Katerina Chatzimeletiou

Comparison of effects of zona drilling by non-contact infrared laser or acid Tyrode's on the development of human biopsied embryos as revealed by blastomere viability, cytoskeletal analysis and molecular cytogenetics

Use of a non-contact infrared laser (IRL) or acid Tyrodes for zona drilling before embryo biopsy was compared by assessing blastomere viability using various fluorescent markers or culture of the single biopsied blastomere, and, by cytoskeletal and molecular cytogenetic analysis of the biopsied embryos following culture to the blastocyst stage. There was no significant difference in the proportion of biopsied embryos that showed no damage in both the biopsied blastomere and in the remaining embryo (acid Tyrodes: 75% versus IRL: 68%), or in the proportion of single biopsied blastomeres that divided in culture (P > 0.05). However, single biopsied blastomeres from laser drilled embryos showed a greater tendency to form miniblastocysts. The proportion of laser or acid Tyrodes biopsied embryos that reached the blastocyst stage by day 6 was similar, although evident earlier (day 5) in the laser biopsied embryos. Spindle abnormalities at the blastocyst stage included tripolar and tetrapolar spindles, but their incidence was not significantly different from controls. In addition, no significant difference was observed in the incidence of chromosomal abnormalities and mosaicism between the two groups. It is concluded that using an IRL at a safe working distance does not cause adverse immediate or longer term effects on the development of human biopsied embryos, although damage can occur if drilling within this distance is unavoidable. Acid Tyrodes drilling can also cause damage, and tended to retard blastocyst development.

The centrosome and early embryogenesis: clinical insights.

In humans and most mammals, with the exception of some rodents notably the mouse, the centrosome, which is the organizing centre of the spindle, is uniparentally (paternally) inherited. The sperm centrosome is transmitted to the egg at fertilization, forming an aster comprising radially arrayed microtubules that brings the male and female pronuclei into close apposition and organizes the first mitotic spindle in the zygote. Each centrosome contains a pair of centrioles that are oriented perpendicular to one another, surrounded by dense fibrillar pericentriolar material, within which functional and regulatory molecules are embedded. The centrosome is a complex organelle with a crucial role to play in human fertility and the ability of human embryos to undergo normal development. Centrosomal defects can lead to failure in fertilization, and cause embryonic arrest through the formation of abnormal spindles and the accumulation of chromosomally abnormal cells that derive from them. This paper is a brief review of the role of the centrosome and the implications of its dysfunction in early embryogenesis, with particular reference to the human embryo.

Cytoskeletal analysis of human blastocysts by confocal laser scanning microscopy following vitrification

BACKGROUND Vitrification of human blastocysts is being used increasingly to cryopreserve supernumerary embryos following IVF. In this study, we investigate the effects of aseptic vitrification on the cytoskeleton and development of human blastocysts, by analysing survival rates and spindle and chromosome configurations by fluorescence and confocal laser scanning microscopy. METHODS A total of 55 fresh blastocysts and 55 day 5 dimethylsulphoxide/ethylene glycol vitrified blastocysts, which were allowed to remain in culture for 24 h post-warming, were rapidly fixed in ice cold methanol, and immunostained with an a-tubulin antibody to visualize microtubules in combination with antibodies against acetylated tubulin (to visualize spindles, poles and mid bodies), gamma tubulin (to identify spindle poles) and 4(6-diamidino-2-phenylindole) to visualize DNA. RESULTS In total, 213 spindles were analysed in the control (fresh) group of which 183/213 (85.9%) were normal, 20/213 (9.4%) were abnormally shaped, 9/213 (4.2%) were multipolar and 1/213 (0.5%) was monopolar. A total of 175 spindles were analysed in the vitrified group, of which 120/175 (68.6%) were normal, 39/175 (22.3%) were abnormally shaped, 10/175 (5.7%) were multipolar and 6/175 (3.4%) were monopolar. The incidence of multipolar spindles was similar in the two groups, but the level of abnormally shaped spindles, often associated with chromosome lagging, or congression failure, was significantly higher in the vitrified group compared with the fresh group (P< 0.05). CONCLUSIONS The high survival rate following thawing and the large proportion of normal spindle/chromosome configurations suggests that vitrification at the blastocyst stage on Day 5 does not adversely affect the development of human embryos and the ability of spindles to form and continue normal cell divisions. However, there was a significantly higher incidence of abnormal spindles in the vitrified group compared with the fresh group, notably of spindles with a focused and an unfocused pole as well as chromosome bridging and disorganized middle spindle fibres at telophase. Further investigation is warranted to elucidate the mitotic stages that are more vulnerable to damage during vitrification, the fate of the abnormal spindles and any potential effects that may be reflected on the chromosomal constitution of the developing blastocysts.

