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Antonio Capalbo,
Laura Rienzi,
Matteo Buccheri,
Roberta Maggiulli,
Fabio Sapienza,
Stefania Romano,
Silvia Colamaria,
Benedetta Iussig,
Maddalena Giuliani,
Antonio Palagiano, Filippo Ubaldi
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ABSTRACT: Cryopreservation of the human embryo has been successfully achieved at the zygote (day 1), cleavage (day 2/3), and blastocyst (day 5) stages; however, each stage presents specific advantages and disadvantages. During the past decades, two major methods have been applied: slow freezing (equilibrium procedure) and vitrification (nonequilibrium procedure). The overwhelming majority of published data prove that the latest vitrification methods induce less cellular trauma and are a more effective cryopreservation technique of human embryos than any other versions of slow freezing. For this reason, fragmented and slow-cleaving embryos that normally would not be recommended may be revaluated for cryopreservation by using the vitrification method. Furthermore, if laser-assisted necrotic blastomere removal is associated with the slow-freezing/thawing procedure, good clinical results can be obtained. Finally, the most proper embryo cleavage stage at which to perform cryopreservation has to be assessed according to clinical indications and laboratory experience.
Annals of the New York Academy of Sciences 03/2011; 1221:32-9. · 3.15 Impact Factor
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ABSTRACT: 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.
In 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.
Between 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).
High 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.
Human Reproduction 02/2010; 25(5):1199-205. · 4.47 Impact Factor
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ABSTRACT: 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.
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.
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 -6.73%; OR: 0.65; 95% CI = 0.33-1.29; P = 0.20) and per inseminated oocyte (absolute difference -4.17%; 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 -0.43%; 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.
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. Clinical Trials Registration number: iSRCTN60158641.
Human Reproduction 10/2009; 25(1):66-73. · 4.47 Impact Factor
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Human Reproduction 03/2009; 24(5):1238-9; author reply 1239-40. · 4.47 Impact Factor
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ABSTRACT: Partially damaged frozen and thawed embryos are currently considered to have a lower viability than intact ones. This study was undertaken to compare the performance of intact frozen and thawed embryos with that of partially damaged embryos after removal of the necrotic blastomeres.
Observational clinical series.
Private hospital.
Three hundred twenty-six infertile couples undergoing frozen embryo transfer.
Removal of necrotic blastomeres from frozen-thawed human embryos.
Pregnancy and implantations rates.
Outcomes of frozen embryo transfer cycles in which all embryos were fully intact (group 1) were compared with those in which all embryos have lost 1-2 blastomeres (group 2) or 3-4 blastomeres (group 3). Laser-assisted hatching was performed in all embryos, and necrotic blastomeres were removed from partially damaged embryos on this occasion. Only embryos that resumed mitotic activity after thawing were transferred. Comparable clinical pregnancy rates (PR) (38.7%, 39.6%, and 29.4%), delivery rates (34.4%, 34.0%, and 29.4%), and implantation rates (21.6%, 21.4%, and 17.2%) were obtained in groups 1, 2, and 3, respectively.
The developmental potential of partially damaged frozen and thawed embryos can be equivalent to fully survived embryos if the necrotic blastomeres are removed from the partially damaged embryos and only those of them that show post-thaw cleavage are selected for transfer.
Fertility and sterility 11/2005; 84(4):888-94. · 3.97 Impact Factor
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ABSTRACT: Most studies examining the use of ICSI for cases of elevated sperm DNA fragmentation report poor pregnancy and implantation rates. ICSI with testicular sperm samples has recently been suggested for these cases. Here we test a less invasive approach based on oral antioxidant treatment prior to ICSI with ejaculated spermatozoa.
Thirty-eight men with an elevated (> or =15%) percentage of DNA-fragmented spermatozoa in the ejaculate were treated with antioxidants (1 g vitamin C and 1 g vitamin E daily) for 2 months after one failed ICSI attempt. In 29 (76%) of these cases this treatment led to a decrease in the percentage of DNA-fragmented spermatozoa, and a second ICSI attempt was performed. Outcomes of the two attempts were compared.
No differences in fertilization and cleavage rates or in embryo morphology were found between the ICSI attempts performed before and after the antioxidant treatment. However, a marked improvement of clinical pregnancy (48.2% versus 6.9%) and implantation (19.6% versus 2.2%) rates was observed after the antioxidant treatment as compared with the pretreatment ICSI outcomes.
Oral antioxidant treatment appears to improve ICSI outcomes in those patiens with sperm DNA damage, in whom this treatment reduces the percentage of damaged spermatozoa.
Human Reproduction 09/2005; 20(9):2590-4. · 4.47 Impact Factor
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ABSTRACT: 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.
