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Gianpiero D Palermo

Publications and source records attributed to Gianpiero D Palermo.

At least 19 recordsLinked to original sources
PubMed2006

Laser-assisted blastocyst dissection and subsequent cultivation of embryonic stem cells in a serum/cell free culture system: applications and preliminary results in a murine model.

BACKGROUND: To evaluate embryonic stem cell (ESC) harvesting methods with an emphasis on derivation of ESC lines without feeder cells or sera. Using a murine model, laser-assisted blastocyst dissection was performed and compared to conventional immunosurgery to assess a novel laser application for inner cell mass (ICM) isolation. METHODS: Intact blastocysts or isolated ICMs generated in a standard mouse strain were plated in medium with or without serum to compare ESC harvesting efficiency. ESC derivation was also undertaken in a feeder cell-free culture system. RESULTS: Although ICM growth and dissociation was comparable irrespective of the media components, an enhanced ESC harvest was observed in our serum-free medium (p < 0.01). ESC harvest rate was not affected by ICM isolation technique but was attenuated in the feeder cell-free group. CONCLUSION: Achieving successful techniques for human ESC research is fundamentally dependent on preliminary work using experimental animals. In this study, all experimentally developed ESC lines manifested similar features to ESCs obtained from intact blastocysts in standard culture. Cell/sera free murine ESC harvest and propagation are feasible procedures for an embryology laboratory and await refinements for translation to human medical research.

Journal Article↗
PubMed2006

Androgen receptor CAG polymorphism (Xq11-12) status and human spermatogenesis: a prospective analysis of infertile males and their offspring conceived by intracytoplasmic sperm injection.

We determined the association of androgen receptor (AR) (CAG)n lengths among fertile and infertile males and offspring conceived by intracytoplasmic sperm injection (ICSI). Assessment of (CAG)n repeats in the AR was performed in a Caucasian population by gene sequencing in fertile men (n=13), infertile men (n=64), boys conceived after ICSI (n=21), and boys conceived naturally (n=11). In the AZF region of the Y chromosome, a total of 22 STSs were analyzed by multiplex PCR; selected spermatozoa were also analyzed by fluorescent in situ hybridization (FISH) for chromosomes 18, 21, X, and Y. The average age was 43.7+/-7 yr for infertile, 44.8+/-7 yr for fertile men, and 5.0+/-0.5 yr for the children. The mean (CAG)n was 22.2+/-3 for the infertile men and 19.3+/-5 for fertile controls. There was a significant difference in CAG repeat length in the severely oligo-/azoo-spermic men vs. controls (p=0.02). An inverse correlation was evident between CAG length and semen parameters. For ICSI male children, the AR (CAG)n lengths were 21.4+/-3.2 vs. 20.8+/-3.4 for boys conceived naturally. While all peripheral karyotypes of fertile and infertile men were normal, de novo gonosomal abnormalities were observed in the ICSI offspring. The incidence of Y microdeletions was 1.6% in infertile men; all the ICSI sons had an intact Y chromosome. In conclusion, severely oligo- and azoospermic men had longer CAG repeat length than fertile controls, suggesting that certain AR gene mutations may have a negative effect on spermatogenesis. An increased incidence of de novo gonosomal abnormalities was found in the ICSI offspring when compared to children conceived naturally. Our assessment of the polymorphic region of the AR gene, in the absence of other specific genomic abnormalities, suggests that the fertility of children conceived by ICSI may be conserved.

Adult↗
PubMed2005

Non-obstructive azoospermia and maturation arrest with complex translocation 46,XY t(9;13;14)(p22;q21.2;p13) is consistent with the Luciani-Guo hypothesis of latent aberrant autosomal regions and infertility.

