Hanna Balakier

Color Doppler assessment of folliculogenesis in in vitro fertilization patients

OBJECTIVES To characterize hemodynamics of human ovarian follicles in the course of their development and to compare the results in relation to the age of patients, pregnancy, location of the ovary, and the quality of the oocytes. DESIGN Serial measurements of peak velocity and resistance index by color Doppler imaging. SETTING The In Vitro Fertilization Clinic, Success Through Assisted Reproductive Technologies, Inc., Toronto, Ontario, Canada. PATIENTS Fifty-two women undergoing hormonal stimulation for IVF. MAIN OUTCOME MEASURES The perifollicular peak velocity and resistance index values. RESULTS The perifollicular peak velocity values were gradually increasing with the increasing size of the follicles. Highly significant elevation of the peak velocity was observed especially after hCG injection. Such rapid rise of blood velocity was greater in the right ovary than in the left in the younger patients (mean age, 34 years) whereas in older patients (mean age, 41 years), the greater values were observed in the left ovary. There was no significant difference in peak velocity or resistance index between pregnant and nonpregnant patients. CONCLUSIONS There is a strong, positive correlation between the size of ovarian follicles and their peak velocity, which suggests an increase of blood flow around developing follicles in the course of the follicular phase. Human chorionic gonadotropin plays an important role in inducing influx of blood within follicles; however, the significance of this event remains obscure. It appears that the resistance index is not a useful parameter for characterization of the intrafollicular flow. Changes in peak velocity did not correlate with oocyte quality and may have no predictive value for IVF outcome.

Sperm-derived WW domain-binding protein, PAWP, elicits calcium oscillations and oocyte activation in humans and mice

Mammalian zygotic development is initiated by sperm‐mediated intracellular calcium oscillations, followed by activation of metaphase II‐arrested oocytes. Sperm postacrosomal WW binding protein (PAWP) fulfils the criteria set for an oocyte‐activating factor by inducing oocyte activation and being stored in the perinuclear theca, the sperm compartment whose content is first released into oocyte cytoplasm during fertilization. However, proof that PAWP initiates mammalian zygotic development relies on demonstration that it acts upstream of oocyte calcium oscillations. Here, we show that PAWP triggers calcium oscillations and pronuclear formation in human and mouse oocytes similar to what is observed during intracytoplasmic sperm injection (ICSI). Most important, sperm‐induced calcium oscillations are blocked by coinjection of a competitive inhibitor, derived from the WWI domain‐binding motif of PAWP, implying the requirement of sperm PAWP and an oocyte‐derived WWI domain protein substrate of PAWP for successful fertilization. Sperm‐delivered PAWP is, therefore, a unique protein with a nonredundant role during human and mouse fertilization, required to trigger zygotic development. Presented data confirm our previous findings in non‐mammalian models and suggest potential applications of PAWP in the diagnosis and treatment of infertility.—Aarabi, M., Balakier, H., Bashar, S., Moskovtsev, S. I., Sutovsky, P., Librach, C. L., and Oko, R., Sperm‐derived WW domain‐binding protein, PAWP, elicits calcium oscillations and oocyte activation in humans and mice. FASEB J. 28, 4434–4440 (2014). www.fasebj.org

Chromosome condensation activity in the cytoplasm of anucleate and nucleate fragments of mouse oocytes

The activity of maturation promoting factor (MPF) which causes chromosome condensation and subsequent oocyte maturation was investigated in mouse oocytes using polyethylene-glycol-mediated cell fusion technique. Fully grown oocytes were bisected at germinal vesicle (GV) stage or shortly after germinal vesicle breakdown (GVBD) into anucleate and nucleate fragments. After 2-3 or 15-17 hr of culture these fragments were fused with interphase blastomeres from two-cell embryos. It was found that almost all the anucleate oocyte fragments cultured for a short term (2-3 hr), regardless of whether they were produced at GV stage or after GVBD, induced premature chromosome condensation in the blastomere nuclei, whereas only about 20% of those cultured for a long term (15-17 hr) could do so. On the other hand, the nucleate fragments always retain the cytoplasmic activity to induce chromosome condensation. Thus we suggested that the MPF initially could appear in mouse oocytes independently of the GV, that the mixing of GV material with the oocyte cytoplasm following GVBD had no effect on the activity of MPF in anucleate fragments, and that oocyte chromosomes or some components associated with them could play a significant role in maintaining the MPF activity.

