Ermanno Greco

Healthy Babies after Intrauterine Transfer of Mosaic Aneuploid Blastocysts

Implantation of 18 mosaic aneuploid blastocysts obtained through in vitro fertilization into 18 women resulted in six pregnancies of normal karyotype. These pregnancies proceeded to term, with the birth of six healthy babies.

Application of next-generation sequencing technology for comprehensive aneuploidy screening of blastocysts in clinical preimplantation genetic screening cycles

STUDY QUESTION Can next-generation sequencing (NGS) techniques be used reliably for comprehensive aneuploidy screening of human embryos from patients undergoing IVF treatments, with the purpose of identifying and selecting chromosomally normal embryos for transfer? SUMMARY ANSWER Extensive application of NGS in clinical preimplantation genetic screening (PGS) cycles demonstrates that this methodology is reliable, allowing identification and transfer of euploid embryos resulting in ongoing pregnancies. WHAT IS KNOWN ALREADY The effectiveness of PGS is dependent upon the biology of the early embryo and the limitations of the technology. Fluorescence in situ hybridization, used to test for a few chromosomes, has largely been superseded by microarray techniques that test all 24 chromosomes. Array comparative genomic hybridization (array-CGH) has been demonstrated to be an accurate PGS method and has become the de facto gold standard, but new techniques, such as NGS, continue to emerge. STUDY DESIGN, SIZE, DURATION The study consisted of a prospective trial involving a double blind parallel evaluation, with both NGS and array-CGH techniques, of 192 blastocysts obtained from 55 consecutive clinical PGS cycles undertaken during the period of September to October 2013. Consistency of NGS-based aneuploidy detection was assessed by matching the results obtained with array-CGH-based diagnoses. Primary outcome measure was accuracy of the chromosomal analysis; secondary outcome measures were clinical outcomes. PARTICIPANTS/MATERIALS, SETTINGS, METHODS Fifty-five patients (median age 39.3 years, range 32-46) undergoing PGS were enrolled in the study. All embryos were cultured to blastocyst stage; trophectoderm biopsy was performed on Day 5 of development or Day 6/7 for slower growing embryos. The method involved whole genome amplification followed by both NGS and array-CGH. The MiSeq control software, real-time analysis and reporter performed on-board primary and secondary bioinformatics analysis. Copy number variation analysis was accomplished with BlueFuse Multi software. MAIN RESULTS AND THE ROLE OF CHANCE A total of 192 blastocysts were blindly evaluated with the NGS-based protocol. Paired comparison between NGS and array-CGH from individual embryos showed concordant results in 191/192 (99.5%) of the blastocysts tested. In total 4608 chromosomes were assessed, 211 (4.6%) of which carried a copy number imbalance. NGS specificity for aneuploidy calling (consistency of chromosome copy number assignment) was 99.98% (4333/4334; 95% confidence interval [95% CI]: 99.87-100) with a sensitivity of 100% (211/211, 95% CI: 99.25-100). Despite one discordant result, NGS specificity and sensitivity for aneuploid embryo calling (24-chromosome diagnosis consistency) were both 100% since the discordant sample presented several other aneuploidies. Clinical application of the NGS-based approach revealed 74/192 (38.5%) euploid blastocysts. Following transfer of 50 embryos in 47 women, 34 women had positive hCG levels: 30 pregnancies continued, confirmed by at least one fetal sac and heart beat (63.8% clinical pregnancy rate/embryo transfer), 3 were biochemical and 1 miscarried. A total of 32 embryos implanted and led to the presence of a fetal sac (64.0% implantation rate). All pregnancies went to term resulting in the birth of 31 healthy babies. LIMITATION, REASON FOR CAUTION Although clinical results reported high pregnancy outcomes following transfer of screened embryos, further data and broad-based clinical application are required to better define the role of NGS in PGS. Before recommending widespread application, a randomized controlled trial confirming its clinical effectiveness is advisable. WIDER IMPLICATION OF THE FINDING This is the first study reporting extensive application of NGS-based comprehensive aneuploidy screening on embryos at blastocyst stage in a clinical setting versus array-CGH as test of reference. NGS has demonstrated a reliable methodology, with the potential to improve chromosomal diagnosis on embryos especially in terms of high-throughput, automation and ability to detect aneuploidy. NGS methodology may represent a valuable alternative to the other comprehensive aneuploidy screening techniques currently available. STUDY FUNDING/COMPETING INTERESTS No external funding was sought for this study. Drs F.K. and C.-E.M. are full-time employees of Illumina, Inc., which provided NGS library and sequencing reagents for the study. All other authors have no conflicts to declare. TRIAL REGISTRATION NUMBER Not applicable.