Use of a non-contact, infrared laser for zona drilling of mouse embryos: assessment of immediate effects on blastomere viability.

To compare the effects of using a non-contact infrared laser with acid Tyrodes for zona drilling, mouse embryos were used to examine both immediate effects on blastomere viability, using fluorescent markers, and development to the blastocyst stage in vitro. Preliminary experiments with isolated blastomeres established that the safe working distance for the lowest setting of the laser (pulse duration 5 ms) was 8 micro m. At the 2-cell stage, when it was possible to drill through the zona while maintaining this safe distance from adjacent blastomeres, no immediate damage was detected. At the 8-cell stage, blastomeres are generally <8 micro m away from the outer surface of the zona and often touch its inner surface. Hence it was not possible to drill the zona while maintaining a safe distance and as a consequence almost all embryos had at least one, and often more, damaged blastomeres. Acid Tyrodes drilling also frequently lysed an adjacent blastomere at this stage and after 24 h in culture cavitation was delayed compared with laser-drilled embryos. Cell numbers at the late morula/early blastocyst stage, analysed by differential labelling of trophectoderm and inner cell mass nuclei, were significantly reduced following zona drilling. With the laser-drilled embryos, this was in proportion to the immediate effects on blastomere viability, whereas with acid Tyrodes, the reduction was greater, indicating an adverse effect on cleavage rate. It is concluded that use of a non-contact laser, at an appropriate pulse duration and distance from adjacent blastomeres, avoids blastomere lysis and adverse effects on development caused by acid Tyrodes zona-drilling.

Corifollitropin alfa compared with follitropin beta in poor responders undergoing ICSI: a randomized controlled trial

STUDY QUESTION Does substituting 150 µg corifollitropin alfa for 450 IU follitropin beta during the first 7 days of ovarian stimulation in proven poor responders, result in retrieval of a non-inferior number (<1.5 fewer) of cumulus oocyte complexes (COCs)? SUMMARY ANSWER A single s.c. dose of 150 µg corifollitropin alfa on the first day of ovarian stimulation, followed if necessary, from Day 8 onwards, with 450 IU of follitropin beta/day, is not inferior to daily doses of 450 IU follitropin beta. The 95% CI of the difference between medians in the number of oocytes retrieved was -1 to +1 within the safety margin of 1.5. WHAT IS KNOWN ALREADY Recent data from retrospective studies suggest that the use of corifollitropin alfa in poor responders is promising since it could simplify ovarian stimulation without compromising its outcome. STUDY DESIGN, SIZE, DURATION Seventy-nine women with previous poor ovarian response undergoing ICSI treatment were enrolled in this open label, non-inferiority, randomized clinical trial (RCT). PARTICIPANTS/MATERIALS, SETTING, METHODS Inclusion criteria were: previous poor response to ovarian stimulation (≤4 COCs) after maximal stimulation, age <45 years, regular spontaneous menstrual cycle, body mass index: 18-32 kg/m(2) and basal follicle stimulating hormone ≤20 IU/l. On Day 2 of the menstrual cycle, patients were administered either a single s.c dose of 150 µg corifollitropin alfa (n = 40) or a fixed daily dose of 450 IU of follitropin beta (n = 39). In the corifollitropin alfa group, 450 IU of follitropin beta were administered from Day 8 of stimulation until the day of human chorionic gonadotrophin (hCG) administration, if necessary. To inhibit premature luteinizing hormone surge, the gonadotrophin releasing hormone antagonist ganirelix was used. Triggering of final oocyte maturation was performed using 250 µg of recombinant hCG, when at least two follicles reached 17 mm in mean diameter. MAIN RESULTS AND THE ROLE OF CHANCE The number of COCs retrieved was not statistically different between the corifollitropin alfa and the follitropin beta groups [Median 3 versus 2, 95% CI 2-4, 2-3, respectively, P = 0.26]. The 95% CI of the difference between medians in the number of oocytes retrieved was -1 to +1. A multivariable analysis adjusting for all the potential baseline differences confirmed this finding. No significant difference was observed regarding the probability of live birth between the corifollitropin alfa and the follitropin beta group (live birth per patient reaching oocyte retrieval: 7.9 versus 2.6%, respectively, difference +5.3%, 95% CI: -6.8 to +18.3). LIMITATIONS, REASONS FOR CAUTION The present study was not powered to test a smaller difference (e.g. 1 COC) in terms of COCs retrieved as well as to show potential differences in the probability of pregnancy. Moreover, it would be interesting to assess whether the continuation of stimulation in the long acting FSH arm, where necessary, with 200 IU instead of 450 IU of follitropin beta would have altered the direction or the magnitude of the effect of the type of FSH, observed on the number of COCs retrieved. WIDER IMPLICATIONS OF THE FINDINGS Corifollitropin alfa simplifies IVF treatment because it is administered in a GnRH antagonist protocol and replaces seven daily FSH injections with a single one of a long acting FSH without compromising the outcome. It could greatly reduce the burden of treatment for poor responders and this deserves further investigation.