Reproductive biomedicine online 06/2005; 10(5):669-81. · 2.04 Impact Factor
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ABSTRACT: 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.
Reproductive biomedicine online 03/2005; 10(2):192-8. · 2.04 Impact Factor
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Filippo Ubaldi,
Laura Rienzi,
Susanna Ferrero,
Elena Baroni,
Marcello Iacobelli,
Fabio Sapienza,
Maria Giulia Minasi,
Luigi Cobellis,
Stefania Romano,
Filomena Scarselli,
Ermanno Greco
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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.
Annals of the New York Academy of Sciences 01/2005; 1034:245-51. · 3.15 Impact Factor
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ABSTRACT: Sperm DNA damage (fragmentation) is a recently discovered cause of male infertility for which no efficient treatment has yet been found. Previous findings have suggested that clinically relevant sperm DNA damage may occur at the post-testicular level. This study was undertaken to assess the clinical usefulness of ICSI with testicular spermatozoa in this indication.
The percentage of spermatozoa with fragmented DNA, assessed by terminal deoxyribonucleotidyl transferase-mediated dUTP nick-end labelling assay, and ICSI outcomes were compared in two sequential attempts performed, respectively, with ejaculated and testicular spermatozoa in 18 men with increased sperm DNA fragmentation.
The incidence of DNA fragmentation was markedly lower in testicular spermatozoa as compared with ejaculated spermatozoa. No differences in fertilization and cleavage rates and in embryo morphological grade were found between the ICSI attempts performed with ejaculated and with testicular spermatozoa. However, eight ongoing clinical pregnancies (four singleton and four twin) were achieved by ICSI with testicular spermatozoa (44.4% pregnancy rate; 20.7% implantation rate), whereas ICSI with ejaculated spermatozoa led to only one pregnancy which was spontaneously aborted.
These data show that ICSI with testicular spermatozoa provides the first efficient assisted reproduction treatment option for men with high levels of sperm DNA damage.
Human Reproduction 01/2005; 20(1):226-30. · 4.47 Impact Factor
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ABSTRACT: Germ cell elimination and sperm DNA fragmentation in men with primary testiculopathies involve apoptosis-related processes whose mechanisms are poorly understood. This study examines the participation of typical (caspase-dependent) and atypical (caspase-independent) pathways in these processes.
Caspase activity and DNA fragmentation were evaluated in Sertoli and germ cells from 63 men with non-obstructive azoospermia and with different histological diagnoses who were undergoing testicular biopsy for an assisted reproduction attempt. In eight of these men, phosphatidylserine externalization was also examined.
The percentage of Sertoli cells showing caspase activity and DNA fragmentation was low and uniform in all diagnoses. In germ cells that remained tightly associated with Sertoli cells despite vigorous mechanical treatment, the incidence of both caspase activity and DNA fragmentation was high, particularly in men with maturation arrest. In Sertoli cell-free germ cells, high incidence of DNA fragmentation contrasted with low incidence of caspase activity and phosphatidylserine externalization.
In men with primary testicular failure, apoptosis of Sertoli cells is insignificant. Some germ cells undergo caspase-dependent apoptosis, show phosphatidylserine externalization and are tightly associated with Sertoli cells. Other germ cells show caspase-independent DNA fragmentation, do not externalize phosphatidylserine and lack a tight association with Sertoli cells.
Human Reproduction 03/2004; 19(2):254-61. · 4.47 Impact Factor
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ABSTRACT: 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.
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.
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.
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.
Journal of Assisted Reproduction and Genetics 09/2003; 20(8):314-7. · 1.84 Impact Factor
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ABSTRACT: To elucidate the relative predictive value of implantation markers at different stages of preimplantation development.
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.
Private infertility center.
A total of 441 couples undergoing infertility treatment.
None.
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.
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.
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.
Fertility and Sterility 08/2003; 80(1):67-74. · 3.56 Impact Factor
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ABSTRACT: To evaluate the usefulness of microfilament disruption before enucleation and nuclear transfer in human oocytes at different stages of maturation.
Prospective experimental study.
Private clinics.
Infertile couples undergoing assisted reproduction attempts.
Oocyte enucleation and nuclear transfer, activation of reconstructed oocytes.
Oocyte survival, nuclear transfer efficacy, activation outcomes.
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.
Microfilament disruption before enucleation is required for germinal vesicle oocytes but not for metaphase II oocytes.
Fertility and Sterility 04/2003; 79 Suppl 1:677-81. · 3.56 Impact Factor
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Fertility and Sterility 02/2003; 79(1):237-8; author reply 238. · 3.56 Impact Factor
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ABSTRACT: To examine whether sperm-borne and oocyte-borne oocyte activation failures can be overcome by mechanical means that entail modifying the ICSI technique.