A morphologic study of unfertilized oocytes and abnormal embryos in human in vitro fertilization

The morphology of human, unfertilized oocytes and abnormal embryos cultured in vitro for 48–72 hr was examined in an attempt to learn more about oocyte maturation and reproductive failure in in vitro fertilization (IVF). About 21% of the unfertilized oocytes were totally degenerated. The majority (56%) of the remaining oocytes was arrested at the metaphase II stage. They contained coherent chromosomal plates and had extruded the first polar body with nuclear material. About 13% of oocytes underwent spontaneous activation. In most of these cases the second polar body was retained and many subnuclei or one big nucleus was formed. Five percent of metaphase II oocytes penetrated by sperm were not activated, likely as a result of oocyte immaturity. The developmental ability of abnormal embryos was poor. Seyeral one-cell-stage zygotes were arrested at the pronuclear stage or at mitosis of the first mitotic division. Poiyspermic embryos, especially those which contained four or more pronuclei, did not divide or formed uneven, multinucleated blastomeres. However, some triploid and tetraploid embryos often appeared normal morpholotisolty despite their lethal chromosomal abnormalities.

The effect of cryopreservation on the development of S- and G2-phase mouse embryos

The survival rate and development of four-cell-stage mouse embryos frozen and thawed in S phase versus G2 phase was compared. Significantly more G2-phase than S-phase embryos survived freezing and thawing. In both groups, disruption of the zona pellucida, fusion of blastomeres, and dispersion of chromosomes were occasionally observed after thawing. Cryopreservation resulted in a longer delay in cleavage from the four-to the eight-cell stage of S (about 5 hr)- and G2-phase embryos (about 3 hr) compared to unfrozen controls. The number of frozen embryos which developed to the blastocyst stage was reduced compared to controls, and in the case of S-phase embryos, formation of the blastocyst cavity was also delayied. However, the average number of cells in the experimental and control embryos was similar. No increased incidence of chromosome abnormalities was seen. Our results suggest that freezing embryos in G2 is superior to freezing in S phase.

Is the zona pellucida thickness of human embryos influenced by women's age and hormonal levels?

OBJECTIVE To evaluate whether zona pellucida thickness (ZPT) of human embryos is correlated with maternal age, patients hormonal status, embryo quality, and IVF outcomes. DESIGN Prospective study. SETTING University-affiliated IVF clinic. PATIENT(S) Couples undergoing IVF-ET cycles. INTERVENTION(S) Zona measurements, clinical data collection. MAIN OUTCOME MEASURE(S) Correlation between the ZPT and maternal age, basal FSH and E(2) levels, stimulation protocols, cause of infertility, embryo quality, and implantation/pregnancy rates. RESULT(S) The measurements of ZPT were collected from 5,184 day 3 human embryos originated from 744 IVF patients. The overall mean ZPT was 16.18 ± 2.00 μm. No significant correlation was observed between the ZPT and the patients age, E(2) values on the day of hCG administration, basal concentration of serum FSH, stimulation protocol, infertility diagnosis, and implantation/pregnancy rates. The ZPT was strongly influenced only by the embryo quality: Embryos with good morphology exhibited considerably thinner ZP compared with those of less favorable morphology (mean 15.87 ± 2.48 μm vs. 16.36 ± 2.57 μm, respectively). The ZPT had no significant impact on the implantation and pregnancy rates. CONCLUSION(S) The thickness of the human ZP of day 3 embryos is not influenced by womens age and hormonal levels. The strong correlation between ZPT and embryo quality suggests that thickness of ZP depends on inherent embryo properties. The overall ZPT is not a good predictive indicator for IVF clinical outcomes.

