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Coticchio G.,Biogenesi Reproductive Medicine Center | Dal Canto M.,Biogenesi Reproductive Medicine Center | Renzini M.M.,Biogenesi Reproductive Medicine Center | Guglielmo M.C.,Biogenesi Reproductive Medicine Center | And 4 more authors.
Human Reproduction Update | Year: 2014

Background: In a growth phase occurring during most of folliculogenesis, the oocyte produces and accumulates molecules and organelles that are fundamental for the development of the preimplantation embryo. At ovulation, growth is followed by a phase of maturation that, although confined within a short temporal window, encompasses modifications of the oocyte chromosome complement and rearrangements of cytoplasmic componentsthat are crucial for the achievement of developmental competence. Cumulus cells (CCs) are central to the process of maturation, providing the oocyte with metabolic support and regulatory cues. Methods: PubMed was used to search the MEDLINE database for peer-reviewed original articles and reviews concerning oocyte maturation in mammals. Searches were performed adopting 'oocyte' and 'maturation' as main terms, in association with other keywords expressing concepts relevant to the subject. The most relevant publications, i.e. those concerning major phenomena occurring during oocyte maturation in established experimental models and the human species, were assessed and discussed critically to offer a comprehensive description of the process of oocyte maturation. Results: By applying the above described search criteria, 6165 publications were identified, of which 543 were review articles. The number of publications increased steadily from 1974 (n = 7) to 2013 (n = 293). In 2014, from January to the time of submission of this manuscript, 140 original manuscripts and reviews were published. The studies selected for this review extend previous knowledge and shed new and astounding knowledge on oocyte maturation. It has long been known that resumption of meiosis and progression to the metaphase II stage is intrinsic to oocyte maturation, but novel findings have revealed that specific chromatin configurations are indicative of a propensity of the oocyte to resume the meiotic process and acquire developmental competence. Recently, genetic integrity has also been characterized as a factor with important implications for oocyte maturation and quality. Changes occurring in the cytoplasmic compartment are equally fundamental. Microtubules, actin filaments and chromatin not only interact to finalize chromosome segregation, but also crucially co-operate to establish cell asymmetry. This allows polar body extrusion to be accomplished with minimal loss of cytoplasm. The cytoskeleton also orchestrates the rearrangement of organelles in preparation for fertilization. For example, during maturation the distribution of the endoplasmic reticulum undergoes major modifications guided by microtubules and microfilaments to make the oocyte more competent in the generation of intracellular Ca2+ oscillations that are pivotal for triggering egg activation. Cumulus cells are inherent to the process of oocyte maturation, emitting regulatory signals via direct cell-to-cell contacts and paracrine factors. In addition to nurturing the oocyte with key metabolites, CCs regulate meiotic resumption and modulate the function of the oocyte cytoskeleton. Conclusions: Although the importance of oocyte maturation for the achievement of female meiosis has long been recognized, until recently much less was known of the significance of this process in relation to other fundamental developmental events. Studies on chromatin dynamics and integrity have extended our understanding of female meiosis. Concomitantly, cytoskeletal and organelle changes and the ancillary role of CCs have been better appreciated. This is expected to inspire novel concepts and advances in assisted reproduction technologies, such as the development of novel in vitro maturation systems and the identification of biomarkers of oocyte quality. © The Author 2015. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology.


Coticchio G.,Biogenesi Reproductive Medicine Center
Human Reproduction | Year: 2016

Oocyte in vitro maturation (IVM) involves the achievement of the process of oocyte maturation in vitro. Clinically, it was introduced several decades ago as a potentially more patient-friendly and less expensive assisted reproductive technology (ART) approach, as immature oocytes collected from mid-antral follicles can be matured in vitro without prior gonadotrophin stimulation. However, IVM oocytes are developmentally less competent compared with oocytes matured in vivo, a fact that has encouraged the use of short FSH priming and/orhCGtriggering in IVM cycles. These alternatives have generated much confusion about the definition and clinical outcome of IVM, also among ART specialists, especially because hCG triggering can support maturation in vivo even in follicles of 10-13 mm in diameter. In a recent manuscript, a team of IVM specialists propose that IVM should include any ARTapproach involving the collection of oocyte from small and intermediate sized follicles even afterhCG or GnRH triggering. It is more than predictable that other scientists and clinicians with an interest in IVM will object to such a definition, believing that semantically and operatively IVM should not be associated with pharmacological interventions aimed at promoting or achieving maturation in vivo, although partially. © The Author 2016.


