Publications by authors named "Antonino Forabosco"

34 Publications

Missense mutations in EDA and EDAR genes cause dominant syndromic tooth agenesis.

Mol Genet Genomic Med 2021 01 18;9(1):e1555. Epub 2020 Nov 18.

Department of Biomolecular Sciences, University of Urbino, Fano, Italy.

Background: Hypohidrotic ectodermal dysplasia (HED) is the most common form of ectodermal dysplasia and is mainly associated with mutations in the EDA, EDAR, and EDARADD responsible for the development of ectodermal-derived structures. HED displays different modes of inheritance according to the gene that is involved, with X-linked EDA-related HED being the most frequent form of the disease.

Methods: Two families with tooth agenesis and manifestations of HED underwent clinical examination and EDA, EDAR, and EDARADD genetic analysis. The impact of the novel variant on the protein was evaluated through bioinformatics tools, whereas molecular modeling was used to predict the effect on the protein structure.

Results: A novel missense variant was identified in the EDAR (c.287T>C, p.Phe96Ser) of a female child proband and her mother, accounting for autosomal dominant HED. The genetic variant c.866G>A (p.Arg289His) in EDA, which has been previously described, was observed in the male proband of another family confirming its role in X-linked HED. The inheritance model of the missense mutation showed a different relationship with X-linked HED and non-syndromic tooth agenesis.

Conclusion: Our findings provide evidence of variable expression of HED in heterozygous females, which should be considered for genetic counseling, and different modes of inheritance related to tooth development.
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http://dx.doi.org/10.1002/mgg3.1555DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7963410PMC
January 2021

Lhx8 ablation leads to massive autophagy of mouse oocytes associated with DNA damage.

Biol Reprod 2018 04;98(4):532-542

Intramural Research Program, National Institute on Aging, National Institutes of Health, Baltimore, Maryland, USA.

Following proliferation of oogonia in mammals, great numbers of germ cells are discarded, primarily by apoptosis, while the remainder form primordial follicles (the ovarian reserve) that determine fertility and reproductive lifespan. More massive, rapid, and essentially total loss of oocytes, however, occurs when the transcription factor Lhx8 is ablated-though the cause and mechanism of germ cell loss from the Lhx8-/- ovaries has been unknown. We found that Lhx8-/- ovaries maintain the same number of germ cells throughout embryonic development; rapid decrease in the pool of oocytes starts shortly before birth. The loss results from activation of autophagy, which becomes overwhelming within the first postnatal week, with extracellular matrix proteins filling the space previously occupied by follicles to produce a fibrotic ovary. Associated with this process, as early as a few days before birth, Lhx8-/- oocytes failed to repair DNA damage-which normally occurs when meiosis is initiated during embryonic development; and DNA damage repair genes were downregulated throughout the oocyte short lifespan. Based on gene expression analyses and morphological changes, we propose a model in which lineage-restricted failure of DNA repair triggers germ cell autophagy, causing premature depletion of the ovarian reserve in Lhx8-/- mice.
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http://dx.doi.org/10.1093/biolre/iox184DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6279113PMC
April 2018

Duplications upstream and downstream of SHOX identified as novel causes of Leri-Weill dyschondrosteosis or idiopathic short stature.

Am J Med Genet A 2016 Apr 24;170A(4):949-57. Epub 2015 Dec 24.

Laboratory of Human Genetics, E.O. Ospedali Galliera, Genoa, Italy.

Leri-Weill dyschondrosteosis is a pseudoautosomal dominantly-inherited skeletal dysplasia ascribed to haploinsufficiency of the SHOX gene caused by deletions, point mutations, or partial duplications of the gene, or to heterozygous deletions upstream or downstream of the intact SHOX gene involving conserved non-coding cis-regulatory DNA elements that show enhancer activity. Recently, two SHOX conserved non-coding element duplications, one upstream and one downstream, were reported in patients referred with idiopathic short stature. To further evaluate the role of these duplications in SHOX-related disorders, we describe seven patients (five with Leri-Weill dyschondrosteosis and two with short stature) all of whom have duplications of part of the upstream or downstream conserved non-coding element regions, identified by multiplex ligation-dependent probe amplification. In addition, we show data from 32 patients with an apparently identical downstream duplication that includes a proposed putative regulatory element (identified by multiplex ligation-dependent probe amplification or array comparative genome hybridization), which results in a variable phenotype from normal to mild Leri-Weill dyschondrosteosis. These additional data provide further evidence that duplications of upstream and downstream long range cis-regulatory DNA elements can result in a SHOX-related phenotype.
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http://dx.doi.org/10.1002/ajmg.a.37524DOI Listing
April 2016

Genetics of the ovarian reserve.

Front Genet 2015 15;6:308. Epub 2015 Oct 15.

Intramural Research Program, National Institute on Aging, National Institutes of Health , Baltimore, MD, USA.