Laser zona thinning in women aged ≤37 years: a randomized study

The objective of this prospective randomized double-blind clinical trial was to evaluate whether laser zona pellucida thinning of human embryos improves clinical outcomes in women <or=37 years old undergoing IVF-ET treatment. The study did not reveal any significant beneficial effect of laser zona thinning on clinical pregnancy (16 out of 45 vs. 18 out of 39) and live birth rates (13 out of 45 vs. 16 out of 39) between the laser-treated and nontreated groups of patients, although there was a trend toward an increased incidence of dichorionic multiple pregnancies in the study group compared with the control group (7 out of 13 vs. 4 out of 16).

Interactions between metaphase and interphase factors in heterokaryons produced by fusion of mouse oocytes and zygotes

The cytoplasmic factor responsible for chromosome condensation was introduced into mouse zygotes at different times after fertilization by fusion of the zygotes with metaphase I oocytes. In 72% of heterokaryons obtained after fusion of early zygotes (14-18 hr post-human chorionic gonadotrophin (HCG) with oocytes, the male and female pronuclei of the zygote decondensed. At the same time, the oocyte chromosomes became enclosed in a nuclear envelope and decondensed to an interphase state. However, in the rest of the heterokaryons, the chromatin of the pronuclei condensed to metaphase chromosomes, thus resulting in three sets of chromosomes. Fusion of zygotes that had begun DNA synthesis (20-22 hr post-HCG) with oocytes induced chromosome condensation of the pronuclei in 76% of the cases. In some heterokaryons, however, the oocyte chromosome decondensed to an interphase state similar to the zygote pronuclei. Fusion between late zygotes (27-29 hr post-HCG) with oocytes resulted in chromosome condensation of the pronuclei in all heterokaryons. On the basis of these results, the formation of the pronuclei and their progression toward mitosis in the zygote may be explained by changing levels of a metaphase factor in the cell, or by a balance between interphase and metaphase factors.

Expression of survivin in human oocytes and preimplantation embryos

OBJECTIVE To determine whether [1] survivin is expressed in human oocytes and embryos; [2] embryos grown in vitro secrete survivin protein; and [3] survivin levels are correlated with embryo cleavage rates. DESIGN Experimental. SETTING University-affiliated IVF clinic. PATIENT(S) Couples undergoing IVF-ET cycles. INTERVENTION(S) Conventional reverse transcriptase-polymerase chain reaction (PCR), real-time PCR, immunohistochemistry, Western blot on oocytes, embryos and control choriocarcinoma JEG-3 cells, and ELISA analysis of conditioned culture media. MAIN OUTCOME MEASURE(S) Detection of survivin mRNA and protein in oocytes and preimplantation embryos and in JEG-3 cancer cells. Detection of survivin concentrations in embryo culture media. RESULT(S) Survivin mRNA and protein were expressed during human oocyte maturation, from germinal vesicle to metaphase II stage, and throughout embryo development, from pronuclear stage to blastocyst stage. Survivin was localized predominantly in the cytoplasm of all cells examined and in the oocytes on the chromatin of metaphase chromosomes and midbodies. Western blot analysis of human oocyte and cancer cell extracts detected a full-length (primary) survivin band of 16.5 kDa. Survivin was also detected in conditioned media samples from embryo cultures and showed a positive correlation with embryo cleavage rates. CONCLUSION(S) Our data have demonstrated for the first time that human oocytes/embryos not only express but also secret survivin, suggesting that survivin may play an important role in human oogenesis and embryogenesis.

Binucleate cells in mouse morulae

SummaryMouse morulae from two strains were examined in whole mounts after dissociation of embryos into single cells and were analysed in serial sections by light and electron microscopy. One or two binucleate cells per embryo were discovered in a statistically significant number of morulae. The frequency of morulae with binucleate cell(s) was higher in older morulae than in younger ones. Binucleate cells were always the outer cells of the embryo. Their ultrastructure did not differ from the ultrastructure of mononucleate cells. It is suggested that cell binuclearity at the morula stage is a possible way to polyploidization of nuclei, resulting in the formation of primary trophoblast giant cells.