The ultrastructural nature of human oocytes' cytoplasmic abnormalities and the role of cytoskeleton dysfunction.

OBJECTIVE: To investigate the structural bases of human oocytes' cytoplasmic abnormalities and the causative mechanism of their emergence. Knowledge of an abnormal oocyte's intracellular organization is vital to establishing reliable criteria for clinical evaluation of oocyte morphology. DESIGN: Laboratory-based study on experimental material provided by a private assisted reproduction clinic. SETTING: University laboratory and imaging center. PATIENTS: A total of 105 women undergoing hormonal stimulation for in vitro fertilization (IVF) donated their spare oocytes for this study. INTERVENTIONS: Transmission electron microscopy (TEM) was used to analyze the fine morphology of 22 dysmorphic IVF oocytes exhibiting different types of cytoplasmic irregularities, namely, refractile bodies; centrally located cytoplasmic granularity (CLCG); smooth endoplasmic reticulum (SER) disc; and vacuoles. A total of 133 immature oocytes were exposed to cytoskeleton-targeting compounds or matured in control conditions, and their morphology was examined using fluorescent and electron microscopy. MAIN OUTCOME MEASURES: The ultrastructural morphology of dysmorphic oocytes was analyzed. Drug-treated oocytes had their maturation efficiency, chromosome-microtubule configurations, and fine intracellular morphology examined. RESULTS: TEM revealed ultrastructural characteristics of common oocyte aberrations and indicated that excessive organelle clustering was the underlying cause of 2 of the studied morphotypes. Inhibition experiments showed that disruption of actin, not microtubules, allows for inordinate aggregation of subcellular structures, resembling the ultrastructural pattern seen in morphologically abnormal oocytes retrieved in IVF cycles. These results imply that actin serves as a regulator of organelle distribution during human oocyte maturation. CONCLUSION: The ultrastructural analogy between dysmorphic oocytes and oocytes, in which actin network integrity was perturbed, suggests that dysfunction of the actin cytoskeleton might be implicated in generating common cytoplasmic aberrations. Knowledge of human oocytes' inner workings and the origin of morphological abnormalities is a step forward to a more objective oocyte quality assessment in IVF practice.

High-Resolution 3D Reconstruction of Human Oocytes Using Focused Ion Beam Scanning Electron Microscopy.

The egg plays a pivotal role in the reproduction of our species. Nevertheless, its fundamental biology remains elusive. Transmission electron microscopy is traditionally used to inspect the ultrastructure of female gametes. However, two-dimensional micrographs contain only fragmentary information about the spatial organization of the complex oocyte cytoplasm. Here, we employed the Focused Ion Beam Scanning Electron Microscopy (FIB-SEM) to explore human oocyte intracellular morphology in three dimensions (3D). Volume reconstruction of generated image stacks provided an unprecedented view of ooplasmic architecture. Organelle distribution patterns observed in nine donor oocytes, representing three maturational stages, documented structural changes underlying the process by which the egg acquires developmental competence. 3D image segmentation was performed to extract information about distinct organelle populations, and the following quantitative analysis revealed that the mitochondrion occupies ∼ 4.26% of the maturing oocyte cytoplasm. In summary, this proof-of-concept study demonstrates the potential of large volume electron microscopy to study rare samples of delicate female gametes and paves the way for applying the FIB-SEM technique in human oocyte research.

In water or on land? Evaluation of perinatal and neonatal outcomes of water births in low-risk women.