Case report series.
Private clinics.
Six infertile couples undergoing ICSI.
Standard ICSI and modified ICSI based on mechanical manipulation that facilitated entry of calcium into the oocyte.
Fertilization rate and pregnancy outcome.
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.
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.
Fertility and Sterility 10/2002; 78(3):619-24. · 3.56 Impact Factor
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ABSTRACT: The respective advantages of day 3 and day 5 embryo transfer are a matter of debate. Previous comparisons did not include pronuclear stage zygote scoring and cumulative success rates (fresh and cryopreserved embryos).
Patients were randomized prospectively for day 3 or day 5 embryo transfer. Day 3 embryos were selected for transfer and cryopreservation by using combined evaluation at the pronuclear and cleavage stages.
There was no difference between day 3 and day 5 fresh embryo transfers as to the rates of pregnancy (58 versus 62%), clinical pregnancy (56 versus 58%), delivery (50 versus 48%), implantation (35 versus 38%) and birth (33 versus 36%) rates. The corresponding values for cryopreserved embryo transfers were also similar. However, day 3 embryo transfer compared favourably with day 5 transfer when the pregnancy (90 versus 66%), clinical pregnancy (85 versus 62%) and delivery (77 versus 52%) rates were calculated per oocyte recovery attempt.
With a selected population of good prognosis patients and our embryo selection criteria, the implantation potential of day 3 and day 5 embryos is equal. Per oocyte recovery attempt, day 3 transfer is more clinically efficient than day 5 transfer, but at least one transfer of cryopreserved embryos is necessary to manifest this superiority.
Human Reproduction 08/2002; 17(7):1852-5. · 4.47 Impact Factor
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ABSTRACT: Caspases are downstream elements of apoptosis-mediating pathways initiated by the Fas ligand/Fas receptor system which is supposed to play a central role in the regulation of apoptosis in the human seminiferous epithelium. However, caspase activity in different cell types of this epithelium has never been addressed.
We evaluated caspase activity and DNA integrity in Sertoli and germ cells within in-vitro cultured segments of human seminiferous tubules after induction of apoptosis by FSH or testosterone withdrawal. FSH withdrawal increased the incidence of DNA fragmentation in meiotic (primary spermatocytes) and post-meiotic (spermatids) germ cells without producing any detectable effect on caspase activity in these cells and without affecting DNA integrity or caspase activity in Sertoli cells. Testosterone withdrawal stimulated caspase activity and produced DNA fragmentation in Sertoli cells, but showed only a weak effect on DNA fragmentation in germ cells and did not alter germ cell caspase activity.
These findings confirm the central role of caspases in apoptosis of Sertoli cells. However, they also suggest that acute apoptosis of germ cells in the adult human testis occurs in a caspase-independent way and is controlled by Sertoli cells via an as yet undetermined mechanism.
Human Reproduction 08/2002; 17(7):1811-9. · 4.47 Impact Factor
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ABSTRACT: Previous studies on mammalian preimplantation embryos have suggested an association between caspase activation, blastomere fragmentation and apoptosis. However, some reports on human embryos questioned the causal relationship between blastomere fragmentation and apoptosis, and information about the presence and activity of caspases in human embryos is lacking.
A fluorochrome-labelled universal caspase inhibitor was used to visualize active caspases in blastomeres and fragments of preimplantation human embryos.
Caspase activity was detected only after fertilization, and was rare in blastomeres but frequent in fragments. The incidence of caspase activity in blastomeres and fragments was stable between the 2-cell and 12-cell stages. Caspase-positive blastomeres were only seen in poor-morphology embryos. The percentage of caspase-positive fragments was increased in embryos with multinucleated blastomeres but was unrelated to embryo morphology. Moreover, caspase-positive fragments detached from healthy blastomeres that were isolated by embryo biopsy and subsequently underwent mitotic division in culture.
These data suggest that caspases in preimplantation human embryos are involved in developmental processes unrelated to cell death.
Human Reproduction 07/2002; 17(6):1584-90. · 4.47 Impact Factor
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[show abstract]
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ABSTRACT: 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.
Prospective pilot study and observational clinical series.
Private hospital.
Two hundred thirty-five infertile couples undergoing frozen embryo transfer.
Removal of necrotic blastomeres from frozen-thawed human embryos.
Pregnancy and implantation rates.
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.
The viability of partially damaged frozen-thawed embryos can be improved by removal of necrotic blastomeres before embryo transfer.
Fertility and Sterility 07/2002; 77(6):1196-201. · 3.56 Impact Factor
