IVF. Pregnancy was achieved following a frozen-thawed single blastocyst transfer. The etiopathogenetic mechanism of the origin of conjoined twins is unknown and several hypotheses exist. The morphokinetics in the present case and morphology of other reported cases will be discussed in this context.

Optimisation of the timing of fertilisation assessment for oocytes cultured in standard incubation: lessons learnt from time-lapse imaging of 78 348 embryos.

STUDY QUESTION: Using time-lapse data, can the current consensus for the timing of fertilisation assessment of oocytes, cultured in standard incubation, be optimised? SUMMARY ANSWER: The optimum time to perform fertilisation assessment for oocytes cultured in standard incubation is 16.5 ± 0.5 h post-insemination (hpi), and the current consensus requires modification in order to minimise the chance of fertilisation being missed. WHAT IS KNOWN ALREADY: Time-lapse incubation allows the embryologist to retrospectively review collated images of oocytes and embryos to capture important embryological observations that may otherwise be missed in standard incubation. According to expert consensus, the optimum time to perform the assessment of fertilisation is 17 ± 1 hpi. STUDY DESIGN, SIZE, DURATION: A retrospective, multicentre analysis utilised data obtained from 54 746 ICSI-derived embryos and 23 602 IVF-derived embryos cultured in time-lapse incubation between January 2011 and November 2019. PARTICIPANTS/MATERIALS, SETTING, METHODS: Using time-lapse imaging (TLI), the precise time of pronuclei appearance and disappearance was recorded, where applicable, and the number of oocytes with two pronuclei observable during 10 30-min intervals from 15 hpi to 20 hpi was determined. MAIN RESULTS AND THE ROLE OF CHANCE: Between 15 and 17.5 hpi, the average number of oocytes exhibiting normal fertilisation, elicited as two pronuclei, was 98.19% with the highest proportion of oocytes having visible pronuclei at 16-16.5 hpi (98.32%). At 18-18.5 hpi, the number of visible pronuclei reduced to 95.53% and continued to fall to 87.02% at 19.5-20 hpi. LIMITATIONS, REASONS FOR CAUTION: The authors' expectation is that these findings are transferable to other settings, however it is possible that, with alternative culture media and incubation environments, calibration of this timing may be required. As data cannot readily be recorded for pronuclear appearance for IVF-derived embryos, it is not possible to determine the optimum time to perform the fertilisation assessment for IVF-derived embryos. WIDER IMPLICATIONS OF THE FINDINGS: By fine-tuning the time at which fertilisation assessment takes place the accuracy of the assessment can be increased to maximise the number of fertilised oocytes identified, thereby increasing the number of usable embryos for the patient. Without TLI and following current consensus guidelines, over 11% (n = 3000) of oocytes would have been marked as unfertilised within this cohort. Further to this, depending on the time of a standard fertilisation assessment, up to 300 embryos which resulted in live births could have been categorised as unfertilised, as they presented no visible pronuclei at the conventional assessment time-point. STUDY FUNDING/COMPETING INTEREST(S): A.C. is a minor shareholder in CARE Fertility Limited. Validated algorithmic time-lapse embryo selection is offered to patients at CARE Fertility at an additional charge as an adjuvant treatment option. TRIAL REGISTRATION NUMBER: N/A.

The paternal toolbox for embryo development and health.

The sperm is essential for reconstitution of embryonic diploidy and highly specialized developmental functions. Immediately after gamete fusion, the sperm-borne PLC-zeta triggers activation, generating intracellular free Ca2+ oscillations. Mutations in the PLC-zeta encoding gene are associated with the absence of this factor in mature sperm and inability to achieve fertilization. Sperm play also a role in the greater game of the choreography of fertilization. In the human, the sperm centrioles are introduced into the oocyte environment with gamete fusion. They interact with the oocyte cytoskeletal apparatus to form a functional pair of centrosomes and ultimately regulate pronuclear juxtaposition in preparation for the first cleavage. As a consequence, the fidelity of chromosome segregation during the first cell divisions depends on the function of sperm centrioles. Sperm DNA integrity is essential for embryo development and health. Damaged DNA does not impact on the sperm fertilization ability following ICSI. However, detrimental effects emerge at pre- and post-implantation stages. Sperm-specific epigenetic factors also play an active role in the regulation of embryonic development, as shown by correlations between reduced embryo morphological quality and incorrect chromatin packaging during spermiogenesis or abnormal methylation of sperm CpG islands. This functional landscape demonstrates that the contribution of the sperm to development goes far beyond its well-established role in fertilization. Clinical studies confirm this view and indicate sperm function as a crucial aspect of research to increase the efficacy of assisted reproduction treatments.

Telomere Length in Metaphase Chromosomes of Human Triploid Zygotes.

