Marcello Iacobelli

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.

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.

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.

Laser-assisted removal of necrotic blastomeres from cryopreserved embryos that were partially damaged

OBJECTIVE To examine whether the developmental potential of embryos that were partially damaged after freezing and thawing can be improved by removal of necrotic blastomeres before embryo transfer. DESIGN Prospective pilot study and observational clinical series. SETTING Private hospital. PATIENT(S) Two hundred thirty-five infertile couples undergoing frozen embryo transfer. INTERVENTION(S) Removal of necrotic blastomeres from frozen-thawed human embryos. MAIN OUTCOME MEASURE(S) Pregnancy and implantation rates. RESULT(S) Removal of necrotic blastomeres from partially damaged frozen-thawed embryos before transfer increased rates of pregnancy (45.7% vs. 17.1%), ongoing pregnancy (40.0% vs. 11.4%) and ongoing implantation (16.2% vs. 4.3%) compared with the control group, in which necrotic blastomeres were not removed. A similarly high implantation rate (16.7%) was seen a subsequent clinical series in which necrotic blastomeres were removed from all partially damaged embryos. CONCLUSION(S) The viability of partially damaged frozen-thawed embryos can be improved by removal of necrotic blastomeres before embryo transfer.

Laser-assisted intracytoplasmic sperm injection

OBJECTIVE To improve oocyte survival and fertilization rates after intracytoplasmic sperm injection (ICSI) in patients with inherent oocyte fragility. DESIGN Pilot feasibility study and case report. SETTING Private hospital. PATIENT(S) Infertile couple with repeated failures of ICSI caused by oocyte degeneration. INTERVENTION(S) Laser-assisted drilling of the zona pellucida followed by ICSI. MAIN OUTCOME MEASURE(S) Oocyte survival, fertilization, and pregnancy. RESULTS In a couple with four previous ICSI failures because of poor oocyte survival (33.3%, 0%, 20%, and 18%), a fifth attempt using laser-assisted ICSI resulted in the survival of 8 oocytes out of 13 injected. Normal fertilization occurred in 5 oocytes, and a clinical pregnancy was established. CONCLUSION(S) Performing ICSI through a laser-drilled hole in the zona pellucida reduces the risk of oocyte damage related to deformation during the initial phase of the microinjection procedure. This modification of ICSI appears to be suitable for patients whose oocyte show inherent fragility and high degeneration rates after the standard ICSI procedure.

Hopes and facts about mild ovarian stimulation

Over the last two decades, easier and less expensive stimulation treatments have been largely replaced by more complex and more demanding protocols. Since the mid-nineties, long-term gonadotrophin-releasing hormone agonist stimulation protocols have been widely used. Such lengthy expensive regimens are not free from short- and long-term risks and complications. Mild stimulation protocols reduce the mean number of days of stimulation, the total amount of gonadotrophins used and the mean number of oocytes retrieved. The proportion of high quality and euploid embryos seems to be higher compared with conventional stimulation protocols and the pregnancy rate per embryo transfer is comparable. Moreover, the reduced costs, the better tolerability for patients and the less time needed to complete an IVF cycle make mild approaches clinically and cost-effective over a given period of time. However, further prospective randomized studies are needed to compare cumulative pregnancy rates between the two protocols. Natural cycle IVF, with minimal stimulation, has been recently proposed as an alternative to conventional stimulation protocols in normo- and poor responder patients. Although acceptable results have been reported, further large prospective randomized studies are needed to better evaluate the efficacy of these minimal regimens compared with conventional stimulation approaches.

Birth of a healthy female after preimplantation genetic diagnosis for Charcot-Marie-Tooth type X.