OBJECTIVE: Evaluate perinatal and neonatal outcomes comparing a water birth to regular childbirth in low-risk women. File and methods: Retrospective analysis of a set of childbirths that took place over a given period of time in the hospital and health center of Havířov. We compared a set of low-risk women that had given a water birth to a selected control group of low-risk women that had given regular childbirth. We evaluated statistical comparability, as well as perinatal and neonatal outcomes in both sets. RESULTS: From 1. 1. 2020 to 28. 2. 2021, 1,083 women gave birth in the delivery department of Havířov hospital; from this set 122 were water births (11.3%). In our study, we only included 101 water deliveries (we designed our study to monitor low-risk births in order to be able to statistically correlate our findings; 21 water deliveries were excluded from our study due to perinatal risk factors - gestational diabetes and induced deliveries). We selected 60 low-risk women for our control group. Both sets of women were compared and we ruled out any statistically significant differences in age, education, body mass index, number of births given, gestation week at time of labor, number of smokers, premature rupture of membranes, women with previous history of one cesarean section, becoming pregnant by in vitro fertilization, presence of streptokoka skupiny B, and fetal weight. Water birth does not affect the Apgar score, neonatal adaptation to extra-uterine life, umbilical blood pH decrease, complications of infection, need of intensive care, and neonatal mortality. In the water birth set, we found increased occurrence of non-infectious conjunctivitis, treatable by regular eye drops without antibio-tics. We have not observed the effect of water birth on duration of the first and second stage of labor, total amount of uterotonics used, blood loss determined by the obstetrician, and uterine hypotonia. In the water birth group, we observed a prolonged third stage of delivery, lesser need for pharmacological stimulation (augmentation) of labor, notably lower use of analgesics, lower occurrence of birth injuries, shorter in-patient time, and more frequent bonding. CONCLUSIONS: We discovered that water birth does not increase the risk for mother and neonate in low-risk women. Despite initial concerns, our outcomes and mother satisfaction have clearly shown that water births are not only a temporary whim, but probably a new integral part of our obstetric care.

Live birth achieved despite the absence of ejaculated spermatozoa and mature oocytes retrieved: a case report.

The most common reason for in vitro fertilization (IVF) cycle cancelation is a lack of quality gametes available for intracytoplasmic sperm injection (ICSI). Here we present the successful fertility treatment of the couple affected by obstructive azoospermia combined with suboptimal response to controlled ovarian stimulation. Since the conventional approach appeared ineffective to overcome both partners' specific problems, the targeted interventions, namely, (1) pharmacological enhancement of sperm motility and (2) polarized light microscopy (PLM)-guided optimization of ICSI time, were applied to rescue the cycle with only immature oocytes and immotile testicular sperm retrieved. The treatment with theophylline aided the selection of viable spermatozoa derived from cryopreserved testicular tissue. When the traditional stimulation protocol failed to produce mature eggs, non-invasive spindle imaging was employed to adjust the sperm injection time to the maturational stage of oocytes extruding a polar body in vitro. The fertilization of 12 late-maturing oocytes yielded 5 zygotes, which all developed into blastocysts. One embryo was transferred into the uterus on day 5 post-fertilization, and another 3 good quality blastocysts were vitrified for later use. The pregnancy resulted in a full-term delivery of a healthy child. This case demonstrates that the individualization beyond the standard IVF protocols should be considered to maximize the chance of poor-prognosis patients to achieve pregnancy with their own gametes.

Egg maturity assessment prior to ICSI prevents premature fertilization of late-maturing oocytes.

PROPOSE: The presence of metaphase II (MII) spindle together with the polar body (PB) indicates completion of oocyte maturation. This study was designed to explore if spindle imaging can be used to optimize timing of intracytoplasmic sperm injection (ICSI). METHODS: The study involved 916 oocytes from 234 conventionally stimulated ICSI cycles with an unexpectedly poor ovarian response. All PB-displaying oocytes were subjected to polarized light microscopy (PLM) prior to ICSI. When MII spindle was absent in the majority of oocytes, ICSI was postponed and performed after additional spindle imaging. Fertilization, embryo development, and clinical outcome were evaluated with respect to the observed spindle pattern. RESULTS: The visible spindle was absent in 32.64% of PB-displaying oocytes. The late-maturing oocytes extruding PB in vitro were less likely to exhibit a spindle signal than in vivo matured MII oocytes (38.86% vs. 89.84%). When fertilization was postponed, 59.39% of initially spindle-negative oocytes developed detectable MII spindle. Spindled eggs had significantly higher developmental potential, and the presence of the spindle has been identified as an independent measure for predicting the formation of the blastocyst. Embryos derived from spindle-positive oocytes also showed a higher chance to implant and develop to term. Notably, 11 children were conceived by finely timed fertilization of late-maturing oocytes which are normally discarded. CONCLUSIONS: The study confirms the prognostic value of spindle imaging and demonstrates that immature oocytes can be clinically utilized and give rise to live births when the timing of ICSI is adjusted to their developmental stage.