Fadini R.,Biogenesi Reproductive Medicine Center | Mignini Renzini M.,Biogenesi Reproductive Medicine Center | Guarnieri T.,Biogenesi Reproductive Medicine Center | Dal Canto M.,Biogenesi Reproductive Medicine Center | And 5 more authors.
Human Reproduction | Year: 2012

STUDY QUESTIONAre the obstetric and perinatal outcomes of deliveries following in vitro maturation (IVM) cycles different from births generated from controlled ovarian stimulation (COS) cycles?SUMMARY ANSWERThe obstetric and perinatal outcomes of births from IVM cycles are comparable with those of ICSI treatments, including the incidence of major and minor abnormalities. WHAT IS KNOWN ALREADYOnly few and numerically small reports on the health of IVM children are currently available. STUDY DESIGN, SIZE AND DURATIONRetrospective cohort study involving 196 babies born from IVM cycles carried out with different priming regimens. Of these children, 79 developed from oocytes matured in vitro after 30 h of culture, while 104 originated from oocytes found mature and inseminated on the day of recovery. Thirteen babies were obtained from embryos developed from both types of oocytes. Data of these births were compared with those of 194 children born from COS ICSI cycles performed during the same period (March 2004 to December 2011). PARTICIPANTS/MATERIALS, SETTING AND METHODSIVM cycles were done in the absence of gonadotrophin administration or with FSH and/or HCG priming. All oocytes were inseminated by microinjection. ICSI and ICSI cycles were chosen as a control group to exclude possible influences of the insemination technique. Couples in which maternal age was >39 years or affected by azoospermia were excluded to rule out major parental effects. MAIN RESULTS AND THE ROLE OF CHANCEIn single births, gestational age at delivery was comparable, but birthweight was significantly higher (P=0. 009) in children from IVM cycles (3091 ± 669 versus 3269 ± 619 g). In a separate analysis of the IVM group, comparing singleton births derived with certainty from oocytes matured in vitro (n=71) or in vivo (n=74), no statistically significant differences were observed in terms of birthweight (3311 ± 637 versus 3194 ± 574 g, respectively) and gestational age (38. 9 ± 2. 4 versus 38. 4 ± 2. 1 weeks, respectively). In twin births, gestational age was lower in IVM cycles, while weight at birth was comparable (ICSI, 2432 ± 540 g; IVM, 2311 ± 577 g). In single births, major and minor abnormalities were 2 (1. 4) and 6 (4. 1) in the ICSI group and 0 (0. 0) and 8 (5. 2) in the IVM category, respectively. In twin children, major and minor abnormalities were 1 (2. 2) and 2 (4. 3) in ICSI babies and 0 (0. 0) and 2 (4. 6) in IVM cycles, respectively. LIMITATIONS AND REASONS FOR CAUTIONThe study is the largest conducted so far. Nevertheless, it is limited by its retrospective nature and the fact that most births of IVM treatments derived from oocytes found mature at recovery in cycles primed with HCG. A more comprehensive appraisal of the health status of IVM children will demand larger prospective studies. WIDER IMPLICATIONS OF THE FINDINGSThe study is consistent with previous reports suggesting a possible role of standard ovarian stimulation in determining a reduced birthweight in children born from COS cycles. STUDY FUNDING/COMPETING INTEREST(S)No external funding was sought to support this work. The authors have no conflicts of interest to declare. TRIAL REGISTRATION NUMBERNot applicable. © 2012 The Author.


Dal Canto M.,Biogenesi Reproductive Medicine Center | Coticchio G.,Biogenesi Reproductive Medicine Center | Mignini Renzini M.,Biogenesi Reproductive Medicine Center | De Ponti E.,San Gerardo Hospital | And 4 more authors.
Reproductive BioMedicine Online | Year: 2012

