Polarity and early human embryo development

Selection of high potential embryos using time-lapse imaging: the era of morphokinetics

Proposed origin of chimeras, mosaics and monochorionic twins

Clinical-pregnancy outcome after vitrification of blastocysts produced from in vitro maturation cycles

To identify and characterize amyloid-like substance (ALS) in human and mouse oocytes and preimplantation embryos. An experimental prospective pilot study. A total of 252 mouse oocytes and preimplantation embryos and 50 immature and in vitro matured human oocytes and parthenogenetic human embryos, from 11 consenting fertility patients, ages 18-45. Fluorescence intensity from immunofluorescent staining and data from confocal microscopy were quantified. Data were compared by one-way analysis of variance, with the least square-MEANS post-test, Pearson correlation coefficients (r), and bivariate analyses (t tests). ALS morphology was verified using transmission electron microscopy. Immunostaining for ALS appears throughout the zona pellucida, as well as in the cytoplasm and nucleus of mouse and human oocytes, polar bodies, and parthenogenetic embryos, and mouse preimplantation embryos. In mouse, 2-cell embryos exhibited the highest level of ALS (69000187.4 ± 6733098.07). Electron microscopy confirmed the presence of ALS. In humans, fresh germinal vesicle stage oocytes exhibited the highest level of ALS (4164.74088 ± 1573.46) followed by metaphase I and II stages (p = 0.008). There was a significant negative association between levels of ALS and patient body mass index, number of days of ovarian stimulation, dose of gonadotropin used, time between retrieval and fixation, and time after the hCG trigger. Significantly higher levels of ALS were found in patients with AMH between 1 and 3ng/ml compared to < 1ng/ml. We demonstrate for the first time the presence, distribution, and change in ALS throughout some stages of mouse and human oocyte maturation and embryonic development. We also determine associations between ALS in human oocytes with clinical characteristics.

Meiotic spindles in living human oocytes can be visualized by the Polscope. This study investigated the relationship between the presence/location of the spindle in metaphase II (MII) oocytes and developmental competence of embryos in vitro. The spindles in 626 MII oocytes were examined by the Polscope and divided into six groups (A-F) based on the presence or absence of the spindles and the angle between the spindle and the first polar body. After ICSI, the fertilization and embryo development were evaluated. Meiotic spindles were imaged in 523 oocytes (83.5%), while 103 (16.5%) did not have a visible spindle (group F). The majority of oocytes (68.8%) had the spindle directly beneath or adjacent to the first polar body (groups A and B: 48.2 and 20.6%). Oocytes in group C (11.2%) had the spindle located between 60 and 120 degrees angle away from the first polar body, those in group D (2.4%) had the spindle located between 120 and 180 degrees angle and those in group E (1.1%) had the spindle located at 180 degrees angle to the first polar body. The fertilization and embryonic development were similar in the oocytes with spindles regardless of spindle position. However, the rate of high quality embryos was significantly higher in the oocytes (64.2%) with visible spindles than in the oocytes (35.9%) without spindle and multipronuclear proportion showed a slight tendency to increase in oocytes without spindles. (10.7 versus 5.9%, P = 0.12; NS). the presence of a bi-refringent meiotic spindle in human oocytes by using the Polscope can predict a higher embryonic developmental competence. However, the relative position of the spindle within the oocyte doesn't appear to influence the developmental potential of embryos.

A total of 297 human oocytes that had failed to fertilize during in-vitro fertilization (IVF) cycles were exposed to the calcium ionophore A23187 to induce parthenogenetic activation. Of these oocytes, 192 (65%) activated, the majority (63%) exhibiting a single pronucleus and extruding a second polar body. The appearance of two pronuclei (18%) was generally associated with a failure to extrude the second polar body. Oocytes obtained from patients who were > or = 35 years had a significantly reduced activation rate (53%). The timing of developmental events, such as extrusion of the second polar body, appearance and disappearance of pronuclei and the first two cleavage divisions, is broadly similar to that seen in fertilized oocytes. However, the developmental potential of human parthenogenetic embryos was reduced, as the majority of those allowed to continue in culture arrested between the 2-cell and 8-cell stages. Measurements of cellular DNA content using a computerized image analysis system showed that activated oocytes with one pronucleus had a DNA content compatible with a haploid number of chromosomes, while those with two pronuclei were diploid. The ability of parthenogenetically activated oocytes to replicate their DNA was also demonstrated.