Semen analysis by electron and fluorescence microscopy in a case of partial hydatidiform mole reveals a high incidence of abnormal morphology, diploidy, and tetraploidy.

OBJECTIVE To perform a highly detailed semen analysis in a man whose wife had a partial mole. DESIGN Case report. SETTING Gynecology departments of two university hospitals and a laboratory of histology/embryology. PATIENT(S) A 32-year-old man whose wife had a partial mole. INTERVENTION(S) Sperm characteristics were examined by light microscopy, morphology was analysed by electron microscopy (TEM), DNA fragmentation was evaluated by TUNEL using fluorescence microscopy, and chromosomal abnormalities were assessed by fluorescence in situ hybridization using probes for chromosomes 13, 15, 16, 18, 21, 22, X, and Y. MAIN OUTCOME MEASURE(S) Sperm count, motility, morphology, DNA fragmentation, and incidence of diploidy, tetraploidy, and aneuploidy. RESULT(S) Sperm concentration was 61 million/mL, with 31% progressive motility and 4% normal morphology. TEM revealed a high incidence of head, neck, and tail abnormalities as well as the presence of phagocytes. DNA fragmentation was within normal limits (11.6%). Aneuploidy levels were low for all chromosomes tested. However, there was a high level of diploidy, with XY, XX, and YY constitution. Tetraploid sperm (XXYY) were also noted. CONCLUSION(S) Semen analysis revealed a high incidence of abnormal morphology and increased diploidy. It may be important to perform FISH testing, to verify increased diploidy in sperm, in men whose wives have had partial moles. These couples could be informed of the option to have preimplantation genetic diagnosis as a means to distinguish between diploid and triploid embryos arising from fertilization of a haploid egg by diploid sperm.

Paternal inheritance of a 16qh-polymorphism in a patient with repeated IVF failure

Polymorphisms of the size of heterochromatic centromeric regions of chromosomes have been well documented in the human. They appear to have no phenotypic effects in the carriers. However, they appear to be over-represented in infertile couples and those with repeated miscarriages, and there is now growing evidence that they are involved in meiotic pairing, spindle fibre attachment and chromosome movement. Here an analysis of inheritance is reported for a couple presenting with repeated IVF failure in which several embryos were identified as carriers of a polymorphism of the centromeric region of chromosome 16 (16qh-) following aneuploidy screening by sequential fluorescence in-situ hybridization (FISH), using probes for chromosomes 13, 16, 18, 21, 22, X and Y. Detailed cytogenetic analysis by high-resolution banding and FISH of both parents and grandparents established that the polymorphism was familial and inherited from the maternal grandfather. Furthermore, complete analysis of all embryonic nuclei from carrier embryos and others rejected for transfer because of aneuploidy revealed no abnormalities in the segregation pattern of chromosome 16.

Effects of vitrification on blastomere viability and cytoskeletal integrity in mouse embryos.