Cleavage kinetics of human embryos is indicative of ability to develop to blastocyst and implant. Recent advances in time-lapse microscopy have opened new and important research opportunities. In this study involving infertile couples requiring standard IVF/intracytoplasmic sperm injection treatment, zygotes were cultured by integrated embryo-culture time-lapse microscopy to analyse cleavage times from the 2- to the 8-cell stages in relation to the ability to develop to blastocyst, expand and implant. In comparison to embryos arresting after 8-cell stage, times of cleavage to 7- and 8-cell stages of embryos developing to blastocyst were shorter (56.5 ± 8.1 versus 58.8 ± 10.4 h, P = 0.03 and 61.0 ± 9.4 versus 65.2 ± 13.0 h, P = 0.0008, respectively). In embryos developing to blastocyst, absence of blastocoele expansion on day 5 was associated with progressive cleavage delay. Implanting embryos developed to 8-cell stage in a shorter period compared with those unable to implant (54.9 ± 5.2 and 58.0 ± 7.2 h, respectively, P = 0.035). In conclusion, cleavage from 2- to 8-cell stage occurs progressively earlier in embryos with the ability to develop to blastocyst, expand and implant. Conventional observation times on days 2 and 3 are inappropriate for accurate embryo evaluation. The speed at which human embryos cleave is known to be suggestive of their ability to develop in vitro to the blastocyst stage and implant after transfer into the uterus. Recent advances in time-lapse microscopy, which allows acquisition of images every 15-20 min, have opened new and important research opportunities. In a retrospective study involving infertile couples requiring standard IVF or intracytoplasmic sperm injection treatment, fertilized oocytes were cultured by an integrated embryo-culture time-lapse microscopy system in order to perform an analysis of cleavage times from the 2- to the 8-cell stage in relation to the ability to develop to blastocyst, expand and implant. In comparison to embryos arresting after the 8-cell stage, times of cleavage to the 7- and 8-cell stage of embryos that developed to blastocyst were significantly shorter (56.5 ± 8.1 h versus 58.8 ± 10.4 h and 61.0 ± 9.4 h versus 65.2 ± 13.0 h, respectively). In embryos developing to the blastocyst stage, absence of blastocoele expansion on day 5 was associated with a progressive cleavage delay. Implanting embryos developed to the 8-cell stage in a shorter period compared to those unable to implant (54.9 ± 5.2 h and 58.0 ± 7.2 h, respectively, P = 0.035). In conclusion, cleavage from the 2- to the 8-cell stage occurs progressively earlier in embryos with the ability to develop to blastocyst, expand and implant. Conventional observation times on day 2 and 3 are appropriate for accurate embryo evaluation. © 2012, Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved.


Coticchio G.,Biogenesi Reproductive Medicine Center
Human reproduction (Oxford, England) | Year: 2013

Are morphometric and morphological parameters of the metaphase II (MII) spindle of human oocytes matured in vivo or in vitro predictive of chromosome alignment on the metaphase plate? Morphometric spindle parameters were very comparable between oocytes matured in vivo and in vitro and were unable to predict chromosome alignment, while a flattened shape of both poles was positively associated with chromosome displacement from the metaphase plate. The relationship between MII spindle morphometry and chromosome alignment has only been sporadically investigated in human oocytes. The possible implications of spindle pole morphology are totally unrecognized. Morphometric and morphological analysis of the MII spindle of donated supernumerary human oocytes (N = 93) aimed at investigating possible associations between novel microtubule parameters and chromosome arrangement. MII oocytes from three sources were analysed: (i) stimulated cycles matured in vivo (ivo-MII), (ii) leftover cumulus-free germinal vesicle oocytes from stimulated cycles matured in vitro (lgv-MII) and (iii) immature cumulus-cell oocyte complexes (COCs) recovered from in vitro maturation (IVM) cycles and matured in vitro (ivm-MII). Oocytes were fixed and stained for tubulin, chromatin and actin. Optical sections were collected at 0.3 μm intervals by high-performance confocal microscopy and three-dimensionally reconstructed for assignment of specific spindle and chromosomal properties. Spindle pole morphology was classified as either focused or flattened depending on whether microtubule ends were more or less convergent, respectively. Optical density measurements were generated to estimate microtubule abundance in chromosome to pole domains proximal and distal to the oolemma. In ivo-MII oocytes, the sizes (mean ± SD) of major and minor axes were 11.8 ± 2.6 and 8.9 ± 1.7 μm, respectively, while maximum projection was 88.8 ± 29.5 μm(2). Very comparable values of these parameters were found in lgv-MII and ivm-MII oocytes. Double-focused spindles were rarely found (3.1%), unlike those with a double-flattened conformation (47.7%). Spindles with both focused and flattened poles amounted to almost half of the sample set (49.2%), but in this subgroup it was very infrequent (4.6%) to observe the flattened pole oriented towards the oolemma. Overall, differences in the relative proportions of pole morphology categories in ivo-MII, lgv-MII and ivm-MII oocytes were not statistically significant. For both the distal and proximal spindle hemidomains, optical intensity profiles were also comparable between ivo-MII, lgv-MII and ivm-MII oocytes. None of the morphometric parameters (major and minor axes, their ratio, maximum projection, distances of the metaphase plate from the poles) was associated with chromosome alignment on the metaphase plate or arrangement inside and outside the spindle. Importantly, a double-flattened outline of pole morphology was positively associated with the displacement of one or more chromosomes from the metaphase plate. Moreover, when a flattened pole was oriented towards the oolemma, a higher rate of chromosome displacement was observed. The findings of the study will require confirmation by further in-depth analysis and extension of the database, especially regarding the relationship between microtubule abundance and chromosome arrangement. Furthermore, considering the high number of comparisons, the observed statistical differences will require future 'ad hoc' analysis. Collectively, this work provides a robust database for future research on the human oocyte cytoskeleton, and contributes to a better definition of oocyte quality in assisted reproduction technology. Also, these data support the notion that IVM does not affect spindle morphometry and morphology. Part of this work was supported by a grant awarded by the Italian Ministry of Labour, Health and Social Policies. The authors have no conflicts of interest to declare. Not applicable.