OBJECTIVE: To describe clinical and histological features observed in the setting of an unusual complex translocation involving three autosomes (9, 13, and 14) identified in an otherwise healthy male referred for infertility consultation. MATERIALS AND METHODS: The patient was age 30 and no family history was available (adopted). Total azoospermia was confirmed on multiple semen analyses. Peripheral karyotype showed a 46,XY t(9;13;14)(p22:q21.2;p13) genotype; no Y-chromosome microdeletions were identified. Cystic fibrosis screening was negative. Bilateral testis biopsy revealed uniform maturation arrest and peritubular fibrosis. RESULTS: Formal genetic counseling was obtained and the extant literature reviewed with the couple. Given the low probability of obtaining sperm on testicular biopsy, as well as the high risk of any retrieved sperm having an unbalanced genetic rearrangement, the couple elected to proceed with fertility treatment using anonymous donor sperm for insemination. CONCLUSION: Although genes mapped to the Y-chromosome have been established as critical to normal testicular development and spermatogenesis, certain autosomal genes are now also recognized as important in these processes. Here we present clinical evidence to support the Luciani-Guo hypothesis (first advanced in 1984 and refined in 2002), which predicts severe spermatogenic impairment with aberrations involving chromosomes 9, 13, and/or 14, independent of Y-chromosome status. Additional study including fluorescent in situ hybridization and molecular analysis of specific chromosomal regions is needed to characterize more fully the contribution(s) of these autosomes to male testicular development and spermatogenesis.

Journal Article↗
PubMed2005

Success of testicular sperm extraction [corrected] and intracytoplasmic sperm injection in men with Klinefelter syndrome.

PURPOSE: The aim of this study was to report the successful fertility treatment of men with Klinefelter syndrome using testicular sperm extraction (TESE) and intracytoplasmic sperm injection (ICSI). METHODS: A total of 42 men with Klinefelter syndrome who underwent 54 TESE procedures were identified. Before TESE, patients with serum testosterone levels less than 15.6 nmol/liter were treated with an aromatase inhibitor. Sperm retrieval rates and results of ICSI, including fertilization and clinical pregnancy, were collected. RESULTS: Mean pretreatment FSH and testosterone levels were 33.2 IU/liter and 9.8 nmol/liter. During medical therapy, the mean testosterone level rose to 17.0 nmol/liter (P < 0.01). Spermatozoa were found during 39 microdissection TESE procedures, on the day before, or day of oocyte retrieval during a programmed in vitro fertilization cycle. The sperm retrieval rate was 72% (39 of 54) per TESE attempt, and 29 of the 42 different men (69%) had adequate sperm found for ICSI. Thirty-three in vitro fertilization cycles yielded embryos for transfer in the 39 (85%) cycles with sperm retrieved. Eighteen clinical pregnancies have resulted in 21 live births [18 of 39 (46%)]. All children had a normal karyotype. CONCLUSION: TESE/ICSI is a successful intervention for the majority of patients with azoospermia and Klinefelter syndrome. Sperm retrieval and ICSI success in men with Klinefelter syndrome are comparable with other men with nonobstructive azoospermia treated at our center.

Adult↗
PubMed2005

Identification and isolation of embryonic stem cells in reproductive endocrinology: theoretical protocols for conservation of human embryos derived from in vitro fertilization.

BACKGROUND: Embryonic stem cells (ESC) are pluripotent cells obtained from the inner cell mass (ICM) of blastocysts derived from in vitro culture associated with reproductive endocrinology therapy. Human ESCs are regarded as highly significant since they retain the capacity to differentiate into any of approximately 200 unique cell types. Human ESC research is controversial because to acquire such cells, the ICM of human blastocysts must be manipulated in a way that renders embryos nonviable and unsuitable for transfer in utero. Techniques to yield competent ESCs with conservation of source blastocysts would satisfy many objections against ESC research, but at present such approaches remain largely untested. RESULTS AND DISCUSSION: We contrast experimental culture of single blastomeres obtained by 1) non-destructive biopsy of embryos destined for transfer, and 2) isolation of karyotypically normal blastomeres from disaggregated ("dead") embryos considered unsuitable for transfer, and evaluate these approaches with regard to production of ESCs. Pluripotency was confirmed by morphological criteria and by quantification of divergent homeodomain proteins specific to undifferentiated cell development. Following ESC isolation and identification, assessment was conducted according to a novel ESC grading system, also proposed here. CONCLUSION: The role of reproductive endocrinology in ESC research remains paramount. In this report, we hypothesize new and expand on existing strategies having the potential to enhance human ESC isolation, identification and in vitro maintenance.