Primordial follicles or non-growing follicles (NGFs) are the functional unit of reproduction, each comprising a single germ cell surrounded by supporting somatic cells. NGFs constitute the ovarian reserve (OR), prerequisite for germ cell ovulation and the continuation of the species. The dynamics of the reserve is determined by the number of NGFs formed and their complex subsequent fates. During the reproductive lifespan, the OR progressively diminishes due to follicle atresia as well as recruitment, maturation, and ovulation. The depletion of the OR is the major determining driver of menopause, which ensues when the number of primordial follicles falls below a threshold of ∼1,000. Therefore, genes and processes involved in follicle dynamics are particularly important to understand the process of menopause, both in the typical reproductive lifespan and in conditions like primary ovarian insufficiency, defined as menopause before age 40. Genes and their variants that affect the timing of menopause thereby provide candidates for diagnosis of and intervention in problems of reproductive lifespan. We review the current knowledge of processes and genes involved in the development of the OR and in the dynamics of ovarian follicles.
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http://dx.doi.org/10.3389/fgene.2015.00308DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4606124PMC
November 2015

FOXL2 modulates cartilage, skeletal development and IGF1-dependent growth in mice.

BMC Dev Biol 2015 Jul 2;15:27. Epub 2015 Jul 2.

Istituto di Ricerca Genetica e Biomedica, Consiglio Nazionale delle Ricerche, Cittadella Universitaria di Monserrato, SS 554 km 4500, Monserrato, 09042, Italy.

Background: Haploinsufficiency of the FOXL2 transcription factor in humans causes Blepharophimosis/Ptosis/Epicanthus Inversus syndrome (BPES), characterized by eyelid anomalies and premature ovarian failure. Mice lacking Foxl2 recapitulate human eyelid/forehead defects and undergo female gonadal dysgenesis. We report here that mice lacking Foxl2 also show defects in postnatal growth and embryonic bone and cartilage formation.

Methods: Foxl2 (-/-) male mice at different stages of development have been characterized and compared to wild type. Body length and weight were measured and growth curves were created. Skeletons were stained with alcian blue and/or alizarin red. Bone and cartilage formation was analyzed by Von Kossa staining and immunofluorescence using anti-FOXL2 and anti-SOX9 antibodies followed by confocal microscopy. Genes differentially expressed in skull vaults were evaluated by microarray analysis. Analysis of the GH/IGF1 pathway was done evaluating the expression of several hypothalamic-pituitary-bone axis markers by RT-qPCR.

Results: Compared to wild-type, Foxl2 null mice are smaller and show skeletal abnormalities and defects in cartilage and bone mineralization, with down-regulation of the GH/IGF1 axis. Consistent with these effects, we find FOXL2 expressed in embryos at 9.5 dpc in neural tube epithelium, in head mesenchyme near the neural tube, and within the first branchial arch; then, starting at 12.5 dpc, expressed in cartilaginous tissue; and at PO and P7, in hypothalamus.

Conclusions: Our results support FOXL2 as a master transcription factor in a spectrum of developmental processes, including growth, cartilage and bone formation. Its action overlaps that of SOX9, though they are antagonistic in female vs male gonadal sex determination but conjoint in cartilage and skeletal development.
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http://dx.doi.org/10.1186/s12861-015-0072-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4489133PMC
July 2015

Dynamics of the ovarian reserve and impact of genetic and epidemiological factors on age of menopause.

Biol Reprod 2015 May 22;92(5):130. Epub 2015 Apr 22.

Intramural Research Program, National Institute on Aging, National Institutes of Health, Baltimore, Maryland.

The narrow standard age range of menopause, ∼50 yr, belies the complex balance of forces that govern the underlying formation and progressive loss of ovarian follicles (the "ovarian reserve" whose size determines the age of menopause). We show here the first quantitative graph of follicle numbers, distinguished from oocyte counts, across the reproductive lifespan, and review the current state of information about genetic and epidemiological risk factors in relation to possible preservation of reproductive capacity. In addition to structural X-chromosome changes, several genes involved in the process of follicle formation and/or maintenance are implicated in Mendelian inherited primary ovarian insufficiency (POI), with menopause before age 40. Furthermore, variants in a largely distinct cohort of reported genes-notably involved in pathways relevant to atresia, including DNA repair and cell death-have shown smaller but additive effects on the variation in timing of menopause in the normal range, early menopause (age <45), and POI. Epidemiological factors show effect sizes comparable to those of genetic factors, with smoking accounting for about 5% of the risk of early menopause, equivalent to the summed effect of the top 17 genetic variants. The identified genetic and epidemiological factors underline the importance of early detection of reproductive problems to enhance possible interventions.
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http://dx.doi.org/10.1095/biolreprod.114.127381DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4645983PMC
May 2015

Constitutively active Foxo3 in oocytes preserves ovarian reserve in mice.

Nat Commun 2013 ;4:1843

Laboratory of Genetics, NIA/NIH-IRP, 251 Bayview blvd, room 10B014, Baltimore, Maryland 21224, USA.