Assisted fertilization and embryonic axis formation in higher primates

Study question Can a machine learning-based model predict successful fertilization of oocyte using first polar body morphology? Summary answer With small imbalance oocyte dataset, the most successful model to predict fertilization of mature oocyte is YOLOv8lcls with 97.47% accuracy, 100.00% specificity, and 94.87% sensitivity. What is known already The development of oocytes is crucial in the development and fertility of embryos in In Vitro Fertilization (IVF). There are several internal and external factors that could affect the fertilization competency of oocytes, one of which is the first polar body (PBI). The importance of the quality of PBI in ICSI has long been studied and has been evaluated by scoring systems regarding Istanbul Consensus. However, there is still a debate regarding the influence of PBI on the developmental competency of oocytes. Study design, size, duration This study involved the analysis of retrospective data including the microscopic light image of meiosis II (MII) oocytes at the time of Intracytoplasmic sperm injection (ICSI), along with polar body scoring by embryologists and the fertilization outcome of 1006 MII oocytes from a single public IVF clinic, between 2020-2022. After ICSI, fertilized oocytes were classified into 2 groups: 2PN and non-2PN (e.g., 0PN, 1PN, &amp;gt; =3PN). Participants/materials, setting, methods Light microscopic images of oocytes in the metaphase of meiosis II (MII) stage are captured, followed by the prediction of the likelihood of 2PN development after fertilization through intra-cytoplasmic sperm injection (ICSI). The image has its PBI segmented by the CNN and then cropped out of the original image. The PBI image is then fed into the prediction CNN to obtain the final result. The model performance was determined by evaluating accuracy, sensitivity, and specificity. Main results and the role of chance There were 1006 images of MII oocytes included in this study. After ICSI, 860 oocytes were fertilized resulting in the 2PN stage embryos and were classified as 2PN, whereas 146 oocytes were not fertilized or abnormally fertilized and were classified as non-2PN. Among these images, 782 oocytes received a PBI score of A, 677 of which are 2PN and 105 are non-2PN. The B grade consists of 224 images, of which are 183 2PN, and 41 non-2PN. The dataset was split into 3 subsets for training (70%), validation (10%), and testing (20%). The images are augmented by horizontal flipping to increase the amount of data and allow the model to observe several positions of the PBI. The YOLOv8 segmentation model is applied to segment the PBI and locate its position. The image is then rotated to move the PBI to the top and the PBI is cropped based on its position. Lastly, the cropped PBI image is used to predict the development by using the YOLOv8 Classify model trained on an augmented dataset. The YOLOv8l-cls model achieves the highest accuracy at 97.47%, specificity at 100.00%, and sensitivity at 94.87%. Limitations, reasons for caution The model does not include information on sperm quality, which also essentially contributes to the fertilization process. 2PN stage prediction is just the initial and surrogate outcome in IVF treatment. The model’s interpretation of the PBI score remains inconclusive. Therefore, additional experiments using dataset with balanced PBI grading are recommended. Wider implications of the findings The developed model is the first step in paving the way to establish and implement tools for assisting embryologists in selecting and prioritizing the high-potential oocyte for fertilization. Additional factors such as patient characteristics, other features of oocytes, and male factors should be further integrated to improve model performance. Trial registration number NA

Human oocyte dysmorphisms attain a large proportion of retrieved oocytes from assisted reproductive technology (ART) treatment cycles. Extracytoplasmic defects involve abnormal morphology of the zona pellucida (ZP), perivitelline space and first polar body. The aim of the present study was to describe a novel dysmorphism affecting the ZP, indented ZP. We also evaluated the clinical, embryological and ultrastructural features of these cases. We evaluated all ART treatment cycles during 7 consecutive years and found 13 treatment cycles (six patients) with all oocytes presenting an indented ZP. In addition, these oocytes presented total or partial absence of the perivitelline space, absence of resistance to ZP and oolemma penetration during microinjection, and low ooplasm viscosity during aspiration. This novel described dysmorphism was recurrent and attained all oocytes in three cases that had more than one treatment cycle. When compared with controls, data showed significant low oocyte maturity (42% versus 81.6%) and high cycle cancellation (30.8% versus 8.5%) rates, normal degeneration (3.4% versus 6.3%) and fertilization rates (69% versus 69.5%), and low pregnancy (15.4% versus 33.3%) and live-birth delivery (7.7% versus 27.7%) rates per cycle. Ultrastructure analysis revealed a zona pellucida structure with large empty electrolucent regions, an outer ZP layer with an indented surface with protuberances and a thick inner ZP that obliterated the perivitelline space. There was evidence of exocytosis of ZP material by the oocyte. In conclusion, oocytes with this novel described dysmorphism (indented ZP) are associated with low maturity, pregnancy and live-birth delivery rates.