Animals↗
PubMed2005

Article processing charges, funding, and open access publishing at Journal of Experimental & Clinical Assisted Reproduction.

Journal of Experimental & Clinical Assisted Reproduction is an Open Access, online, electronic journal published by BioMed Central with full contents available to the scientific and medical community free of charge to all readers. Authors maintain the copyright to their own work, a policy facilitating dissemination of data to the widest possible audience without requiring permission from the publisher. This Open Access publishing model is subsidized by authors (or their institutions/funding agencies) in the form of a single pound330 article processing charge (APC), due at the time of manuscript acceptance for publication. Payment of the APC is not a condition for formal peer review and does not apply to articles rejected after review. Additionally, this fee is waived for authors whose institutions are BioMed Central members or where genuine financial hardship exists. Considering ordinary publication fees related to page charges and reprints, the APC at Journal of Experimental & Clinical Assisted Reproduction is comparable to costs associated with publishing in some traditional print journals, and is less expensive than many. Implementation of the APC within this Open Access framework is envisioned as a modern research-friendly policy that supports networking among investigators, brings new research into reach rapidly, and empowers authors with greater control over their own scholarly publications.

Journal Article↗
PubMed2005

Construction and fertilization of reconstituted human oocytes.

Construction of artificial gametes may be made possible by transferring somatic cells into enucleated oocytes and inducing chromosomal halving of their nuclei. This study examines the possibility of constructing viable human gametes, and their potential for participation in normal fertilization. Spare germinal vesicle-stage oocytes were donated by consenting patients undergoing intracytoplasmic sperm injection (ICSI). Approximately 62% of in-vitro matured oocytes survived enucleation and subsequent cumulus cell injection. Following micromanipulation and subsequent activation, about 40% of the reconstituted oocytes yielded two pronuclear-like entities. This was not accompanied by extrusion of a polar body, but resulted in the formation of two 'putative haploid' pronuclei. Therefore selective removal of a female pronucleus marker was required to restore a balanced ploidy. Male pronuclei were identified by association with sperm mitochondria. Additional pronuclei were then removed, allowing further cleavage. Zygotes derived were 'putatively haploid' in approximately 38% of cases with a limited number of chromosomes assessed. However, on karyotypic analysis, blastomeres isolated from cleaving embryos showed a chaotic distribution of chromosomes. Oocytes could induce 'putative haploidization' of transplanted somatic cell nuclei independently of donor cell gender. Fertilization of artificial oocytes was followed by embryonic cleavage despite blastocyst development and chromosomal content possibly being compromised.

Cell Culture Techniques↗
PubMed2004

Effect of treating induced mitochondrial damage on embryonic development and epigenesis.

Germinal vesicle transplantation (GVT) has been proposed as a possible treatment to correct age-related oocyte aneuploidy caused by dysfunctional ooplasm. How healthy ooplasm regulates normal meiosis and subsequent development has yet to be elucidated, but impaired mitochondrial metabolism may be attributable to incomplete segregation of the oocyte chromosomes. In the present study, after ooplasmic mitochondrial damage by photoirradiating chloromethyl-X-rosamine, examination of the oocyte nuclei's ability to survive after transfer into healthy ooplasts was performed. To assess their fertilizability and potential for development, GVT oocytes were fertilized by intracytoplasmic sperm injection (ICSI) and transferred to foster mice. Condition of the offspring at birth was assessed, and epigenetic analysis was performed. Photosensitization consistently inhibited oocyte maturation. However, after GVT of photosensitized nuclei into healthy ooplasts, 67.2% were reconstituted, and 76.2% of these matured normally, with an overall rate of 51.2%, much higher than that (6.0%) in the mitochondrially injured oocytes. After ICSI, 65.8% (52/79) of GVT oocytes were fertilized normally, and 21.1% (11/52) eventually reached the blastocyst stage. The transfer of 132 two-cell GVT embryos into the oviducts of pseudopregnant females resulted in 17 apparently healthy live offspring. For some key developmental genes, a high level of expression was identified in the GVT and "rescue"-derived fetal adnexa. Thus, one can induce in oocyte mitochondria a photosensitization-based type of damage, which consistently inhibits GV breakdown, meiotic spindle formation, chromosomal segregation, and polar body extrusion. Germinal vesicle transplanted and rescued oocytes were able to undergo maturation, fertilization, and embryonic cleavage and, ultimately, to develop to term. This approach may provide a model with which to study the age-related ooplasmic dysfunction seen in human oocytes.