Vitrification is widely used to cryopreserve supernumerary embryos following in vitro fertilization (IVF). The mouse model was used to investigate the effects of vitrification on blastomere viability, using viability markers, and on the cytoskeleton, by analysing spindle/chromosome configurations, using confocal scanning microscopy. Ninety cleavage and morula stage dimethyl sulphoxide (DMSO)/EG vitrified mouse embryos were either processed immediately following warming for viability assessment by labelling with the fluorescent markers carboxyfluorescein-diacetate succinimidylester (CFSE) and propidium iodide (PI) or were cultured to the blastocyst stage and immunostained with α-tubulin antibody to visualize microtubules and DAPI or PI to visualize DNA. Sixty-five fresh embryos were also used as the control. Vitrified embryos showed high survival rates following warming, but they had a higher incidence of damaged blastomeres compared with fresh embryos. Most mitotic spindles examined in all groups were normal, but multivariable analysis revealed that the proportion of abnormal spindles was significantly higher in vitrified/warmed embryos (P < 0.05). This study is the first to examine the immediate effects of vitrification on blastomere viability, using fluorescent markers and shows that although vitrification results in a higher incidence of damaged blastomeres, vitrified embryos may compensate for this limited number of damaged/abnormal cells, as development to the blastocyst stage was not compromised.

Is the time interval between HCG administration and oocyte retrieval associated with oocyte retrieval rate

The aim of this study was to evaluate whether prolongation of the time interval between HCG administration and oocyte retrieval, from 36 h to 38 h, affects oocyte retrieval rate in women undergoing ovarian stimulation with gonadotrophins and GnRH antagonists for IVF. One hundred and fifty-six normo-ovulatory women were randomized to have oocyte retrieval performed 36 h (n = 78) or 38 h (n = 78) following HCG administration. Oocyte retrieval rate was defined as number of cumulus-oocyte-complex (COC) retrieved/follicle ≥ 11 mm present on day of HCG administration. No significant differences were observed between the groups regarding baseline characteristics. Moreover, no significant difference was observed between the groups regarding oocyte retrieval rate (difference: + 1.2%, 95% CI for difference between medians: -4.5 to +12.1). The median (95% CI for the median) was not significantly different between the groups regarding number of cumulus-oocyte-complexes (COCs) retrieved: 5.5 (5.0-7.0) versus 6.0 (5.0-6.2), respectively, and fertilization rates: 57.7% (50.0-66.7) versus 50.0% (44.8-65.5), respectively. Live birth rates were similar between the groups (20.5% versus 16.7%, RD: + 3.8%, 95% CI: -8.5 to +16.1, respectively). Prolongation of time interval between HCG administration and oocyte retrieval from 36 h to 38 h does not affect oocyte retrieval rate.

Artificial oocyte activation: physiological, pathophysiological and ethical aspects

ABSTRACT Infertile couples with low oocyte yield in combination with abnormal semen parameters may experience intra-cytoplasmic sperm injection (ICSI) failure. An established factor associated with ICSI failure is oocyte activation deficiency (AOD). The latter originates from seminal contributors, such as phospholipase C-zeta (PLCζ) that is not adequate to produce calcium (Ca2+) oscillations for oocyte activation. Apart from this natural activator, other stimulants, such as A23187, ionomycin, strontium chloride or even electric pulses, have been used in embryological laboratories to overcome AOD and ICSI failure. The aim of the present narrative review is to discuss the role of Ca+2 oscillations in oocyte activation and summarize the evidence concerning the use of oocyte activators as agents for artificial oocyte activation (AOA). Studies in humans and animals have emerged many physiological, pathophysiological and ethical aspects of AOA. In conclusion, in mammalian eggs, the cytosolic Ca+2 oscillations derive from a periodic release of Ca+2 from intracellular pools. PLCζ, as well as artificial stimulants, have been used to produce Ca+2 oscillations for AOA. As the latter may increase the risk of epigenetic induced malformations, further studies are required to clarify whether AOA constitutes an effective and safe method to overcome ICSI failure. Abbreviations: AOA: artificial oocyte activation; AOD: oocyte activation deficiency; Ca+2: Calcium; CAMKII: Ca+2/calmodulin-dependent protein kinase II; CICR: calcium-induced calcium-release; DAG: diacylglycerol; GM-CSF: granulocyte-macrophage colony-stimulating factor; ICSI: intra-cytoplasmic sperm injection; InsP3R: inositol-trisphosphate receptor; IP3: inositol 1,4,5-trisphosphate; IVF: in vitro fertilization; MAP: mitogen-activated protein; MII: metaphase II; NADP: nicotinic acid adenine dinucleotide phosphate; NO: nitric oxide; PAWP: post-acrosomal WW-binding domain protein; PIP2: phosphatidylinositol 4,5-bisphosphate; PLC: phospholipase C; PLCζ: phospholipase C-zeta; SOAFs: spermatozoon-released oocyte-activating factors; Sr+2: strontium; TFF: total fertilization failure