Significance of morphological attributes of the early embryo

There are many morphological transformations during development of human embryos that mainly involve phenomena that can be easily assessed in living embryos by simple non-invasive microscopical observation. A clear correlation between pronuclear morphology and the ability of the resulting embryo to continue developing and to implant has been described. There is also general agreement that a positive relationship exists between early embryo morphology and implantation rate. The parameters classically involved in embryo evaluation are: cleavage rate, blastomere symmetry, cytoplasmic appearance, extent of fragmentation and blastomere nuclear status. In this paper, morphological features that have been related to embryo developmental potential are described. Furthermore, the ability of a cumulative classification scheme developed in the laboratory to predict blastocyst formation and implantation is analysed.

Use of a modified intracytoplasmic sperm injection technique to overcome sperm-borne and oocyte-borne oocyte activation failures

OBJECTIVE To examine whether sperm-borne and oocyte-borne oocyte activation failures can be overcome by mechanical means that entail modifying the ICSI technique. DESIGN Case report series. SETTING Private clinics. PATIENT(S) Six infertile couples undergoing ICSI. INTERVENTION(S) Standard ICSI and modified ICSI based on mechanical manipulation that facilitated entry of calcium into the oocyte. MAIN OUTCOME MEASURE(S) Fertilization rate and pregnancy outcome. RESULT(S) In three cases of sperm-borne and three cases of oocyte-borne oocyte activation deficiencies, the modified ICSI technique enabled normal fertilization and development of embryos with good morphology. In terms of fertilization, the efficacy of modified ICSI was similar to use of a calcium ionophore, without producing extensive embryo fragmentation during postfertilization development. Term pregnancies resulting in the birth of normal children were achieved with the modified ICSI technique in five cases. CONCLUSION(S) Sperm-borne and oocyte-borne oocyte activation failures can be overcome by modifying the ICSI technique. The modification obviates the need to use insufficiently tested and potentially harmful drugs.

Significance of metaphase II human oocyte morphology on ICSI outcome

OBJECTIVE To evaluate the influence of specific oocyte morphologic features (morphotypes) on intracytoplasmic sperm injection (ICSI) outcome. The identification of oocyte quality markers is particularly important when a low number of oocytes can be used for IVF. DESIGN Retrospective analysis. SETTING Medical center. PATIENT(S) Five hundred sixteen consecutive ICSI cycles. Only couples affected by severe male factor infertility were excluded. INTERVENTION(S) A total of 1,191 metaphase II (MII) oocytes (1-3 per patient) were randomly selected from the cohort of oocytes obtained from each patient and evaluated for morphologic appearance. MAIN OUTCOME MEASURE(S) Fertilization, pronuclear morphology, embryo quality, pregnancy rate. RESULT(S) There was a presence of vacuoles, abnormal I polar body, and large perivitelline space related to a lower fertilization rate. Pronuclear morphology was effected by the presence of a large perivitelline space, diffused cytoplasmic granularity, and/or centrally located granular area. The latter characteristic also negatively related to day 2 embryo quality. According to the odds ratios obtained for each oocyte morphotype to reach at least one outcome, an MII oocyte morphologic score (MOMS) was calculated. A significant relationship was found between MOMS and female age, female basal FSH, and clinical outcome. CONCLUSION(S) Morphologic evaluation before ICSI helps to identify MII oocytes with higher developmental potential.

Management of poor responders in IVF.

Correct controlled ovarian stimulation is of paramount importance in assisted reproductive technologies. Therefore, analysis of the ovarian reserve of the patient is mandatory to tailor the best ovarian stimulation regimen. When the ovarian reserve is reduced, the induction of a multifollicular growth remains a challenge. Several factors could be associated with reduced ovarian response. However, reduced ovarian reserve either in older patients or in young patients represents the most frequent aetiological factor. Whatever is the aetiology, one of the main problems is how to predict a reduced ovarian response, and although several tests have been suggested, no very accurate predictive test is available. A variety of different stimulation protocols have been suggested but the lack of any large-scale, prospective, randomized, controlled trials of the different management strategies and the lack of a uniform definition of the population may result in comparisons of heterogeneous groups of patients, making it difficult to draw any definitive conclusions. Natural cycle IVF may represent an easy and cheap approach in the management of this group of patients. Although no controlled large prospective randomized studies are available to compare the natural IVF procedure with ovarian stimulation IVF in poor responder patients, the efficacy of natural cycle IVF is hampered by high cancellation rates mainly due to untimely LH surge. The use of gonadotrophin-releasing hormone antagonists in the late follicular phase, which reduces the premature LH rise rate, and the improvements in laboratory conditions and fertilization techniques, increase the embryo transfer rates, making this procedure more cost-effective.