Animals↗
PubMed2004

Journal of Experimental & Clinical Assisted Reproduction: shaping the future of research and practice in reproductive endocrinology/infertility.

Journal of Experimental & Clinical Assisted Reproduction is an open access, online, peer-review journal publishing papers on all aspects of research into reproductive endocrinology, infertility, bioethics and the advanced reproductive technologies. The journal reports on important developments impacting the field of human reproductive medicine and surgery. The field exists as a sub-specialty of obstetrics & gynecology, focusing on the diagnosis and treatment of complex human reproductive problems. The continued growth of this relatively new field depends on quality research by proven scientists as well as junior investigators who, together, make contributions to this area of medical and surgical practice. The publishing revolution made possible by internet technology presages a bright future for continued interdisciplinary collaboration among researchers. Against this background, Journal of Experimental & Clinical Assisted Reproduction exists for the scientific community to facilitate this scholarly dialogue.

Journal Article↗
PubMed2004

A successful model to assess embryo development after transplantation of prophase nuclei.

BACKGROUND: Germinal vesicle transplantation (GVT) provides a means of investigating interactions between karyoplasts and cytoplasts isolated from different cells. Technically, GVT can be accomplished with a high degree of efficiency without compromising the maturation of either the human or mouse oocyte nucleus. Although maturation, fertilization and preimplantation development have been established using GVT, full-term development has been reported only after supplementation with fresh mature ooplasm. In this study, we assess the ability of immature oocytes collected from gonadotrophin-primed ovaries to mature in vitro after GVT and develop to full-term. METHODS: GV oocytes were retrieved from either non-stimulated or pregnant mare's serum gonadotrophin (PMSG)-primed female mice. Microsurgically isolated GV karyoplasts were transplanted into previously enucleated oocytes. Oocytes successfully reconstituted by electrofusion were cultured for 14 h to allow nuclear maturation. Metaphase II oocytes were subjected to Piezo-ICSI, and those fertilized normally were cultured to the blastocyst stage. Some such embryos were transferred to pseudopregnant female mice to examine their potential for normal development. Cumulus-denuded non-manipulated oocytes that were matured in vitro served as controls. RESULTS: The reconstitution and maturation rates were comparable in oocytes isolated from PMSG-primed and from unstimulated ovaries. The rate of normal fertilization in oocytes from primed ovaries was significantly higher than that of their non-primed counterparts (63.5 versus 39.6%; P < 0.01). This difference was also confirmed in terms of blastocyst development (31.8 versus 7.9%; P < 0.01). Of a total of 70 embryos transferred to the oviduct of five recipient mice, 21.4% developed to normal live offspring. All developed as normal adults and proved to be fertile. The live birth rate was comparable to that obtained using non-manipulated control oocytes (22.3%). CONCLUSIONS: Higher rates of fertilization and blastocyst formation were obtained after GVT of mouse oocytes isolated from PMSG-primed ovaries compared with their non-primed counterparts. These represent the first mouse offspring derived from in vitro matured, cumulus-denuded oocytes treated by allo-GVT and fertilized by ICSI. Thus, GVT appears not to impair oocyte maturation, fertilization and pre- and post-implantation development and, after gonadotrophin priming, allows generation of healthy mouse offspring without mature ooplasm supplementation.

