anna pia ferraretti - Academia.edu (original) (raw)

Papers by anna pia ferraretti

Research paper thumbnail of DEBATE The Bologna

criteria for the definition of poor ovarian responders: is there a need for revision?

Research paper thumbnail of Luca Gianaroli1'3, M.Cristina Magli1

Reducing the time of sperm-oocyte interaction in human in-vitro fertilization improves the implan... more Reducing the time of sperm-oocyte interaction in human in-vitro fertilization improves the implantation rate

Research paper thumbnail of Sperm chromosome abnormalities in patients with normal karyotype and in translocation carriers: clinical relevance for assisted reproductive technology

Reproductive BioMedicine Online, 2020

Protein kinase C signals thromboxane induced increases in fibronectin synthesis and TGF.p bioacti... more Protein kinase C signals thromboxane induced increases in fibronectin synthesis and TGF.p bioactivity in mesangial cells. Previous studies have demonstrated that thromboxane (TX) stimulates matrix protein synthesis in mesangial cells (MC), and that this action is signalled by receptor mediated activation of protein kinase C (PKC). In the present study, we examined the hypothesis that activation of PKC by TX signals increases in transforming growth factor /3 (TGF-/3) bioactivity, which in turn induces enhanced matrix protein synthesis. In cultured rat MC, the TXAJ prostaglandin endoperoxide analogue U-46619, but not exogenous human platelet TGF-/31, activated PKC as reflected by enhanced in situ phosphorylation of MARCKS protein, an endogenous substrate of PKC. U-46619 and TGF-/31 stimulated fibronectin (Fn) synthesis in MC, as shown by [35S]methionine incorporation into immunoprecipitable Fn. Pan-specific rabbit anti-TGF-f3 antibody blocked the increases in Fn synthesis induced by exogenous TGF-/3 and those induced by U-46619 at 24 to 72 hours after addition. Anti-TGF-/3 antibody did not block the small increases in Fn synthesis observed six hours after addition of U-46619, suggesting that this acute response was not dependent on TGF-/3. Anti-TGF-13 antibody also failed to block activation of PKC by U-46619. U-46619 and 50 n of the PKC agonist phorbol dibutyrate (PDBu) significantly increased both the active fraction and total (latent plus active) TGF-/3 in MC culture media, as assayed with the mink lung epithelial cell bioassay system. PKC inhibition with bisindolylmaleimide GF 109203X (GFX) or down-regulation of PKC in MC by prior exposure to a high concentration (0.5 j.tM) of phorbol myristate acetate (PMA) blocked increases in TGF-/3 bioactivity induced by either U-46619 or PDBu. PKC down-regulation in MC also blocked increases in Fn synthesis induced by U-46619. By contrast, exogenous TGF-/3 stimulated Fn synthesis in both intact MC and in MC with down-regulated PKC. The findings indicate that activation of PKC by U-46619 signals an increase in TGF-j3 bioactivity, which in turn stimulates Fn synthesis in MC by processes not dependent on PKC. examined the possibility that TX activation of PKC is a signal to increase TGF-/3 bioactivity in MC, which in turn mediates the sustained increases in Fn synthesis observed in response to TX. Methods Rat MC culture

Research paper thumbnail of Cinnoxicam andl-Carnitine/Acetyl-l-Carnitine Treatment for Idiopathic and Varicocele-Associated Oligoasthenospermia

