Yannis Prapas

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.

Open versus closed oocyte vitrification system: a prospective randomized sibling-oocyte study

Vitrification has been successfully applied in the cryopreservation of oocytes and embryos. It can be achieved either by direct (open system) or indirect (closed system) contact with liquid nitrogen. Unlike embryo vitrification, few reports have been published regarding oocyte vitrification in closed systems. In order to validate the effectiveness of a closed and aseptic vitrification approach for oocyte cryopreservation, a prospective, randomized study was performed. Sibling oocytes donated from the same donor were randomly and equally assigned into closed or open vitrification groups. A total of 75 vitrification-warming cycles were performed in each group. Apart from the survival rate (82.9% versus 91.0%, P<0.05), no statistically significant differences were observed in pregnancy (β-human chorionic gonadotrophin positive) (42.7% versus 33.3%), clinical pregnancy (36.0% versus 28.0%), implantation (13.8% versus 10.1%), ongoing pregnancy (33.3% versus 24.0%) and live birth (36.0% versus 24.0%) rates between the closed and open groups, and 27 and 18 healthy babies were born, respectively. This study shows that the replacement of the open vitrification system by a closed system has no impact on clinical pregnancy and implantation rates. Therefore, the closed vitrification system provides an aseptic alternative to the open method for oocyte vitrification.

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.

GnRH antagonists and endometrial receptivity in oocyte recipients: a prospective randomized trial

The effect that gonadotrophin-releasing hormone (GnRH) antagonists exert on endometrial receptivity has not yet been elucidated. GnRH antagonists might directly affect oocytes, the embryo and/or the endometrium. The aim of this study was to investigate the direct effect of GnRH antagonists on the endometrium in oocyte donation cycles. In an oocyte donation programme, oocytes from each donor (n = 49), stimulated with gonadotrophins and a GnRH antagonist, were equally shared between two different matched recipients. Recipients were randomly allocated to either receive a GnRH antagonist concomitant to donor during their endometrial priming with oestradiol (group I, n = 49) or to solely continue with their endometrial preparation (group II, n = 49). Pregnancy rate was 55.1% in group I and 59.1% in group II. Implantation rate was 26.1% in group I and 24.4% in group II. Endometrial thickness was also similar between the two groups on the day of human chorionic gonadotrophin injection to the donor. In conclusion, GnRH antagonist administration during the proliferative phase at a dose of 0.25 mg per day does not appear to adversely affect endometrial receptivity in oocyte recipients.

Open versus closed vitrification of blastocysts from an oocyte-donation programme: a prospective randomized study

The use of open carriers for embryo vitrification has raised safety concerns and therefore vitrification in closed systems has been proposed. However, the drop in the cooling rate emerges as a major drawback. The objective of the present study was to compare the efficiency of vitrification in open versus closed conditions. Blastocysts were randomly allocated either to open ultra-rapid vitrification (group I) or closed aseptic vitrification (group II). In group I, blastocysts were exposed to two solutions of ethylene glycol/dimethylsulphoxide (10%/10% and 20%/20%), while in group II, blastocysts were pretreated with a solution of lower concentration (5%/5%). A total of 208 and 224 vitrification-warming cycles were performed for groups I and II, respectively. Both groups were equal in terms of maternal age, sperm parameters and number and quality of blastocysts vitrified, warmed and transferred per cycle. Importantly, there was no significant difference between the groups in the analysed outcomes; embryo survival rate (84.1% versus 82.1%), clinical pregnancy rate (45.9% versus 42.4%), implantation rate (25.6% versus 24.5%), cycle cancellation rate (6.7% versus 8.5%) and live birth rate (41.2% versus 41.0%). These data suggest that ultra-rapid vitrification may be replaced by aseptic vitrification without affecting clinical efficiency.

GnRH antagonist versus long GnRH agonist protocol in poor IVF responders: a randomized clinical trial.

OBJECTIVE To compare the efficacy of the long GnRH agonist and the fixed GnRH antagonist protocols in IVF poor responders. STUDY DESIGN This was a randomized controlled trial performed in the Iakentro IVF centre, Thessaloniki, from January 2007 to December 2011, concerning women characterised as poor responders after having 0-4 oocytes retrieved at a previous IVF cycle. They were assigned at random, using sealed envelopes, to either a long GnRH agonist protocol (group I) or a GnRH antagonist protocol (group II). RESULTS Overall 364 women fulfilled the inclusion criteria and were allocated to the two groups: finally 330 participated in our trial. Of these, 162 were treated with the long GnRH agonist protocol (group I), and 168 with the fixed GnRH antagonist protocol (group II). Numbers of embryos transferred and implantation rates were similar between the two groups (P=NS). The overall cancellation rate was higher in the antagonist group compared to the agonist group, but the difference was not significant (22.15% vs. 15.2%, P=NS). Although clinical pregnancy rates per transfer cycle were not different between the two groups (42.3% vs. 33.1%, P=NS), the clinical pregnancy rate per cycle initiated was significantly higher in the agonist compared to the antagonist group (35.8% vs. 25.6%, P=0.03). CONCLUSIONS Although long GnRH agonist and fixed GnRH antagonist protocols seem to have comparable pregnancy rates per transfer in poor responders undergoing IVF, the higher cancellation rate observed in the antagonist group suggests the long GnRH agonist protocol as the first choice for ovarian stimulation in these patients.