Animals↗
PubMed2004

AZF microdeletions of the Y chromosome and in vitro fertilization outcome.

OBJECTIVE: To determine whether the presence of a Y microdeletion confers any adverse effects on in vitro fertilization or intracytoplasmic sperm injection (IVF/ICSI) outcome. DESIGN: Retrospective case-control study. SETTING: Academic infertility center. PATIENT(S): A total of 17 patients with Y microdeletions who attempted IVF/ICSI cycles at our center between March 1996 and March 2002 were studied. Study patients were analyzed in two groups: those who underwent testicular sperm extraction (TESE) and those for whom ejaculated sperm was used. INTERVENTION(S): The two patient study groups were matched to controls treated at the same time who had either nonobstructive severe oligozoospermia or azoospermia with normal Y chromosomes. Controls were matched for age of the female partner, sperm concentration, and number of embryos transferred. MAIN OUTCOME MEASURE(S): Fertilization and clinical pregnancy rates. RESULT(S): Sperm was only obtained from patients with azoospermic factor (AZF)c microdeletions (and from one patient with a partial AZFb microdeletion). A trend toward lower fertilization rates in patients with Y microdeletions was noted, which did not reach statistical significance. Clinical pregnancy rates per cycle and per transfer were similar to those for controls. CONCLUSION(S): Patients with AZFc microdeletions seem to have IVF/ICSI outcomes comparable to those of controls with normal Y chromosomes.

Case-Control Studies↗
PubMed2004

Genetic and epigenetic modifications associated with human ooplasm donation and mitochondrial heteroplasmy - considerations for interpreting studies of heritability and reproductive outcome.

The mitochondrial heteroplasmy present in offspring from IVF and human ooplasm donation is troublesome and merits further exploration in a debate that is already complex and controversial. Improving the understanding of mitochondrial genomics in this context is important because mitochondriopathies can impact crucial cellular processes in renal, cardiovascular, central nervous, and endocrine systems. Relevant epigenetic consequences of mitochondrial heteroplasmy include associated abnormalities in mitochondrial translation products. Furthermore, as transmission and inheritance patterns of mtDNA are species-specific, it remains to be proven if findings derived from animal studies are applicable to human offspring. As an alternative to gamete research and proteomics based on animal experimentation, continued molecular characterization of the de novo human mitochondriopathies is posed to offer further insights regarding mitochondrial heteroplasmy. In this context, because knowledge of human mitochondrial genetics remains limited and the risks associated with ooplasm donation cannot be quantified, we do not favor its use for our patients at present. However, the small number of infants already conceived from this experimental approach warrant careful longitudinal evaluation. In particular, observational study of the few children born after ooplasm donation could provide opportunities to assess human mtDNA transmission and inheritance. Such findings could help identify features distinguishing natural mtDNA heteroplasmy from heteroplasmy observed after ooplasm donation. Future investigations should also quantify the degree any such heteroplasmy can exist innocuously. Disclosure of mtDNA mutations potentially affecting children conceived from IVF and ooplasm donation must be included during patient education at centers contemplating such treatment.

Animals↗
PubMed2004

Y chromosome assessment and its implications for the development of ICSI children.

The aetiology of compromised spermatogenesis is often genetic in nature. There are only a few reports of father/son cohorts that have been evaluated to assess heritability of mutations associated with male factor infertility and the psychological well-being of the children. In the present study, multiple tissues were sampled from consenting male patients and their sons derived from intracytoplasmic sperm injection (ICSI) and underwent chromosomal and genetic analyses. Paediatric and psychological examinations were also conducted. In 87 men and 47 boys, 22 sequence tagged sites (STS) were analysed by multiplex PCR and deletion breakpoints were defined with additional loci. In one patient, the breakpoints map to the highly unstable palindrome-rich region within AZFb and proximal AZFc was investigated. A total of 86 blood, 26 semen, and 73 cheek cells samples were collected from adults, and 36 blood samples and 44 cheek cell specimens were obtained from the boys. Though all of the fathers had normal karyotypes, the incidence of chromosomal abnormalities in the somatic cells of male progeny was 8.3% (3/36). The incidence of germ line aneuploidy in these men was 0.5-2.8%. A CF mutation (Delta508) was detected in one of 87 men (1.2%) and microdeletions in Yq AZF were detected in 3.4% of 87 adults and in 2.1% of their sons (n = 47). In conclusion, screening for Y chromosome microdeletions provides crucial information in the counselling of couples seeking infertility treatment. Moreover, DNA extraction and Y deletion assessments of cheek cells provide a non-invasive approach. Inheritance of Yq deletions appears not to affect the psychological and physical development of children derived from ICSI.

