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Fiorentino F.,GENOMA Molecular Genetics Laboratory | Spizzichino L.,GENOMA Molecular Genetics Laboratory | Bono S.,GENOMA Molecular Genetics Laboratory | Biricik A.,GENOMA Molecular Genetics Laboratory | And 6 more authors.
Human Reproduction | Year: 2011

Background: Fluorescence in situ hybridization (FISH) is the most widely used method for detecting unbalanced chromosome rearrangements in preimplantation embryos but it is known to have several technical limitations. We describe the clinical application of a molecular-based assay, array comparative genomic hybridization (array-CGH), to simultaneously screen for unbalanced translocation derivatives and aneuploidy of all 24 chromosomes. Methods: Cell biopsy was carried out on cleavage-stage embryos (Day 3). Single cells were first lysed and DNA amplified by whole-genome amplification (WGA). WGA products were then processed by array-CGH using 24sure arrays, BlueGnome. Balanced/normal euploid embryos were then selected for transfer on Day 5 of the same cycle. Results: Twenty-eight consecutive cycles of preimplantation genetic diagnosis were carried out for 24 couples carrying 18 different balanced translocations. Overall, 187/200 (93.5) embryos were successfully diagnosed. Embryos suitable for transfer were identified in 17 cycles (60.7), with transfer of 22 embryos (mean 1.3 ± 0.5). Twelve couples achieved a clinical pregnancy (70.6 per embryo transfer), with a total of 14 embryos implanted (63.6 per transferred embryo). Three patients delivered three healthy babies, during writing, the other pregnancies (two twins and seven singletons) are ongoing beyond 20 weeks of gestation. Conclusions: The data obtained demonstrate that array-CGH can detect chromosome imbalances in embryos, also providing the added benefit of simultaneous aneuploidy screening of all 24 chromosomes. Array-CGH has the potential to overcome several inherent limitations of FISH-based tests, providing improvements in terms of test performance, automation, sensitivity and reliability. © 2011 The Author.

Vajta G.,BGI Shenzhen | Vajta G.,Central Queensland University | Rienzi L.,Genera Center For Reproductive Medicine | Ubaldi F.M.,Genera Center For Reproductive Medicine
Reproductive BioMedicine Online | Year: 2015

Vitrification is now the dominant approach for cryopreservation of human oocytes and embryos; however, serious disagreement persists, particularly about biosafety issues. Techniques are categorized as either 'open' or 'closed' according to occurrence of direct contact between the medium and liquid nitrogen during cryopreservation. Advocates of closed systems emphasize the potential danger of disease transmission mediated through liquid nitrogen, and praise the safety of their approach; those who use the open systems refer to the lack of evidence of disease transmission and regard their systems as more consistent and efficient. The purpose of this review is to clarify whether open and closed systems are really open and closed; if closed systems are safe and free of any danger of contamination; if closed systems are equally efficient as open ones for cryopreservation of human embryos and oocytes by considering overall outcome; and finally, if ethical and legal concerns are sound when risks and benefits are considered in a broader sense. On the basis of these answers, implementation of rational measures to lower the theoretical danger of disease transmission are proposed while maintaining the achievements in cryopreservation that have contributed substantially to the advancement in assisted reproduction techniques during the past decade. © 2014 Reproductive Healthcare Ltd. All rights reserved.

Rienzi L.,Genera Center For Reproductive Medicine | Vajta G.,BGI Shenzhen | Ubaldi F.,Genera Center For Reproductive Medicine
Placenta | Year: 2011

During the past decades, improvements in culture of preimplantation embryos have contributed substantially in the success of human assisted reproductive techniques. However, most efforts were focused on optimization of media and gas components, while the established physical conditions and applied devices have remained essentially unchanged. Very recently, however, intensive research has been started to provide a more appropriate environment for the embryos and to replace the rather primitive and inappropriate devices with more sophisticated and practical instruments. Success has been reported with simple or sophisticated tools (microwells or microchannels) that allow accumulation of autocrine factors and establishment of a proper microenvironment for embryos cultured individually or in groups. The microchannel system may also offer certain level of automation and increased standardization of culture parameters. Continuous monitoring of individual embryos by optical or biochemical methods may help to determine the optimal day of transfer, and selection of the embryo with highest developmental competence for transfer. This advancement may eventually lead to adjustment of the culture environment to each individual embryo according to its actual needs. Connection of these techniques to additional radical approaches as automated ICSI or an ultimate assisted hatching with full removal of the zona pellucida after or even before fertilization may result in devices with high reliability and consistency, to increase the overall efficiency and decrease the work-intensity, and to eliminate the existing technological gap between laboratory embryology work and most other fields of biomedical sciences. © 2011 Elsevier Ltd. All rights reserved.