The human lifespan is strongly influenced by telomere length (TL) which is defined in a zygote-when two highly specialised haploid cells form a new diploid organism. Although TL is a variable parameter, it fluctuates in a limited range. We aimed to establish the determining factors of TL in chromosomes of maternal and paternal origin in human triploid zygotes. Using Q-FISH, we examined TL in the metaphase chromosomes of 28 human triploid zygotes obtained from 22 couples. The chromosomes' parental origin was identified immunocytochemically through weak DNA methylation and strong hydroxymethylation in the sperm-derived (paternal) chromosomes versus strong DNA methylation and weak hydroxymethylation in the oocyte-derived (maternal) ones. In 24 zygotes, one maternal and two paternal chromosome sets were identified, while the four remaining zygotes contained one paternal and two maternal sets. For each zygote, we compared mean relative TLs between parental chromosomes, identifying a significant difference in favour of the paternal chromosomes, which attests to a certain "imprinting" of these regions. Mean relative TLs in paternal or maternal chromosomes did not correlate with the respective parent's age. Similarly, no correlation was observed between the mean relative TL and sperm quality parameters: concentration, progressive motility and normal morphology. Based on the comparison of TLs in chromosomes inherited from a single individual's gametes with those in chromosomes inherited from different individuals' gametes, we compared intraindividual (intercellular) and interindividual variability, obtaining significance in favour of the latter and thus validating the role of heredity in determining TL in zygotes. A comparison of the interchromatid TL differences across the chromosomes from sets of different parental origin with those from PHA-stimulated lymphocytes showed an absence of a significant difference between the maternal and paternal sets but a significant excess over the lymphocytes. Therefore, interchromatid TL differences are more pronounced in zygotes than in lymphocytes. To summarise, TL in human zygotes is determined both by heredity and parental origin; the input of other factors is possible within the individual's reaction norm.

Migration speed of nucleolus precursor bodies in human male pronuclei: a novel parameter for predicting live birth.

PURPOSE: To study the relationship between the migration speed of nucleolus precursor bodies (NPBs) in male and female pronuclei (mPN; fPN) and human embryo development during assisted reproduction. METHODS: The migration speed of 263 NPBs from 47 zygotes was quantitated, and embryonic development was observed until the blastocyst stage. The central coordinates of mPN, fPN, and NPBs were noted at multiple timepoints. Then, the distance traveled by the NPBs between two sequential images was measured, and migration speed was calculated. Additionally, we investigated the relationship between NPB migration speed and ploidy status (N = 33) or live birth/ongoing pregnancy (LB/OP) (N = 60) after assisted reproduction. RESULTS: The NPB migration speed in both mPN and fPN was significantly faster in the zygotes that developed into blastocysts (N = 25) than that in the zygotes that arrested (N = 22). The timing of blastulation was negatively correlated with NPB migration speed in the mPN. Faster NPB migration was significantly correlated with LB/OP. In multivariate logistic analysis, NPB migration speed in the mPN was the only morphokinetic parameter associated with LB/OP. In a receiver-operating characteristic curve analysis of LB/OP by the NPB migration speed in the mPN, the cut-off value was 4.56 µm/h. When this cut-off value was applied to blastocysts with preimplantation genetic testing for aneuploidy, 100% of the blastocysts faster than or equal to the cut-off value were euploid. CONCLUSION: The NPBs migrated faster in zygotes having the potential to develop into a blastocyst, and eventually into a baby. This predictor could be an attractive marker for non-invasive embryo selection.

Oocyte Spontaneous Activation: An Overlooked Cellular Event That Impairs Female Fertility in Mammals.

In mammals, including humans, mature oocytes are ovulated into the oviduct for fertilization. Normally, these oocytes are arrested at metaphase of the second meiosis (MII), and this arrest can be maintained for a certain period, which is essential for fertilization in vivo and oocyte manipulations in vitro, such as assisted reproduction in clinics and nuclear/spindle transfer in laboratories. However, in some species and under certain circumstances, exit from MII occurs spontaneously without any obvious stimulation or morphological signs, which is so-called oocyte spontaneous activation (OSA). This mini-review summarizes two types of OSA. In the first type (e.g., most rat strains), oocytes can maintain MII arrest in vivo, but once removed out, oocytes undergo OSA with sister chromatids separated and eventually scattered in the cytoplasm. Because the stimulation is minimal (oocyte collection itself), this OSA is incomplete and cannot force oocytes into interphase. Notably, once re-activated by sperm or chemicals, those scattered chromatids will form multiple pronuclei (MPN), which may recapitulate certain MPN and aneuploidy cases observed in fertility clinics. The second type of OSA occurs in ovarian oocytes (e.g., certain mouse strains and dromedary camel). Without ovulation or fertilization, these OSA-oocytes can initiate intrafollicular development, but these parthenotes cannot develop to term due to aberrant genomic imprinting. Instead, they either degrade or give rise to ovarian teratomas, which have also been reported in female patients. Last but not the least, genetic models displaying OSA phenotypes and the lessons we can learn from animal OSA for human reproduction are also discussed.