Adult↗
PubMed2004

Implications of cloning technique for reproductive medicine.

The birth of Dolly following the transfer of mammary gland nuclei into enucleated eggs established cloning as a feasible technique in mammals, but the moral implications and high incidence of developmental abnormalities associated with cloning have induced the majority of countries to legislate against its use with human gametes. Because of such negative connotations, restrictive political reactions could jeopardize the therapeutic and scientific promise that certain types of cloning may present. For example, in addition to its proposed use as a way of generating stem cells, the basic technique of nuclear transplantation has proven useful in other ways, including its application to immature eggs as a new approach to the prevention of the aneuploidy common in older women, and for some recent advances in preimplantation genetic diagnosis. Thus, while attempts at reproductive cloning in man would seem premature and even dangerous at present, this field will require rational rather than emotional reactions as a basis for legislation if the therapeutic promise of stem cell research and the experimental potential of nuclear transplantation techniques are to be fully realized.

Cloning, Organism↗
PubMed2004

Medical follow-up study of 5-year-old ICSI children.

Children born after intracytoplasmic sperm injection (ICSI) are still a matter of concern. The purposes of the present study were to investigate the physical outcome in 5-year-old children born after ICSI and compare them with children born after spontaneous conception. Three hundred singleton children from Belgium, Sweden and the USA, born after ICSI, were matched by maternal age, child age and gender. In one centre, matching was also performed for maternal education. The main end-point was growth. Secondary end-points were general health, e.g. common diseases, chronic illnesses, surgical interventions and physical/neurological examinations. Standard deviation scores assessed growth. Growth assessed as stature at follow-up was similar in the two groups, despite a higher rate of preterm birth and low birth weight in the ICSI children. Common diseases and chronic illnesses occurred at similar rates in both groups. More ICSI children underwent surgical interventions and required other therapy e. g. physiotherapy and dietary therapy. Physical/neurological examinations revealed few abnormalities in either group. In conclusion, infertility treatment by ICSI does not adversely affect growth during childhood. The children's general health seems satisfactory.

Abnormalities, Multiple↗
PubMed2004

Does ICSI require acrosomal disruption? An ultrastructural study.

BACKGROUND: Aggressive immobilization of sperm prior to ICSI significantly improves fertilization rates, but the mechanism of this effect is not yet clear. This study was performed in order to assess the characteristics of mechanically immobilized human sperm by transmission electron microscope (TEM). METHODS: Sperm obtained from ejaculated semen samples from three different donors were immobilized in a standard manner for ICSI. They were then injected into the perivitelline space of mouse oocytes in order to be able to locate them by TEM. Intact motile sperm injected subzonally served as controls (n=160). Finally, the 'carrier' oocytes were fixed and processed for TEM. RESULTS: A total of 300 sperm were mechanically immobilized and inserted into the perivitelline space of mouse oocytes. Ultrathin sections revealed consistent alterations in the acrosomal region including disruption of the plasma membrane, and disruption, vesiculation or even loss of the acrosome. Thus, all of the sperm assessed had undergone some disorganization of the head, in contrast to a majority of control sperm. CONCLUSIONS: Immobilization of sperm for ICSI by compressing and rolling the sperm tails induces a variable disruption and sometimes loss of the acrosome. This could well be a reason for the higher success rates when ICSI is performed using immobilized sperm.

Acrosome↗