Vajta G.,James Cook University | Rienzi L.,Genera Center For Reproductive Medicine | Bavister B.D.,Wayne State University
Reproductive BioMedicine Online | Year: 2010

Sporadic reports published during the previous decade have documented pregnancies achieved with transfer of zona-free human embryos. Although the overall efficiency seems to be good and some authors have suggested systematic application for special infertility problems, there have been only a few attempts to compare the benefits of zona-free embryo culture and transfer with the traditional approach using zona-intact embryos. So far, the majority of instances in which zona-free culture has been applied have occurred accidentally. This review summarizes the known functions of the zona pellucida, analyses natural and artificial situations where its function is compromised, including zona hardening and difficult hatching that seem to be related to in-vitro embryo culture, and discusses possible methods and timing for artificial zona removal. With the availability of in-vitro systems capable of replacing important functions of the zona pellucida, routine use of zona-free culture for the whole in-vitro period, after or even before fertilization, is a realistic possibility with potential additional benefits. Based on the increasing amount of animal studies, a systematic comparison is suggested that may eventually diminish the handicaps of the in-vitro situation and lead to simplification of manipulations as well as higher success rates after embryo transfer. © 2010, Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved.

Ubaldi F.,Genera Center For Reproductive Medicine | Anniballo R.,Andrology Center John od | Romano S.,Genera Center For Reproductive Medicine | Baroni E.,Genera Center For Reproductive Medicine | And 6 more authors.
Human Reproduction | Year: 2010

BACKGROUND: Recent advancement of minimum volume vitrification methods has resulted in a dramatic increase in the efficiency of the process. The aim of this study was to estimate the cumulative reproductive outcome of a cohort of infertile couples undergoing ICSI and oocyte vitrification in restrictive legal conditions, where only a limited number of oocytes could be inseminated per cycle and embryo selection and cryopreservation were forbidden.METHODSIn this prospective longitudinal cohort study, the cumulative ongoing pregnancy rates obtained by the insemination of fresh and vitrified oocytes from the same cohort were calculated as primary outcome measures. Moreover, the effect of basal and cycle characteristics on clinical outcomes were assessed.RESULTSBetween September 2008 and May 2009, 182 ICSI cycles were performed where oocyte vitrification was possible. A total of 104 first and 11 second oocyte warming cycles were then performed in non-pregnant patients of the same cohort. The overall ongoing pregnancy rates obtained in the fresh, and first and second warming cycles were 37.4, 25.0 and 27.3, respectively. The overall cumulative ongoing clinical pregnancy rate observed per stimulation cycle was 53.3. Maternal age was the only characteristic found to influence the reproductive outcome, with an inverse correlation between the age >40 and the ongoing pregnancy rates (P = 0.04, by Cox regression analysis).CONCLUSIONSHigh cumulative ongoing pregnancy rates can be obtained with transfers of embryos derived from fresh and cryopreserved oocytes in a typical infertile population. Female age significantly affects outcomes in this system. © 2010 The Author.

Rienzi L.,Genera Center For Reproductive Medicine | Romano S.,Genera Center For Reproductive Medicine | Albricci L.,Genera Center For Reproductive Medicine | Maggiulli R.,Genera Center For Reproductive Medicine | And 5 more authors.
Human Reproduction | Year: 2010