During female reproductive life, ovarian follicle reserve is reduced by maturation and atresia until menopause ensues. Foxo3 is required to maintain the ovarian reserve in mice. Here we show that overexpression of constitutively active FOXO3 can increase ovarian reproductive capacity in mice. We find increased follicle numbers and decreased gonadotropin levels in aging FOXO3-transgenic mice compared with wild-type littermates, suggesting maintenance of a greater ovarian reserve. Based on cumulative progeny in aging animals, we find 31-49% increased fertility in transgenic females. The gene expression profile of Foxo3-/- knockout ovaries appears older than that of wild-type littermates, and the transgene induces a younger-looking profile, restoring much of the wild-type transcriptome. This is the first gain-of-function model of augmented reproductive reserve in mice, thus emphasizing the role of Foxo3 as a guardian of the ovarian follicle pool in mammals and a potential determinant of the onset of menopause.
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http://dx.doi.org/10.1038/ncomms2861DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4504230PMC
December 2013

Germ cell formation from embryonic stem cells and the use of somatic cell nuclei in oocytes.

Ann N Y Acad Sci 2011 Mar;1221:18-26

Laboratory of Genetics, National Institute on Aging/NIH Intramural Research Program, Baltimore, Maryland, USA.

Embryonic stem cells (ESCs) have remarkable properties of pluripotency and self-renewal, along with the retention of chromosomal integrity. Germ cells function as a kind of "transgenerational stem cells," transmitting genetic information from one generation to the next. The formation of putative primordial germ cells (PGCs) and germ cells from mouse and human ESCs (hESCs) has, in fact, been shown, and the apparent derivation of functional mouse male gametes has also been described. Additionally, investigators have successfully reprogrammed somatic nuclei into a pluripotent state by inserting them into ESCs or oocytes. This would enable the generation of ESCs genetically identical to the somatic cell donor and their use in cell therapy. However, these methodologies are still inefficient and their mechanisms poorly understood. Until full comprehension of these processes is obtained, clinical applications remain remote. Nevertheless, they represent promising tools in the future, enhancing methods of therapeutic cloning and infertility treatment.
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http://dx.doi.org/10.1111/j.1749-6632.2011.05982.xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3059193PMC
March 2011

Unexpected phenotype in a boy with trisomy of the SHOX gene.

J Pediatr Endocrinol Metab 2010 Jan-Feb;23(1-2):159-69

Department of Pediatrics, University of Modena & Reggio Emilia, Modena, Italy.

The assessment that heterozygous SHOX mutations leading to SHOX haploinsufficiency play a role in patients with idiopathic short stature (ISS) is already documented in the literature as well as the suggestion that additional copies of SHOX are strongly implicated in a condition of tall stature. However, we report the first case of short stature in a male associated with the presence of three copies of the SHOX gene. Through chromosomal analysis, using Multiplex Ligation-dependent Probe Amplification method of SHOX salsa P018B kit and microsatellite analysis, we identify a new interstitial isolated duplication of the SHOX gene and its enhancer caused by a larger duplication of the PAR1 region in a boy with ISS. Consequently, we propose the hypothesis that this chromosome re-arrangement disrupts the regular interaction between the enhancer and promoter, resulting in a transcription block, thus producing a lack of gene activation, causing the clinical feature of short stature.
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http://dx.doi.org/10.1515/jpem.2010.23.1-2.159DOI Listing
May 2010

Determination and stability of gonadal sex.

J Androl 2010 Jan-Feb;31(1):16-25. Epub 2009 Oct 29.

Laboratory of Genetics, National Institute on Aging-IRP, Baltimore, MD 21224, USA.

The discovery that the SRY gene induces male sex in humans and other mammals led to speculation about a possible equivalent for female sex. But females are proving to be more complicated. Several master genes appear to be autonomously involved, and female sex determination seems to remain relatively labile. Partial loss of function of the transcription factor FOXL2 leads to premature ovarian failure in women; and in animal models, Foxl2 is required for folliculogenesis as well as for maintenance, and possibly induction, of female sex determination. In the germ line, oocytes apparently form normally even in the absence of Foxl2, dependent on genes that include female-specific factors such as Fig-alpha, Nobox, etc. In the soma, ablation of Foxl2 or the independently expressed gene Wnt4 (likely downstream of Rspo1) can produce partial testis differentiation in XX mice, and the double knockout results in the formation of tubules and spermatogonia. This indicates that at least 2 autonomous ovarian pathways are required to antagonize testis differentiation in females, a finding that is being increasingly corroborated by studies in goats and nonmammalian vertebrates. In recent expression profiling of mouse ovaries that lack Foxl2 alone or in combination with Wnt4 or Kit/c-Kit, we found that following Foxl2 loss, early testis genes (including the downstream effector of Sry, Sox9) and several novel ovarian genes were consistently dysregulated during embryo-fetal development. The results support the proposal of dose-dependent Foxl2 function and antitestis action. A partial working model for somatic development and sex determination is presented in which Sox9 is a direct antagonist of Foxl2 in the supporting cell lineage.
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http://dx.doi.org/10.2164/jandrol.109.008201DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2882171PMC
June 2010

Foxl2 functions in sex determination and histogenesis throughout mouse ovary development.

BMC Dev Biol 2009 Jun 18;9:36. Epub 2009 Jun 18.

Laboratory of Genetics, NIA/NIH-IRP, Baltimore, MD, USA.