History of endometriosis may adversely affect the outcome in menopausal recipients of sibling oocytes.

Due to the known adverse effect of endometriosis on gamete quality, it has always been difficult to demonstrate a direct effect of endometriosis on implantation. In order to eliminate these confounding effects, this prospective comparative study studied a population of menopausal recipients with and without endometriosis sharing sibling oocytes coming from the same donor. The aim was to understand the impact of endometriosis on implantation, pregnancy and live birth rates in menopausal recipients. A total of 240 menopausal recipients of donated sibling oocytes, were divided in two groups. Group I consisted of 120 recipients diagnosed with endometriosis and group II consisted of 120 controls. The implantation and pregnancy rates were significantly lower in the endometriosis group compared with the control group (23.81% versus 31.48%, P=0.019; 45.00% versus 58.33%, P=0.039, respectively). In oocyte donation cycles, a recipients history of endometriosis might have a negative impact on implantation, pregnancy and live birth rates, even in menopausal women. Infertility in endometriosis may be due to poor oocyte quality or embryos with decreased ability to implant due to impaired fertilization. There are no conclusive data on the impact of endometriosis on implantation. The already-known adverse effect of endometriosis on gamete quality makes it more difficult to demonstrate a direct effect of endometriosis on implantation. In order to eliminate these confounding effects we studied a population of menopausal recipients with and without endometriosis sharing sibling oocytes coming from the same oocyte donor. The oocyte donation model was used in an attempt to understand whether the endometrium, the oocytes or both are affected by endometriosis. The aim of the present study was to understand the impact of endometriosis on implantation, pregnancy and live birth rates in menopausal recipients. A total of 240 menopausal recipients of donated sibling oocytes were divided into two groups. Group I consisted of 120 recipients diagnosed with endometriosis and group II consisted of 120 controls. The pregnancy and implantation rates were significantly lower in the endometriosis group compared to the control group (45.00% versus 58.33%, P=0.039) and (23.81% versus 31.48%, P=0.019) respectively. In oocyte donation cycles, a recipients history of endometriosis might have a negative impact on implantation, pregnancy and live birth rates, even in menopausal women.

Low-dose human chorionic gonadotropin during the proliferative phase may adversely affect endometrial receptivity in oocyte recipients

The effect of low-dose human chorionic gonadotropin (hCG) administration in the proliferative phase of oocyte recipients was investigated in a prospective randomized trial. Sibling oocytes from the same donor were shared at random among two different recipients. In group I oocyte recipients received 750 IU of hCG every three days concomitant to endometrial preparation with estradiol until hCG injection to the donor, whereas in group II recipients received no hCG during endometrial priming with estradiol. Endometrial thickness was significantly lower in group I compared with group II, although similar endometrial thickness was detected during the mock cycle. Pregnancy rates were significantly lower in group I than in group II (13.6% vs. 45.4%, p<0.05). Implantation rates were also significantly lower in group I (1.7% vs. 22.4%, p<0.01). The study was discontinued prematurely for ethical reasons when 22 cycles were completed, as pregnancy rates were very low in group I. In conclusion, hCG administration in the proliferative phase might directly affect endometrial proliferation and receptivity.

Cryptic sperm defects may be the cause for total fertilization failure in oocyte donor cycles.

In conventional IVF cycles with total fertilization failure, rescue intracytoplasmic sperm injection (ICSI) performed 24h after insemination has yielded poor results. However, when ICSI is used, total fertilization failure is a rare event. The aim of the present study is to investigate the degree of sperm contribution to fertilization failures using the egg-sharing model in oocyte donor cycles. The study included only the oocyte donor cycles of sibling oocytes with total fertilization failure in at least one of the matched recipients. Oocytes from 49 oocyte donor cycles were equally shared among 98 recipients undergoing conventional IVF. Due to total fertilization failure in half of the recipients, rescue ICSI was carried out. Compared with the conventional IVF only group, the rescue ICSI group had a lower pregnancy rate (30.61% versus 71.43%), clinical pregnancy rate (28.57% versus 67.35%) and ongoing pregnancy rate (28.57% versus 63.27%) (all P<0.01). Cryptic sperm defects in apparently normal spermatozoa may be the cause of total fertilization failure, indicating the need for simple routine tests to detect them.