Background: A successful oocyte cryopreservation programme is of utmost importance where a limited number of oocytes can be inseminated per cycle, to overcome legal and ethical issues related to embryo storage, for oocyte donation programmes and for fertility preservation (especially for cancer patients). Vitrification has been recently proposed as an effective procedure for this purpose. methods: In order to validate the effectiveness of oocyte vitrification a non-inferiority trial was started on sibling metaphase II (MII) oocytes. To demonstrate the non-inferiority based on an absolute difference of 17% in the fertilization rate per sibling oocyte, a minimum of 222 oocytes were required. After oocyte denudation, MII oocytes with normal morphology were randomly allocated to fresh ICSI insemination or to vitrification procedure. If pregnancy was not obtained a subsequent ICSI cycle was performed with warmed oocytes of the same cohort. In both groups, three oocytes were inseminated per cycle by ICSI procedure. Primary end-points were fertilization rates calculated per warmed and per injected oocytes. Secondary end-points were zygote and embryo morphology. results: A total of 244 oocytes were involved in this study. Of the 120 fresh sibling oocytes inseminated, 100 were fertilized (83.3%). Survival rate of sibling vitrified oocytes was 96.8% (120/124 oocytes). Fertilization rate after ICSI was 76.6% (95/124) per warmed oocyte and 79.2% (95/120) per survived/inseminated oocyte. No statistical difference in fertilization rates was observed between the two groups when calculated per sibling oocytes (absolute difference 26.73%; OR: 0.65; 95% CI = 0.33-1.29; P = 0.20) and per inseminated oocyte (absolute difference 24.17%; OR: 0.76; 95% CI = 0.37-1.53; P = 0.50). Embryo development was also similar in both treatment groups up till Day 2. The percentage of excellent quality embryos was 52.0% (52/100) in the fresh group and 51.6% (49/95) in the vitrification group (absolute difference 20.43%; OR: 0.98; 95% CI = 0.53-1.79; P = 0.9). The mean age of the 40 patients included in this study was 35.5+4.8 years (range 26-42). Fifteen clinical pregnancies were obtained in the vitrification cycles of 39 embryo transfers performed (37.5% per cycle, 38.5% per embryo transfer), with an implantation rate of 20.2% (19/94). Three spontaneous miscarriages occurred (20%). Twelve pregnancies are ongoing (30.0% per cycle, 30.8% per embryo transfer) beyond 12 weeks of gestation. conclusions: Our results indicate that oocyte vitrification procedure followed by ICSI is not inferior to fresh insemination procedure, with regard to fertilization and embryo developmental rates. Moreover, ongoing clinical pregnancy is compatible with this procedure, even with a restricted number of oocytes available for insemination. The promising clinical results obtained, in a population of infertile patients, need to be confirmed on a larger scale.

Rienzi L.,Genera Center For Reproductive Medicine | Cobo A.,IVI | Paffoni A.,Fondazione Ca Granda Ospedale Maggiore Policlinico | Scarduelli C.,Fondazione Ca Granda Ospedale Maggiore Policlinico | And 5 more authors.
Human Reproduction | Year: 2012

Background: An efficient method for cryopreservation of human oocytes may offer solutions to legal and ethical problems in routine infertility programs and may also be used for fertility preservation for medical and social reasons. Methods We conducted an observational longitudinal cohort multicentric study to investigate the efficacy and reproducibility of oocyte cryopreservation outcomes in IVF/ICSI cycles. Moreover, the effects of patient and cycle characteristics on the delivery rate (DR) were analyzed. Results In 486 cycles performed in 450 couples, 2721 oocytes were warmed and 2304 of them survived cryopreservation (84.7). Of the 2182 oocytes subjected to ICSI, the rates of fertilization and development to top-quality embryos were 75.2 and 48.1, respectively. A total of 128 deliveries were obtained (26.3 per cycle and 29.4 per transfer) for 450 patients (28.4) and 147 babies were live born from 929 embryos transferred (15.8). The forward logistic regression analysis on a per patient basis showed that female age [odds ratio (OR): 0.93, 95 confidence interval (CI): 0.880.98], number of vitrified oocytes (OR: 1.08, 95 CI: 1.011.17) and the day of transfer (OR: 1.97, 95 CI: 1.143.42) influenced DR. By recursive partitioning analysis, it can be estimated that more than eight oocytes vitrified are required to improve the outcome (22.6 versus 46.4 DR, respectively). When fewer oocytes are available in women aged >38 years, Results are dramatically reduced (12.6 versus 27.5 DR, respectively). Conversely, when >8 oocytes are available, blastocyst culture represents the most efficient policy (62.1 DR; data from one center only). Conclusions Oocyte vitrification is an efficient and reliable approach, with consistent Results between centers and predictable DRs. It should be applied routinely for various indications. A predictive model is proposed to help patient counselling and selection. © 2012 The Author.