Background: Partial loss of function of the transcription factor FOXL2 leads to premature ovarian failure in women. In animal models, Foxl2 is required for maintenance, and possibly induction, of female sex determination independently of other critical genes, e.g., Rspo1. Here we report expression profiling of mouse ovaries that lack Foxl2 alone or in combination with Wnt4 or Kit/c-Kit.

Results: Following Foxl2 loss, early testis genes (including Inhbb, Dhh, and Sox9) and several novel ovarian genes were consistently dysregulated during embryonic development. In the absence of Foxl2, expression changes affecting a large fraction of pathways were opposite those observed in Wnt4-null ovaries, reinforcing the notion that these genes have complementary actions in ovary development. Loss of one copy of Foxl2 revealed strong gene dosage sensitivity, with molecular anomalies that were milder but resembled ovaries lacking both Foxl2 alleles. Furthermore, a Foxl2 transgene disrupted embryonic testis differentiation and increased the levels of key female markers.

Conclusion: The results, including a comprehensive principal component analysis, 1) support the proposal of dose-dependent Foxl2 function and anti-testis action throughout ovary differentiation; and 2) identify candidate genes for roles in sex determination independent of FOXL2 (e.g., the transcription factors IRX3 and ZBTB7C) and in the generation of the ovarian reserve downstream of FOXL2 (e.g., the cadherin-domain protein CLSTN2 and the sphingomyelin synthase SGMS2). The gene inventory is a first step toward the identification of the full range of pathways with partly autonomous roles in ovary development, and thus provides a framework to analyze the genetic bases of female fertility.
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http://dx.doi.org/10.1186/1471-213X-9-36DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2711087PMC
June 2009

Short stature homeoboxcontaining gene and idiopathic short stature.

Expert Rev Endocrinol Metab 2009 May;4(3):241-250

g Chair of Genetics, University of Modena and Reggio Emilia, Via del Pozzo, 71-41.100 Modena, Italy.

The term idiopathic short stature (ISS) refers to patients who are short due to various unknown reasons. Although it is clear that multiple factors contribute to final height, genetic factors play a crucial role. Mutations of a human homeobox gene, short stature homeobox-containing (SHOX) gene, have been shown to be associated with the short stature phenotype in patients with Turner syndrome, most patients with Leri-Weill dyschondrosteosis and some cases of ISS. The prevalence of SHOX anomalies in subjects previously recognized as having ISS has been estimated at 2.4% in a large series of ISS individuals. This review focuses on the functional properties of the SHOX gene and its linkage to ISS.
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http://dx.doi.org/10.1586/eem.09.5DOI Listing
May 2009

Incidence of non-age-dependent chromosomal abnormalities: a population-based study on 88965 amniocenteses.

Eur J Hum Genet 2009 Jul 21;17(7):897-903. Epub 2009 Jan 21.

Department of Medical Genetics, University of Modena, Modena, Italy.

Current knowledge about the incidence of chromosomal abnormalities in the general population comes from studies in newborns carried out in the 70s, before the era of widespread prenatal diagnosis. In the following years, data on frequency of chromosomal abnormalities in the second trimester of pregnancy have been used in conjunction with the data on the natural history of chromosomally abnormal fetuses to infer maternal age-specific rates of cytogenetic abnormalities in live-born infants. Starting from the data gathered in 1995-1996 from all Italian cytogenetic laboratories (with 92% compliance to the study), we have compared the frequency of chromosomal abnormalities at amniocentesis in cases with maternal age of >or=35 years (51,758 individuals) and cases with maternal age of <35 years (37,207 cases). The comparison confirmed the age-dependency of aneuploidies, whereas none of the structural abnormalities showed age-related differences. Furthermore, among the mosaic aneuploidies, trisomy 21 and 45,X/46,XX were found with a significantly higher incidence in older women. Chromosomal abnormalities that showed no significant difference between the two groups were summed for the overall national cohort, providing a general estimate of the incidence in the second trimester of pregnancy. The data provide critical background information for prenatal genetic counseling and for the planning of health care policy.
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http://dx.doi.org/10.1038/ejhg.2008.265DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2986487PMC
July 2009

Loss of Wnt4 and Foxl2 leads to female-to-male sex reversal extending to germ cells.

Hum Mol Genet 2007 Dec 29;16(23):2795-804. Epub 2007 Aug 29.

Laboratory of Genetics, NIA/NIH-IRP, Baltimore, USA.

The discovery that the SRY gene induces male sex in humans and other mammals led to speculation about a possible equivalent for female sex. However, only partial effects have been reported for candidate genes experimentally tested so far. Here we demonstrate that inactivation of two ovarian somatic factors, Wnt4 and Foxl2, produces testis differentiation in XX mice, resulting in the formation of testis tubules and spermatogonia. These genes are thus required to initiate or maintain all major aspects of female sex determination in mammals. The two genes are independently expressed and show complementary roles in ovary morphogenesis. In addition, forced expression of Foxl2 impairs testis tubule differentiation in XY transgenic mice, and germ cell-depleted XX mice lacking Foxl2 and harboring a Kit mutation undergo partial female-to-male sex reversal. The results are all consistent with an anti-testis role for Foxl2. The data suggest that the relative autonomy of the action of Foxl2, Wnt4 and additional ovarian factor(s) in the mouse should facilitate the dissection of their respective contributions to female sex determination.
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http://dx.doi.org/10.1093/hmg/ddm235DOI Listing
December 2007

Transcriptional control of ovarian development in somatic cells.