Franciosi F.,University of Milan | Coticchio G.,Biogenesi Reproductive Medicine Center | Lodde V.,University of Milan | Tessaro I.,University of Milan | And 6 more authors.
Biology of Reproduction | Year: 2014

Oocyte in vitro maturation (IVM) has become a valuable technological tool for animal breeding and cloning and the treatment of human infertility because it does not require the administration of exogenous gonadotropin to obtain fertilizable oocytes. However, embryo development after IVM is lower compared to in vivo maturation, most likely because oocytes collected for IVM are heterogeneous with respect to their developmental competencies. Attempts to improve IVM outcome have relied upon either prematuration culture (PMC) or two-step maturation strategies in the hope of normalizing variations in developmental competence. Such culture systems invoke the pharmacological arrest of meiosis, in theory providing oocytes sufficient time to complete the acquisition of developmental competence after cumulus-enclosed oocytes isolation from the follicle. The present study was designed to test the efficiency of natriuretic peptide precursor C (NPPC) as a nonpharmacologic meiosis-arresting agent during IVM in a monoovulatory species. NPPC has been shown to maintain meiotic arrest in vivo and in vitro in mice and pigs; however, the use of this molecule for PMC has yet to have been explored. Toward this end, meiotic cell cycle reentry, gap-junction functionality, and chromatin configuration changes were investigated in bovine cumulus-enclosed oocytes cultured in the presence of NPPC. Moreover, oocyte developmental competence was investigated after IVM, in vitro fertilization, and embryo culture and compared to standard IVM-in vitro fertilization protocol without PMC. Our results suggest that NPPC can be used to delay meiotic resumption and increase the developmental competence of bovine oocytes when used in PMC protocols. © 2014 by the Society for the Study of Reproduction, Inc.


Brambillasca F.,Biogenesi Reproductive Medicine Center | Guglielmo M.C.,Biogenesi Reproductive Medicine Center | Coticchio G.,Biogenesi Reproductive Medicine Center | Mignini Renzini M.,Biogenesi Reproductive Medicine Center | And 2 more authors.
Journal of Assisted Reproduction and Genetics | Year: 2013

Oocyte cryopreservation represents an important tool for assisted reproductive technology. It offers the opportunity to preserve fertility in women at risk of loss of the ovarian function for various pathologies. It also represents a treatment alternative for couples that cannot benefit from embryo cryopreservation because of moral, religious, or legal constrains. On the other hand, in vitro oocyte maturation has a range of applications. It can be applied in patients with a contraindication to ovarian stimulation to prevent ovarian hyperstimulation syndrome or to eliminate the risk of stimulation of hormone-sensitive tumours in cancer patients. However, while mature oocyte cryopreservation has found wide-spread application and oocyte in vitro maturation has a place for the treatment of specific clinical conditions, data on the efficiency of freezing of immature or in vitro matured oocytes are poorer. In this review we will focus on the combination of oocyte in vitro maturation with oocyte cryopreservation with particular emphasis on the biological implications of the cryopreservation of immature or in vitro matured oocytes. The two cryopreservation approaches, slow freezing and vitrification, will be discussed in relation to possible cryodamage occurring to subcellular structures of the oocyte and the functional interaction between oocyte and cumulus cells. © 2013 Springer Science+Business Media New York.