Journal of Andrology, 2004

The objective of this study was to detect a therapy for idiopathic and varicocele-associated olig... more The objective of this study was to detect a therapy for idiopathic and varicocele-associated oligoasthenospermia (OAT). Idiopathic and varicocele OAT patients were randomized into 3 groups. Each group was composed of varying degrees of left varicoceles (graded into 5 grades with echo-color Doppler) and of idiopathic OATs. Group 1 used a placebo, group 2 used oral L-carnitine (2 g/d) + acetyl-L-carnitine (1 g/d), group 3 used L-carnitine/acetyl-L-carnitine + 1 x 30-mg cinnoxicam suppository every 4 days. Drugs were administered for 6 months. The groups were composed as follows: group 1, 71 varicoceles and 47 idiopathic OATs; group 2, 62 varicoceles and 39 idiopathic OATs; group 3, 62 varicoceles and 44 idiopathic OATs. Sperm concentration, motility, and morphology before during and after treatments were assessed. Pregnancy rates and side effects were recorded. Group 1 did not have modified sperm patterns during treatment. Group 2 had significantly increased sperm patterns at 3 and 6 months into therapy in idiopathic patients and in patients with grades I, II, and III varicocele, but not in grades IV and V. Group 3 had significantly increased sperm parameters in all patients, with the exception of grade V varicocele. Group 3 sperm patterns proved significantly higher during therapy than group 2. All sperm patterns fell to baseline after therapy suspension. Minor side effects occurred. Pregnancy rates were 1.7% (group 1), 21.8% (group 2), and 38.0% (group 3) (P <.01). L-carnitine/acetyl-L-carnitine + cinnoxicam suppositories proved a reliable treatment for low-grade varicoceles and idiopathic OATs.

Research paper thumbnail of The Bologna criteria for the definition of poor ovarian responders: is there a need for revision?

Human reproduction (Oxford, England), 2014

The Bologna criteria were published by the European Society of Human Reproduction and Embryology ... more The Bologna criteria were published by the European Society of Human Reproduction and Embryology (ESHRE) in 2011 to help address the lack of a clear definition of poor ovarian responders. Since its publication, aspects of the criteria have been cited several times, whilst others have been criticized. In this debate, we re-examine the criteria (which address age, the number of oocytes retrieved and the results of ovarian reserve tests) following new evidence produced and we analyse the criticism received in an attempt to determine if revisions are needed.

Research paper thumbnail of Mitochondrial DNA analysis and numerical chromosome condition in human oocytes and polar bodies

Molecular human reproduction, 2015

To investigate the mitochondrial DNA (mtDNA) segregation in human oocytes, the level of heteropla... more To investigate the mitochondrial DNA (mtDNA) segregation in human oocytes, the level of heteroplasmy in the three products of meioses, polar bodies (PBs) and corresponding oocytes, was assessed by studying the hypervariable region I (HVRI) of the D-loop region. The DNA from 122 PBs and 51 oocytes from 16 patients was amplified by whole genome amplification (WGA). An aliquot of the WGA product was used to assess aneuploidy, and another aliquot to study mtDNA. The HVRI was amplified and sequenced with an efficiency of 75.4 and 63%, respectively, in PBs, and of 100% in oocytes. The comparison with the mtDNA sequences from blood of the individual donors showed full correspondence of polymorphisms with the matching oocytes, whilst in PBs the degree of concordance dropped to 89.6%. Haplogroups were inferred for all 16 patients. Of the 89 diagnosed PBs from the 13 patients belonging to macrohaplogroup R, 23 were euploid and 66 aneuploid. The incidence of total anomalies was significantly l...

Research paper thumbnail of Effect of inner myometrium fibroid on reproductive outcome after IVF

Reproductive BioMedicine Online, 2005

To evaluate the influence of inner myometrium fibroids (myomas) on the outcome of IVF cycles, a r... more To evaluate the influence of inner myometrium fibroids (myomas) on the outcome of IVF cycles, a retrospective agematched controlled study was performed at SISMeR Reproductive Medicine Unit. The study group included 129 IVF/intracytoplasmic sperm injection cycles in 75 patients with one or more intramural and/or submucosal fibroids, while the control group consisted of 129 cycles in 127 patients without fibroids. The two groups were similar for mean oestradiol concentration at human chorionic gonadotrophin administration (1205.16 ± 874 versus 1395 ± 821 pg/ml), mean number of transferred embryos (2.02 ± 0.4 versus 2.14 ± 0.6) and clinical pregnancy rate (34.9 versus 41.1%). Conversely, the implantation rate was significantly lower in the study group (18.0%) than in the control group (26.5%; χ 2 = 4.81, P < 0.05), whereas the rate of spontaneous abortion demonstrated an opposite trend (40 versus 18.9%; χ 2 = 4.34, P < 0.05). Further research should be aimed at classifying fibroids on the basis of their location, especially when they are positioned in the junctional zone of the myometrium. Whether this classification will be superior in predicting the impact of fibroids on the reproductive outcome should be elaborated in a large multicentric study.