Semin Reprod Med 2007 Jul;25(4):252-63

Laboratory of Genetics, National Institute on Aging, Baltimore, MD, USA.

Developmental transitions of the bipotential gonad to the embryonic ovary and thence to the follicle-filled mature ovary are expected to be coordinated by sets of transcription factors. We infer candidate lists here, focusing on somatic cell fate and function. For the mouse, developmental stages of ovary differentiation are relatively discretely phased, and provide a unique tool to investigate the intricate mechanisms that lead to the acquisition of female reproductive competence. Cross-platform gene expression profiles supplement functional studies of specific genes and comparative information about human biology. Available data suggest that: (1) peak transcription activity just precedes the two most decisive steps of early ovary differentiation (i.e., entry into meiosis and follicle formation); (2) alternating peak gene activities in oocytes and somatic cells may reflect reciprocal interactions; and (3) in addition to stable states of chromatin modification associated with morphogenesis, some features of differentiation are labile, contingent on the expression state of critical factors. Examples are the maintenance of somatic sex determination by continued Foxl2 action and the reversible maintenance of follicles in a quiescent state by nuclear Foxo3.
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http://dx.doi.org/10.1055/s-2007-980219DOI Listing
July 2007

Establishment of ovarian reserve: a quantitative morphometric study of the developing human ovary.

Fertil Steril 2007 Sep 16;88(3):675-83. Epub 2007 Apr 16.

Medical Genetics Unit, Dipartimento Materno Infantile, Facoltà di Medicina e Chirurgia, Università degli Studi di Modena e Reggio Emilia, Modena, Italy.

Objective: To assess directly the dynamics of the formation of the ovarian reserve in the normal human ovary by evaluating the total number of follicles in developing ovaries when folliculogenesis occurs.

Design: Histomorphometry-based follicle counts in complete serial tissue sections.

Setting: Functional Anatomy Research Center, University of Milano.

Patient(s): Thirteen fetuses, neonates, and one 8-month-old infant.

Intervention(s): Fifteen ovaries were completely cut, obtaining serial sections. Ovarian volume, volume fractions, density and total number of follicles per ovary were calculated using quantitative morphometric methods.

Main Outcome Measure(s): Age-related dynamics of the establishment of ovarian reserve in human developing ovary at the end of the organogenesis.

Result(s): The ovarian reserve (100,000 follicles at 15 weeks of postconceptional age) increased progressively to 680,000 follicles at 34 weeks. At 8 months of postnatal age the pool was still about 680,000 primordial follicles.

Conclusion(s): The consistence of the primordial follicle pool during organogenesis shows an exponential increase until month 8 of prenatal life and it is subsequently maintained without modifications at least until month 8 of postnatal life.
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http://dx.doi.org/10.1016/j.fertnstert.2006.11.191DOI Listing
September 2007

Age-specific risk of fetal loss post second trimester amniocentesis: analysis of 5043 cases.

Prenat Diagn 2007 Feb;27(2):180-3

Prenatal Medicine Unit, Department of Obstetrics and Gynecology, Modena and Reggio Emilia University, Italy.

Objectives: To assess the risk of fetal loss attributable to second trimester amniocentesis in singleton pregnancies through a cross-sectional study.

Methods: Records of 5043 consecutive second trimester amniocentesis, performed by a single operator between 1997 and 2003, were analyzed. Fetal loss post amniocentesis was calculated by grouping pregnant women in age classes and assessing observed/expected (O/E) rate.

Results: Total fetal losses were 40 (0.81%): 33 cases (0.67%) occurred before the 24th week, 37 cases (0.76%) before the 28th gestational week, and 3 cases (0.06%) after the 28th week of pregnancy. An age-dependent increase of the rate of fetal loss, not statistically significant (Chi-Square = 0.349, p = 0.505) was observed. The total O/E ratio values did not show any statistically significant risk (O/E ratio = 1.25, CI = 0.86-1.64). The analysis of the single age classes did not detect any statistical significance. The excess fetal loss rate associated with amniocentesis was 0.16%.

Conclusions: No effect of the 2nd trimester amniocentesis was noted on fetal loss.
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http://dx.doi.org/10.1002/pd.1647DOI Listing
February 2007

Determination and stability of sex.

Bioessays 2007 Jan;29(1):15-25

Laboratory of Genetics, National Institute on Aging, Baltimore, MD 21224, USA.

How is the embryonic bipotential gonad regulated to produce either an ovary or a testis? In males, transient early activation of the Y chromosome Sry gene makes both germ cells and soma male. However, in females, available evidence suggests that the process of ovary sex determination may take place independently in the germline and somatic lineages. In addition, in contrast to testis, in ovary somatic cells, female-to-male gonadal sex reversal can occur at times throughout ovary development and maturation. We suggest that a single gene pathway, likely hinging on the Foxl2 transcription factor, both initiates and maintains sex differentiation in somatic cells of the mammalian ovary.
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http://dx.doi.org/10.1002/bies.20515DOI Listing
January 2007

Detection of a novel dystrophin gene mutation through carrier analysis performed during prenatal diagnosis in a case with intragenic recombination.