The X-linked dominant form of Charcot-Marie-Tooth syndrome (CMTX) is a clinically and genetically heterogeneous hereditary disorder of the peripheral nerves caused by mutations in the GJB1 gene that encodes a gap junction protein named connexin 32 (Cx32). Clinically, CMTX is characterized by peripheral motor and sensory deficit with muscle atrophy. A couple with a previous history of pregnancy termination after being diagnosed positive for CMTX by chorionic villus sampling, was referred for preimplantation genetic diagnosis (PGD). The female partner carried the causative H94Q, characterized by a C-->G substitution in codon 94 of exon 2 of the GJB1 gene. Embryos obtained after intracytoplasmic sperm injection (ICSI) were evaluated for the presence of the mothers mutation using polymerase chain reaction (PCR), followed by mutation analysis performed using the minisequencing method. Amelogenin sequences on the X and Y chromosomes were also co-amplified to provide a correlation between embryo gender and mutation presence. A single PGD cycle was performed, involving nine fertilized oocytes, five of which developed into good quality embryos useful for biopsy. Two unaffected embryos were transferred, resulting in a singleton pregnancy followed by the birth of a healthy female.

Natural In Vitro Fertilization Cycles

Abstract: Although the first in vitro fertilization (IVF) baby was born after a natural IVF cycle, very soon this procedure was almost abandoned mainly because of the very high cancellation rates, and controlled pharmacological ovarian hyperstimulation became the standard treatment in IVF cycles of normoresponder patients. However, in poor‐responder patients, where only very few follicles can be recruited and very few oocytes, if any, can be retrieved after controlled ovarian hyperstimulation, natural IVF cycles may offer a comparable number of follicles, reduced costs, and less discomfort for the patients. In this group of patients, natural IVF cycle is a cost‐effective approach.

Blastomere cytoplasmic granularity is unrelated to developmental potential of day 3 human embryos.

AbstractPurpose: To examine whether the increase in cytoplasmic granularity observed in some human embryos on day 3 of development is of any predictive value as to embryo developmental potential. Methods: Retrospective study comparing outcomes of treatment attempts in three groups of patients after day 3 embryo transfer. Attempts in which only embryos with clear cytoplasm were transferred form Group I, those in which only embryos with granulated cytoplasm were transferred constitute Group II, and Group III consists of cases with mixed transfer combining both types of embryos. Each group was further divided according to the female age. Results: Clinical pregnancy rates in Groups I (314 attempts), II (173 attempts), and III (323 attempts) were 33.8, 36.4, and 31.3%, respectively. Implantations rates for Groups I–III were 17.0, 17.3, and 14.8%, respectively. No significant differences between groups concerning these and other values, including the number of oocytes and of metaphase II oocytes recovered, fertilization and cleavage rates were found. The proportion of good-morphology embryos was also similar between the different groups (74.3, 72.7, and 70.2% respectively). The representation of women of advanced age (>36 years) was also similar in each group, and intergoup differences remained insignificant were only younger or only older women were taken into account. Conclusion(s): These data show that the appearance of cytoplasmic granulation in blastomeres of day 3 human embryos is of no prognostic value as to embryo quality and appears to be unrelated to the female age.

Microfilament disruption is required for enucleation and nuclear transfer in germinal vesicle but not metaphase II human oocytes

OBJECTIVE To evaluate the usefulness of microfilament disruption before enucleation and nuclear transfer in human oocytes at different stages of maturation. DESIGN Prospective experimental study. SETTING Private clinics. PATIENT(S) Infertile couples undergoing assisted reproduction attempts. INTERVENTION(S) Oocyte enucleation and nuclear transfer, activation of reconstructed oocytes. MAIN OUTCOME MEASURE(S) Oocyte survival, nuclear transfer efficacy, activation outcomes. RESULT(S) Survival rate and nuclear transfer efficacy of germinal vesicle oocytes exposed to the microfilament disrupting agent cytochalasin B before enucleation were 88% and 80%, respectively. These figures dropped, respectively, to 8% and 2% when cytochalasin treatment was omitted. By contrast, cytochalasin-treated and -untreated metaphase II oocytes showed similar survival rate (87% vs. 90%) and nuclear transfer efficacy (78% vs. 87%). This also applied to metaphase II oocytes matured in vitro from the germinal vesicle stage. Cytochalasin treatment did not affect activation rate of reconstructed oocytes, but it increased the occurrence of oocytes with multiple female pronuclei. CONCLUSION(S) Microfilament disruption before enucleation is required for germinal vesicle oocytes but not for metaphase II oocytes.