Three hypotheses were tested: (i) the distance between first and second polar bodies (PB) may relate to embryo morphology, (ii) that the orientation of pronuclei (PN) relative to PB may relate to embryo morphology, (iii) that the placement of a spermatozoon in a fixed plane relative to the first PB [intracytoplasmic sperm injection (ICSI)] may alter PN/PB orientation relative to in-vitro fertilization (IVF). A total of 251 two pronuclear (2PN) embryos (124 ICSI, 127 IVF) from 64 patients was studied. Angles were measured between the PN axis and the nearest PB (alpha), the furthest PB (beta), and between the two PB (gamma). On day 2, the morphological grades of embryos were recorded. gamma ranged from 0 to 150 degrees and was not significantly different for ICSI or IVF embryos of different grades; however, an unusual distribution of gamma suggested different populations of oocytes. The first hypothesis was rejected. alpha and beta ranged from 0 to 90 degrees : alpha did not relate significantly to embryo grade, but beta increased significantly with decreasing quality of ICSI embryos (P < 0.05) and the total group (P < 0.01), supporting hypothesis (ii). The difference in beta between ICSI and IVF embryos was not significant, so hypothesis (iii) was unproven. Significant differences between ICSI and IVF embryos in PN positions, irregular cleavage, and cleavage failure were noted.

Treatment of aged human oocytes by puromycin allows a high rate of parthenogenetic activation and development until the first cleavage division. This technique was used for the study of the chromosome complement of oocytes which remained unfertilized after in vitro fertilization. Three hundred four unfertilized oocytes were treated with 10 micrograms/ml puromycin for 6-8 hr and further cultured for 12-15 hr. Activation occurred in 90.5% of the oocytes. Heterozygous diploids with two pronuclei predominated (61%), which is in contrast to the mouse, where the majority of oocytes activated by puromycin are uniform haploids (89%). Therefore we conclude that puromycin treatment induces retention of the second polar body in human oocytes, unlike in mouse oocytes treated in the same way. Chromosome analysis performed on 182 oocytes suggested a nondisjunction (ND) rate for the second meiotic division of 12.7%. This is a low figure considering the fact that puromycin itself has been reported to induce nondisjunction. For the first meiotic division a ND rate of only 5.6% was found. This rate is lower than the one found in metaphase II arrested oocytes and we believe that this difference is due to the technical differences between the study of meiotic and that of mitotic chromosomes.

Fertilisation and development of dysmorphic human oocytes recovered from hyperstimulated ovaries have been evaluated following intracytoplasmic sperm injection (ICSI) for treatment of male infertility. A total of 2968 oocytes at metaphase II of meiosis were injected, of which 806 (27.2%) were dysmorphic at the light microscopic level. Cytoplasmic abnormalities included granularity, areas of necrosis, organelle clustering, vacuoles, and accumulating saccules of smooth endoplasmic reticulum. Anomalies of the first polar body and zona pellucida, as well as non-spherical shapes of oocytes, were also noted. Contrary to previous findings linking some dysmorphisms to non-assisted fertilisation failure, in this study no single abnormality led to a reduction in the fertilisation rate, nor was fertilisation compromised in oocytes with multiple abnormalities. The incidence of normal fertilisation (two pronuclei and two polar bodies) was 69% in both the dysmorphic and non-dysmorphic oocytes. While overall pregnancy and implantation results were not altered in the group of patients (n = 242) in whom at least one dysmorphic oocyte was injected, exclusive replacement of embryos which originated from dysmorphic oocytes led to a higher incidence of early pregnancy loss. It is concluded that aberrations in the morphology of human oocytes--most probably a product of controlled ovarian stimulation--are of little or no consequence to fertilisation or early cleavage after ICSI. It is possible, however, that these embryos have a reduced potential for implantation and further development.