Prenat Diagn 2005 Nov;25(11):1011-4

Unità di Genetica Medica, Università di Modena, Italy.

Objectives: To report a multi-technical approach to Duchenne muscular dystrophy (DMD) mutation testing through carrier analysis, in the prenatal diagnosis of a male foetus without a known mutation segregating in the family and with inconclusive results of linkage analysis.

Methods: Haplotype analysis with the DMD region markers for assigning the carrier status of the mother and for prenatal diagnosis of foetal DNA; semiquantitative multiplex analysis of maternal and foetal DNA for the promoter and for 34 exons of the DMD gene; sequencing analysis of the maternal and foetal DNA for confirmation of the results.

Results: Because of an intragenic recombination of the DMD gene in foetal DNA, haplotype analysis gave inconclusive results. Semiquantitative PCR analysis displayed a pattern compatible with a heterozygous exon 60 mutation in the mother's DNA, while foetal DNA showed a normal migration pattern. Sequencing analysis confirmed the presence of a novel 7 base-pair deletion in exon 60 of the DMD gene in the mother and excluded the deletion in the foetus.

Conclusion: Semiquantitative PCR results allowed the DMD mutation detection in the mother and the exclusion in the foetus, showing its crucial importance in prenatal diagnosis in those cases where linkage analysis is not conclusive.
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http://dx.doi.org/10.1002/pd.1238DOI Listing
November 2005

Foxl2 is required for commitment to ovary differentiation.

Hum Mol Genet 2005 Jul 8;14(14):2053-62. Epub 2005 Jun 8.

Laboratory of Genetics, National Institute on Aging, Baltimore, MD 21224, USA.

Genetic control of female sex differentiation from a bipotential gonad in mammals is poorly understood. We find that mouse XX gonads lacking the forkhead transcription factor Foxl2 form meiotic prophase oocytes, but then activate the genetic program for somatic testis determination. Pivotal Foxl2 action thus represses the male gene pathway at several stages of female gonadal differentiation. This suggests the possible continued involvement of sex-determining genes in maintaining ovarian function throughout female reproductive life.
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http://dx.doi.org/10.1093/hmg/ddi210DOI Listing
July 2005

Gonadoblastoma in Turner syndrome and Y-chromosome-derived material.

Am J Med Genet A 2005 Jun;135(2):150-4

Department of Pediatrics, Pediatric Clinic, S.Orsola-Malpighi Hospital, University of Bologna, Bologna, Italy.

The identification of Y-chromosome material is important in females with Ullrich-Turner syndrome (UTS) due to the risk of developing gonadoblastoma or other gonadal tumors. There is controversy regarding the frequency of the Y-chromosome-derived material and the occurrence of gonadoblastoma in these patients. The aim of our study was to evaluate a large number of patients with UTS, followed before and during the pubertal age for the prevalence of Y-chromosome derived material, the occurrence of gonadoblastoma, and the incidence of possible neoplastic degeneration. An unselected series of 171 patients with UTS (1-34 years old), diagnosed cytogenetically, was studied for Y-chromosome markers (SRY and Y-centromeric DYZ3 repeats). The follow-up was of 2-22 years; 101 of these patients were followed during pubertal age. Y-chromosome material was found in 14 patients (8%): 12 of these were gonadectomized (2.8-25.9 years). A gonadoblastoma was detected in four patients under 16 years of age: in two, Y-material was detected only at molecular analysis (at conventional cytogenetic analysis, one was included in the 45,X group and one in the X + mar group) and one had also an immature teratoma and an endodermal sinus carcinoma. The prevalence of gonadoblastoma in our series of gonadectomized UTS patients with Y-positive material was of 33.3% (4/12). Our data suggest that the age of appearance and the possibility of malignant degeneration of gonadoblastoma can occur early in life. These patients, in particular those with 45,X or a marker chromosome may benefit from molecular screening to detect the presence of Y-chromosome material; PCR is a rapid and inexpensive technique. At the moment, laparoscopy and preventive gonadectomy performed as soon as possible remain the procedures of choice for patients with UTS, when Y-chromosome has been identified, as we are still unable to predict a future malignant evolution of gonadoblastoma.
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http://dx.doi.org/10.1002/ajmg.a.30569DOI Listing
June 2005

Mouse ovary developmental RNA and protein markers from gene expression profiling.

Dev Biol 2005 Mar;279(2):271-90

Laboratory of Genetics, Gerentalogy Research Centre, National Institute on Aging, Suite 3000, 333 Cassell Drive, Baltimore, MD 21224, USA.