Rienzi L.,Genera Center For Reproductive Medicine | Capalbo A.,Genera Center For Reproductive Medicine | Stoppa M.,Genera Center For Reproductive Medicine | Romano S.,Genera Center For Reproductive Medicine | And 6 more authors.
Reproductive BioMedicine Online | Year: 2015

Recent studies involving a limited number of patients have indicated a correlation between aneuploidy and various morphokinetic parameters during preimplantation development. The results among different groups, however, have been inconsistent in identifying the parameters that are able to predict chromosomal abnormalities. The aim of this study was to investigate whether aneuploidy of human blastocysts was detectable by specific morphokinetic parameters in patients at increased risk of aneuploidy because of advanced maternal age, history of unsuccessful IVF treatments, or both. A longitudinal cohort study was conducted using 455 blastocysts from 138 patients. Morphokinetic features of preimplantation development were detected in a timelapse incubator. Blastocysts were subjected to trophectodermal biopsy and comprehensive chromosomal screening. Analyses were conducted by means of logistic mixed-effects models, with a subject-specific intercept. No statistical correlation between 16 commonly detected morphokinetic characteristics of in-vitro embryo development and aneuploidy was found. Results suggest that morphokinetic characteristics cannot be used to select euploid blastocysts in poor-prognosis patients regarded as candidates for pre-implantation genetic screening. © 2014 Reproductive Healthcare Ltd. Published by Elsevier Inc. All rights reserved.

Rienzi L.,Genera Center For Reproductive Medicine | Vajta G.,BGI | Ubaldi F.,Genera Center For Reproductive Medicine
Human Reproduction Update | Year: 2011

Background: Non-invasive selection of developmentally competent human oocytes may increase the overall efficiency of human assisted reproduction and is regarded as crucial in countries where legal, social or religious factors restrict the production of supernumerary embryos. The purpose of this study was to summarize the predictive value for IVF success of morphological features of the oocyte that can be obtained by light or polarized microscopic investigations. Methods: Studies about oocyte morphology and IVF/ICSI outcomes were identified by using a systematic literature search. Results: Fifty relevant articles were identified: 33 analysed a single feature, 9 observed multiple features and investigated the effect of these features individually, 8 summarized the effect of individual features. Investigated structures were the following: meiotic spindle (15 papers), zona pellucida (15 papers), vacuoles or refractile bodies (14 papers), polar body shape (12 papers), oocyte shape (10 papers), dark cytoplasm or diffuse granulation (12 papers), perivitelline space (11 papers), central cytoplasmic granulation (8 papers), cumulus-oocyte complex (6 papers) and cytoplasm viscosity and membrane resistance characteristics (2 papers). None of these features were unanimously evaluated to have prognostic value for further developmental competence of oocytes. Conclusions: No clear tendency in recent publications to a general increase in predictive value of morphological features was found. These contradicting data underline the importance of more intensive and coordinated research to reach a consensus and fully exploit the predictive potential of morphological examination of human oocytes. © The Author 2010. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology.

Vajta G.,Cairns Fertility Center | Rienzi L.,Genera Center For Reproductive Medicine | Cobo A.,University of Valencia | Yovich J.,Cairns Fertility Center
Reproductive BioMedicine Online | Year: 2010

Culture of preimplantation-stage embryos has always been a key element of laboratory embryology and has contributed substantially to the success of many assisted reproduction procedures. During the past decade, its importance has increased as extended in-vitro embryo culture and single blastocyst transfer have become indispensable parts of the approach to decreasing the chance of multiple pregnancy while preserving the overall efficiency of the treatment. However, in spite of the scientific and commercial challenge stimulating research worldwide to optimize embryo culture conditions, a consensus is missing even in the basic principles, including composition and exchange of media, the required physical and biological environment and even the temperature of incubation. This review attempts to summarize the controversies, demonstrate the fragility of some widely accepted dogmas and generate an open-minded debate towards rapid and efficient optimization. New approaches expanding the traditional frames of mammalian embryo culture are also discussed. Although some researchers suppose that the efficiency of the presently applied in-vitro culture systems have already approached the biological limits, authors are confident that substantial improvement may be achieved that may expand considerably the possibilities of future assisted reproduction in humans. © 2010, Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved.

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