Morphokinetic analysis of pronuclei using time-lapse cinematography in bovine zygotes.

The morphokinetics of pronuclei (PN) are considered crucial factors affecting embryogenesis in mammals. Whereas, since bovine zygotes contain a large number of cytosolic lipid droplets, detailed observation of PN has not been performed. In this study, we visualized PN using time-lapse cinematography (TLC) with light microscopy for the first time in delipidated bovine zygotes. The proportions of 0 PN, 1PN, 2PN, and multi-PN in delipidated bovine zygotes were 10.1%, 6.5%, 72.7%, and 10.8%, respectively. Abnormal fertilization, including 1 PN and multi-PN, was observed in 15.6% of blastocysts. The times from IVF to PN appearance, PN fading, and first cleavage in 2 PN bovine zygotes that developed into blastocysts were 10.4, 25.5, and 27.6 h, respectively, which were similar to PN morphokinetics in humans. The 2 PN zygotes showed that the prolonged time from IVF to the appearance of PN and from the fading of PN to the first cleavage negatively affected blastocyst formation. The time from appearance to fading of PN in multi-PN zygotes that developed into blastocysts was longer than that in multi-PN zygotes that did not develop into blastocysts. Besides, among zygotes that developed into blastocysts, the time from appearance to fading of PN in multi-PN zygotes was longer than that in 2 PN and 1 PN zygotes. These results suggest that PN morphokinetic abnormalities are associated with subsequent embryonic development. Observation of PN in bovine zygotes by using non-invasive visible light TLC by delipidation could be a powerful tool to clarify the relationship between PN morphokinetics and developmental competence.

Abnormal fertilization in ICSI and its association with abnormal semen parameters: a retrospective observational study on 1855 cases.

Intracytoplasmic sperm injection (ICSI) efficiently addresses male factor infertility. However, the occurrence of abnormal fertilization, mainly characterized by abnormal pronuclei (PN) patterns, merits investigation. To investigate abnormal fertilization patterns following ICSI and identify their respective associations with abnormal parameters in semen analysis (SA), a retrospective observational study including 1855 cycles was performed. Male infertility diagnosis relied on the 2010 WHO criteria. The population was divided into groups based on their SA results. The presence of 2PNs and extrusion of the second polar body (PB) indicated normal fertilization. A Kruskal-Wallis test along with a Wilcoxon post hoc evaluation and Bonferroni correction was employed for comparison among the groups. For the pregnancy rate, logistic regression was employed. No correlation was established between the SA abnormalities and the 1PN or 3PN formation rates. The highest and lowest 0PN rates were reported for the oligoasthenoteratozoospermic and normal groups, respectively. The lowest cleavage formation rates were identified in the oligoasthenozoospermic and oligoasthenoteratozoospermic groups. The aforementioned groups along with the oligoteratozoospermic group similarly presented the lowest blastocyst formation rates. For the clinical pregnancy rate, no statistically significant difference was observed. In conclusion, the incidence of two or more abnormal SA parameters - with the common denominator being oligozoospermia - may jeopardize normal fertilization, cleavage, and blastocyst rates. Once the developmental milestone of achieving blastocyst stage status was achieved, only oligoasthenozoospermia and oligoasthenoteratozoospermia were associated with lower rates. Interestingly, following adjustment for the number of blastocysts, no statistically significant differences were observed.

Mutation analysis of tubulin beta 8 class VIII in infertile females with oocyte or embryonic defects.

Variants of tubulin beta 8 class VIII (TUBB8) have been shown to be associated with female infertility characterized by oocyte or embryonic defects. To further investigate the mutational spectrum of TUBB8 and the prevalence of variants, we performed Sanger sequencing of TUBB8 on a total of 115 infertile females who had undergone repeated in vitro fertilization cycles with oocyte or embryonic defects and 200 healthy controls. A total of 31 variants which were absent from the controls were identified in 36 unrelated individuals, accounting for a large proportion of this cohort (31.3%). All of the variants including heterozygous/homozygous missense variants and a heterozygous frameshift insertion variant were at conserved sites and predicted to be deleterious. Besides, these variants had diverse phenotypic effects, including not only oocyte maturation arrest, fertilization failure, and early embryonic arrest, but also multi-pronuclei (MPN) formation, which is a new phenotype associated with TUBB8 variants. Overall, this study reveals a large number of variants of the TUBB8 gene in infertile females with oocyte or embryonic defects. Our results not only broaden the mutational and phenotypic spectra of TUBB8 variants, but also further confirm the critical role of TUBB8 in oocyte maturation, fertilization, and early embryonic development.