To identify genes involved in morphogenetic events during mouse ovary development, we started with microarray analyses of whole organ RNA. Transcripts for 60% of the 15,000 gene NIA panel were detected, and about 2000 were differentially expressed in nascent newborn compared to adult ovary. Highly differentially expressed transcripts included noncoding RNAs and newly detected genes involved in transcription regulation and signal transduction. The phased pattern of newborn mouse ovary differentiation allowed us to (1) extend information on activity and stage specificity of cell type-specific genes; and (2) generate a list of candidate genes involved in primordial follicle formation, including podocalyxin (Podxl), PDGFR-beta, and a follistatin-domain-encoding gene Flst1. Oocyte-specific transcripts included many (e.g., Deltex2, Bicd2, and Zfp37) enriched in growing oocytes, as well as a novel family of untranslated RNA's (RLTR10) that is selectively expressed in early stage follicles. The results indicate that global expression profiling of whole organ RNA provides sensitive first-line information about ovarian histogenesis for which no in vitro cell models are currently available.
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http://dx.doi.org/10.1016/j.ydbio.2004.11.029DOI Listing
March 2005

Aging of oocyte, ovary, and human reproduction.

Ann N Y Acad Sci 2004 Dec;1034:117-31

NIA/NIH IRP, Laboratory of Genetics, Suite 3000, Triad Technology Center, 333 Cassell Drive, Baltimore MD 21224, USA.

We review age-related changes in the ovary and their effect on female fertility, with particular emphasis on follicle formation, follicle dynamics, and oocyte quality. The evidence indicates that the developmental processes leading to follicle formation set the rules determining follicle quiescence and growth. This regulatory system is maintained until menopause and is directly affected in at least some models of premature ovarian failure (POF), most strikingly in the Foxl2 mouse knockout, a model of human POF with monogenic etiology (blepharophimosis/ptosis/epicanthus inversus syndrome). Several lines of evidence indicate that if the ovarian germ cell lineage maintains regenerative potential, as recently suggested in the mouse, a role in follicle dynamics for germ stem cells, if any, is likely indirect or secondary. In addition, age-related variations in oocyte quality in animal models suggest that reproductive competence is acquired progressively and might depend on parallel growth and differentiation of follicle cells and stroma. Genomewide analyses of the mouse oocyte transcriptome have begun to be used to systematically investigate the mechanisms of reproductive competence that are altered with aging. Investigative and therapeutic strategies can benefit from considering the role of continuous interactions between follicle cells and oocytes from the beginning of histogenesis to full maturation.
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http://dx.doi.org/10.1196/annals.1335.015DOI Listing
December 2004

The use of non-physiological conditions to isolate fetal cells from maternal blood.

Exp Cell Res 2005 Jan;302(2):153-61

Department of Medicine Policlinico San Matteo and University of Pavia, Italy.

Fetal cells are always present in maternal blood starting in the first trimester of pregnancy, however a rapid, simple, and consistent procedure for their isolation for prenatal non-invasive genetic investigation is still lacking. Sensitivity and recovery of fetal cells is jeopardized by the minute amount of circulating fetal cells and their loss during the enrichment procedure. We report here a single-step approach to isolate fetal cells from maternal blood which relies on the use of non-physiological conditions to modify cell densities before their separation in a density gradient and in a newly developed cell separation device. Isolated fetal cells have been investigated using cytochemistry, Soret band absorption microscopy, monoclonal antibodies for epsilon- and gamma-chain-Hb, monoclonal antibody for i-antigen, and by fluorescence in situ hybridization (FISH). Fetal cells were always detected in all 105 maternal blood samples investigated and fetal aneuploidies were correctly diagnosed by FISH, in a pilot study of pathological pregnancies, in fetal cells isolated from maternal blood obtained either before or after invasive procedure.
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http://dx.doi.org/10.1016/j.yexcr.2004.08.039DOI Listing
January 2005

Growth patterns of human ovarian volume during intrauterine and postnatal organogenesis.

Early Hum Dev 2004 Oct;80(1):7-17

Functional Anatomy Research Center (FARC), Dipartimento di Morfologia Umana, Facoltà di Medicina e Chirurgia and Facoltà di Scienze Motorie, Università degli Studi di Milano, via Mangiagalli 31, I-20133 Milan, Italy.

Background: Ovarian volumes during pre-pubertal, reproductive or menopausal life in a healthy female have been studied by in vivo and anatomical methods. In contrast, conclusive studies on normal human ovarian volume during intrauterine and postnatal organogenesis, when the folliculogenesis occurs, do not exist.

Aim: To assess the growth patterns of ovarian volume during human ovarian definitive histogenesis using morphometric methods.

Subjects: Twenty-five left ovaries removed from fetuses, newborns and children with karyotype 46,XX, ranging from 15 to 74 weeks of development.

Outcome Measures: The ovaries were completely cut obtaining serial sections. Ovarian volume was calculated both with the ellipsoid method, using its length, maximum and minimum diameters, and the Cavalieri quantitative morphometric method. The age-related pattern of ovarian volume was studied using linear regression analyses.

Results: Ovarian volume calculated with the Cavalieri method increased from 16 mm3 at the 15th week of development to over 300 mm3 at the end of the process, with a 20-fold increase compared to the initial volume. Ovarian volume calculated with the ellipsoid method ranged between 25 to over 380 mm3, with a 15-fold increase. On average, ellipsoidal volume over-estimated the true Cavalieri volume (mean difference -14%, S.D. 29, p=0.014, Student's t-test for paired samples). Linear regression curves were developed to predict ovarian volume at any age. Overall, the approximation was better for Cavalieri volume (approximately, 72% of variance explained by the linear regression) than for ellipsoid volume (59% of explained variance).