Employing the carp, Cyprinus carpio L. as the meterial, the processes of maturation of the egg and the phenomena of fertilization were microscopically observed in this study. Observations were carried on in the sections of the eggs, fixed at different intervals ranging from five minutes to 2 hours after fertilization. The majority of eggs were fixed in Carothers' solution and some were preserved in Gilson's fluid. For sectioning the Gilson material proved to be better than the Carothers material. Sections were made following the usual paraffin method. They were stained with Ehrlich's haematoxylin. The results of observations are briefly outlined as follows:1) The first maturation division of the egg is completed prior to ovulation within the ovary. After the extrusion of the first polar body, the chromosomes remaining in the egg are immdiately drawn into the equatorial plate forming the metaphase spindle of the second maturation division (Fig. 4).2) The spawning and fertilization take place in the water at the same time.3) At the time of insemination the second maturation division of the egg persists in the stage of metaphase, and then it gradually advances in further course of division (Fig. 8). About 25 to 30 minutes after insemination the second division is completed, resulting in the formation of the second polar body (Fig. 9).4) During the ovulation process the course of the second division does not proceed being arrested in metaphase. Stages subsequent to the metaphase of the second division take place only after the entry of the spermatozoon into the egg. This fact emphasizes that insemination is a necessary antecedent to the second maturation division to advance further from metaphase.5) Only a single spermatozoon enters an egg (Fig. 7). Thus the carp egg is monospermic in fertilization under the normal condition.6) About 40 to 50 minutes after insemination there is found at the animal pole of the egg the already metamorphosed female pronucleus. It assumes a spherical shape lying in naked state in the ooplasm (Fig. 13).7) As early as 50-60 minutes after entrance, the head of the spermatozoon is converted into the male pronucleus attaining nearly spherical form (Figs. 10-12). At this stage there is no visible demarkation between the male and female pronuclei, so far as their structure and size are concerned. But there is present a ramarkable character peculiar to the male pronucleus. That is the pronounced appearance of the sperm-aster.8) After completion both the pronuclei begin to move towards the deeper part of the animal pole of the egg to conjugate with each other (Fig. 14). The conjugation of the pronuclei generally takes place about 1 hour after insemination occurs (Figs. 15-17).9) After the two pronuclei conjugate together, they do not actually fuse, but lie side by side in closs contact with the nuclear membrance intact (Figs. 15-16). Thus the maternal and paternal chromosome materials are separated in distinct groups during the stages prior to the first cleavage division. The chromosomes are formd independently in each vesicle (Fig. 17).10) In the egg about 11/2 hours after fertilization, the first cleavage spindle is generally formed (Fig. 18).

The human oocyte appears to be particularly prone to meiotic errors, and the incidence of these errors is strongly influenced by maternal age. We have initiated studies of human oocytes from unstimulated ovaries and have observed age-related effects on the meiotic process in oocytes from unselected antral follicles. Specifically, in oocytes obtained from donors over the age of 35 years, the majority of oocytes that extruded a first polar body in culture and arrested at second meiotic metaphase had aberrations in spindle formation and chromosome alignment. Similarly, observations of a limited number of oocytes at first meiotic metaphase suggest disturbances at this stage of meiosis as well. Finally, preliminary results of non-disjunction studies suggest that the frequency of errors in chromosome segregation at the first meiotic division is influenced by donor age in in-vitro matured oocytes as it is in oocytes undergoing meiotic maturation in vivo. These data provide direct evidence that the meiotic competence of oocytes from unstimulated ovaries declines with donor age. Similarly, studies of in-vitro fertilization (IVF) pregnancies in older women indicate that the developmental competence of the human oocyte declines with age. Since both meiotic and developmental competence are acquired during the late stages of oocyte growth, we postulate that an age-related decline in the process of folliculogenesis results in reduced oocyte quality and that the well characterized age-related increase in meiotic non-disjunction is one symptom of compromised oocyte growth.

Five chromosome segregation in polar bodies and the corresponding oocyte