Pronuclear morphology evaluation with subsequent evaluation of embryo morphology significantly increases implantation rates

OBJECTIVE To elucidate the relative predictive value of implantation markers at different stages of preimplantation development. DESIGN Correlation of pronuclear morphology with embryo morphology and implantation rates in retrospective and prospective analysis of in vitro fertilization/intracytoplasmic sperm injection (IVF-ICSI) treatment cycles. SETTING Private infertility center. PATIENT(S) A total of 441 couples undergoing infertility treatment. INTERVENTION(S) None. MAIN OUTCOME MEASURE(S) Size of pronuclei and distance between them, the number and polarization of nucleolus precursor bodies (NPB) at the one-cell stage, embryo cleavage and fragmentation rates on days 2 and 3, and pregnancy and implantation rates. RESULT(S) Polarization of the NPB in both pronuclei had a statistically significant correlation with normal membrane breakage during ICSI (40%, compared with 33% easy, and 31% difficult membrane breakage) and also with faster cleavage and lower fragmentation rates of embryos. Sixty-one percent of implanting embryos had polarization of the NPB in both pronuclei compared with 37% for all embryos. Larger distance between pronuclei and their unequal size had a statistically significant correlation with slower cleavage and inferior embryo quality. Embryo selection based on only pronuclear morphology or on only day-3 embryo morphology yielded implantation rates of 15.1% and 12.1%, respectively. Embryo selection based on sequential evaluation of both pronuclear morphology and embryo morphology on day 3 resulted in a 21.1% implantation rate. CONCLUSION(S) Polarization of NPB in both pronuclei is as reliable marker of implantation as embryo morphology on day 3. However, pronuclear morphology assessment improves embryo selection only when it is combined with embryo morphology evaluation on day 3.

Caesarean scar pregnancy: a review of management options.

Purpose of review The current review aims to provide an overview of the already available and emerging treatment modalities for caesarean scar pregnancy (CSP). Recent findings CSP is a type of ectopic gestation associated with a high risk of serious complications. The cause of this condition and the best management are still unclear. However, some medical and surgical treatment modalities have been suggested. The main objectives in the clinical management of CSP should be the prevention of massive blood loss and the conservation of the uterus to maintain further fertility, womens health and quality of life. Current data suggest that expectant management should not be recommended, whereas there are accumulating data suggesting that early diagnosis offers single or combined medical and surgical treatment options avoiding uterine rupture and haemorrhage, thus preserving the uterus and fertility. Summary No universal treatment guidelines for management of CSP have been published up to now. The lack of data on the best evidence should encourage any individual case report and further multicentre studies for recommendation establishment.

Meiotic spindle visualization in living human oocytes

A computer-assisted polarization microscopy system (polscope) has made it possible to analyse the meiotic spindle of oocytes subjected to intracytoplasmic sperm injection (ICSI) without affecting their viability. It has been shown that the presence of a detectable birefringent meiotic spindle inside the oocyte cytoplasm of human metaphase II (MII) prepared for ICSI is an indicator of oocyte quality, such as fertilization and developmental ability. Meiotic spindle imaging has also shown that this structure, when detectable, is not always aligned with the first polar body (PB1) in fresh MII oocytes. The relationship between the degree of meiotic spindle deviation from the PB1 location and ICSI outcomes is discussed in this paper. When the meiotic spindle of in-vitro matured oocytes is analysed, it is always found to be aligned with the PB1, suggesting that the misalignment observed in the oocytes matured in vivo results from the PB1 displacement during the manipulations for the cumulus and corona removal. Furthermore, polscope analysis of meiotic spindle changes in living MII oocytes subjected to freezing and thawing procedures has shown that the current techniques of oocyte cryopreservation cause meiotic spindle destruction. The polscope system may assist in the selection of fresh and thawed oocytes for ICSI.

Metabolomic assessment of oocyte viability

The aim of the current study was to evaluate whether near-infrared (NIR) spectroscopy-generated metabolomic data obtained from oocyte culture samples would correlate with nuclear maturity status and derived embryo development. A total of 412 oocyte culture samples were collected from 43 patient cycles. Metabolomic profiles of metaphase I and II oocytes were obtained by NIR spectroscopy and were significantly different from each other and from profiles of prophase I (germinal vesicle) oocytes (P +/- 0.001 at the 95% confidence interval). Additionally, NIR spectroscopic analysis of culture medium of oocytes that developed to grade A embryos on day 3 demonstrated significantly higher viability indices (0.62 +/- 0.23) than those that developed to grades C/D (0.42 +/- 0.26; P < 0.006); and on day 5 grade A (0.37 +/- 0.20) was also higher than grades C/D (0.14 +/- 0.21; P < 0.02). Metabolomic profiles of oocytes that resulted in pregnancy had higher viability indices (0.87 +/- 0.27) than those that did not (0.44 +/- 0.17; P < 0.0001). The results of the current study demonstrate that metabolomic profiling from spent culture medium of the oocyte is related to nuclear maturity, is able to predict embryo development at day 3 and day 5 stages, and relates to embryo viability.