Conclusions: During the intrauterine and postnatal organogenesis, the normal growth rate of human ovary was almost linear. The calculation of ovarian volume using Cavalieri method does not depend on assumptions about the organ shape and it appears to be more appropriate.
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http://dx.doi.org/10.1016/j.earlhumdev.2004.05.005DOI Listing
October 2004

First-trimester prenatal screening for the common 35delG GJB2 mutation causing prelingual deafness.

Prenat Diagn 2004 Aug;24(8):631-4

Laboratorio di Genetica Medica, Istituti Clinici di Perfezionamento, Milan, Italy.

Objective: This study evaluates the prevalence of 35delG GJB2 mutation, the most common genetic mutation causing prelingual deafness, and its screening feasibility and acceptability in pregnant women undergoing first-trimester CVS for chromosomal abnormality investigation.

Methods: Samples were taken from 5786 pregnant women who requested CVS for chromosomal analysis. The samples were split into two aliquots for chromosome and DNA analysis, respectively. The results of foetal karyotyping were provided 7 days after sampling, at which time the fully informed couple decided whether or not to undergo DNA testing.

Results: Of the 5449 eligible candidates, 2997 (55.0%) chose to undergo 35delG testing. Among them, 67 proved to be carriers of the mutation, resulting in a prevalence rate of 1:44.7 (2.23%). There were no homozygous foetuses, but two double heterozygous foetuses were found, and in one case the couple chose to terminate the pregnancy.

Conclusions: The results confirm the high frequency of 35delG mutation in the Italian population. The study shows that prenatal screening for GJB2 mutations in pregnant women with no retrospective risk for deafness appears to be feasible and highly acceptable. Consequently, given evidence that early evaluation and treatment significantly improve speech and language skills, as well as social and emotional well-being in affected children, 35delG mutation analysis in pregnant women booking CVS primarily for chromosomal investigation could be considered a useful addition to more comprehensive population screening strategies.
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http://dx.doi.org/10.1002/pd.954DOI Listing
August 2004

Foxl2 disruption causes mouse ovarian failure by pervasive blockage of follicle development.

Hum Mol Genet 2004 Jun 31;13(11):1171-81. Epub 2004 Mar 31.

Laboratory of Genetics, National Institute on Aging, Baltimore, MD 21224, USA.

FOXL2 mutations cause gonadal dysgenesis or premature ovarian failure (POF) in women, as well as eyelid/forehead dysmorphology in both sexes (the 'blepharophimosis-ptosis-epicanthus inversus syndrome', BPES). Here we report that mice lacking Foxl2 recapitulate relevant features of human BPES: males and females are small and show distinctive craniofacial morphology with upper eyelids absent. Furthermore, in mice as in humans, sterility is confined to females. Features of Foxl2 null animals point toward a new mechanism of POF, with all major somatic cell lineages failing to develop around growing oocytes from the time of primordial follicle formation. Foxl2 disruption thus provides a model for histogenesis and reproductive competence of the ovary.
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http://dx.doi.org/10.1093/hmg/ddh124DOI Listing
June 2004

Position of follicles in normal human ovary during definitive histogenesis.

Early Hum Dev 2003 Oct;74(1):27-35

Functional Anatomy Research Center, Dipartimento di Anatomia Umana, Facoltà di Medicina e Chirurgia and Facoltà di Scienze Motorie, Università degli Studi di Milano, via Mangiagalli 31, Milan 20133, Italy.

Background: Follicular growth in the mammalian ovary follows a geographically determined pattern, but no exhaustive data about their spatial localization in the cortex of human ovary exists.

Aim: The aim of this study is to assess the spatial position of the follicles during human ovarian definitive histogenesis using morphometric methods.

Subjects: Ovaries removed from four fetuses, five newborns and one 8-month-old child with 46,XX karyotype was used.

Outcome Measures: The position of the different follicle categories (primordial, primary, secondary and antral) was estimated as percentage distance of the centroid of the follicle from the cortico-medullary boundary.

Results: In normal ovaries, during definitive histogenesis, the primordial follicles progressively occupy more peripheral parts of the cortex (on average, 41% of cortical length at 20 weeks of fetal development, 53% at birth, 68% at 8 postnatal months). Primary follicles were crowded in the inner part of the cortex (newborn: 30-40% of cortical length; 8 months: 52% of cortical length). Secondary and antral follicles were sampled in all neonatal specimens and in the 8-month old specimen; they were grouped next to the cortico-medullary boundary.

Conclusions: Ovarian development corresponded to a migration of the maximum crowding of follicles from a position next to the medulla towards a more peripheral location. The control of the primordial follicle assembly, recruitment and development are coordinated by locally produced paracrine factors. The action of these factors seems to follow a negative gradient from the cortex toward the medulla.
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http://dx.doi.org/10.1016/s0378-3782(03)00081-1DOI Listing
October 2003