Barberi M.,University of Rome La Sapienza | Di Paolo V.,University of Rome La Sapienza | Latini S.,University of Rome La Sapienza | Guglielmo M.C.,University of Rome La Sapienza | And 3 more authors.
Molecular and Cellular Endocrinology | Year: 2013

Pituitary adenylate cyclase-activating polypeptide (PACAP) and its receptor PAC1-R (PACAP type 1 receptor) are transiently expressed in granulosa cells (GCs) of mouse preovulatory follicles and affect several parameters associated with the ovulatory process. We investigated the expression of PACAP and its receptors in cumulus cells (CCs) after the LH surge and their role on cumulus expansion/apoptosis and oocyte maturation. PACAP and PAC1-R expression increased in CCs isolated at different times after treatment with human chorionic gonadotropin (hCG). Moreover, PACAP was able to reverse the inhibition of oocyte meiotic maturation caused by hypoxantine in cumulus cell-oocyte complexes (COCs) and efficiently promoted male pronuclear formation after fertilisation. PACAP was also able to induce cumulus expansion and prevent CC apoptosis. Our results demonstrated the induction of PACAP and its receptors in CCs by LH and EGF, suggesting that PACAP may play a significant role in the complex interactions of gonadotropin and growth factors during ovulation and fertilisation. © 2013 Elsevier Ireland Ltd.


Grondahl M.L.,Copenhagen University | Nielsen M.E.,University of Southern Denmark | Dal Canto M.B.,Biogenesi Reproductive Medicine Center | Fadini R.,Biogenesi Reproductive Medicine Center | And 4 more authors.
Reproductive BioMedicine Online | Year: 2011

This study evaluated whether anti-Müllerian hormone (AMH) was differentially expressed in cumulus (CC) and granulosa (GC) cells from large antral and pre-ovulatory follicles collected from individual follicles in women undergoing in-vitro maturation (IVM) or IVF treatment. Expression studies of AMH, AMH receptor 2, FSH receptor, aromatase and androgen receptor were performed in CC in IVM patients where cumulus-oocyte-complex had expanded, CC in IVM patients where cumulus-oocyte-complex remained compacted, GC from immature follicles and CC and GC from IVF patients. Microarray data on corresponding GC and CC from follicles from IVF patients was included. AMH expression was significantly higher in CC than in GC from both mature and immature follicles and in CC from immature follicles than in CC from pre-ovulatory follicles from IVF patients (P < 0.05). AMH expression was significantly higher in CC that remained compacted compared with those that had expanded (P < 0.008). AMH was correlated to the expression of FSH receptor, androgen receptor and AMH receptor 2 but not to aromatase expression. The expression pattern of AMH receptor 2 reflected that of AMH. AMH may exert intrafollicular functions even in human large antral and pre-ovulatory follicles and may be related to follicular health. © 2010, Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved.


Coticchio G.,Biogenesi Reproductive Medicine Center | Guglielmo M.C.,Biogenesi Reproductive Medicine Center | Albertini D.F.,University of Kansas | Dal Canto M.,Biogenesi Reproductive Medicine Center | And 3 more authors.
Molecular Human Reproduction | Year: 2014

In mature mammalian oocytes, cortical f-actin distribution is polarized, as evidenced by a prominent cap subtended by the metaphase II (MII) spindle. Formation of a polarized actin cap is a consequence of a complex actomyosin-driven contractile process that directs polar body extrusion. Human mature oocytes also display a network of suboolemmal actin, but so far there has been no suggestion of an actin-rich domain in the vicinity of the spindle. By high-resolution confocal microscopy, we generated semi-quantitative data of the actin cytoskeleton in human mature and immature oocytes, with the aim to better understand the characteristics and remodelling of this cytoskeletal component in the female gamete. In mature MII oocytes, the cortical domain near the spindle showed a more intense actin signal in comparison to the opposite cortical domain (177.2 ± 59.0 versus 126.8 ± 61.0, P < 0.0001; data expressed in arbitrary units). The extent of cortical f-actin polarity was comparable between in vivo and in vitro matured oocytes. However, both the degree of polarity and relative abundance of signal were diminished with increasing maternal age. Mean intensity of cytoplasmic actin was significantly higher in oocytes matured in vitro derived from in vitro maturation (IVM) cycle, in comparison to oocytes matured in vivo or in vitro obtained from controlled ovarian stimulation cycles (35.0 ± 8.0, 21.1 ± 12.4 and 25.9 ± 8.6, respectively; P = 0.025). In germinal vesicle (GV)-stage oocytes obtained from both IVM and controlled ovarian stimulation cycles, cortical actin did not appear polarized, irrespective of whether the GV was located centrally or asymmetrically. These data indicate that, during maturation, cortical actin acquires a polarized distribution involving an accumulation in the domain adjacent the spindle. They also propose new questions concerning the existence of cytoplasmic actin in mature oocytes. Finally, they are suggestive of an influence of maternal age on the actin cytoskeleton. © The Author 2013. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved.

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