Research paper thumbnail of L- and T-Type Voltage-Gated Ca2+Channels in Human Granulosa Cells: Functional Characterization and Cholinergic Regulation

The Journal of Clinical Endocrinology & Metabolism, 2005

Using the whole-cell configuration of the patch-clamp technique, we have characterized two types ... more Using the whole-cell configuration of the patch-clamp technique, we have characterized two types of ionic currents through voltage-dependent Ca 2؉ channels in human granulosa cells. One is long-lasting, activates at approximately ؊20 mV, reaches the peak at approximately ؉20 mV, has an inactivation time constant of 132.5 ؎ 5.6 msec at 20 mV, and is sensitive to dihydropyridines. The other is transient, activates at approximately ؊40 mV, peaks at approximately ؊10 mV, has an inactivation time constant of 38.8 ؎ 1.8 msec at ؊10 mV, displays a voltage-dependent inactivation, and is sensitive to 100 M Ni 2؉ , but not to dihydropyridines. Biophysical and pharmacological properties of these currents indicate that they are gated through Land T-type calcium channels, respectively. The cholinergic receptor agonist carbachol (50 M) reduces the amplitude of the currents through both L-type (؊34.7 ؎ 6.4%; n ‫؍‬ 10) and T-type (؊52.6 ؎ 7.4%; n ‫؍‬ 8) channels, suggesting a possible role of these channels in the cholinergic regulation of human ovarian functions.

Research paper thumbnail of Positive outcome after preimplantation diagnosis of aneuploidy in human embryos *

Human Reproduction, 1999

Chromosomal abnormalities are responsible for a great deal of embryo wastage, which is reflected,... more Chromosomal abnormalities are responsible for a great deal of embryo wastage, which is reflected, at least partially, in decreased implantation and increased miscarriage in older women. To address this problem the transfer of only chromosomally normal embryos previously selected by preimplantation genetic diagnosis (PGD) has been proposed. We designed a multi-centre in-vitro fertilization (IVF) study to compare controls and a test group that underwent embryo biopsy and PGD for aneuploidy. Patients were matched retrospectively, but blindly, for average maternal age, number of previous IVF cycles, duration of stimulation, oestradiol concentrations on day ⍣1, and average mature follicles. All these parameters were similar in test and control groups. Only embryos classified as normal for those chromosomes were transferred after PGD. The results showed that the rates of fetal heart beat (FHB)/embryo transferred between the control and test groups were similar. However, spontaneous abortions, measured as FHB aborted/FHB detected, decreased after PGD (P < 0.05), and ongoing pregnancies and delivered babies increased (P < 0.05) in the PGD group of patients. Two conclusions were obtained: (i) PGD of aneuploidy reduced embryo loss after implantation; (ii) implantation rates were not significantly improved, but the proportion of ongoing and delivered babies was increased.

Research paper thumbnail of Elective cryopreservation of all pronucleate embryos in women at risk of ovarian hyperstimulation syndrome: efficiency and safety

Human Reproduction, 1999

In a prospective randomized study, we analysed 125 patients at risk of ovarian hyperstimulation s... more In a prospective randomized study, we analysed 125 patients at risk of ovarian hyperstimulation syndrome (OHSS), selected in the period between January 1996 and July 1997. All the patients had blood oestradiol concentration ജ1500 pg/ml on the day of human chorionic gonadotrophin (HCG) administration and ജ15 oocytes were collected. The patients were matched in two groups: group A, control group (n ⍧ 67), had fresh embryo transfers; group B (n ⍧ 58) had cryopreservation of all obtained pronucleate embryos. Pregnancy, live birth rates and the incidence of OHSS were compared between the two groups. There were no significant differences in terms of pregnancies per patient (46.3 versus 48.3%) and live birth rates (38.8 versus 39.6%). No cases of OHSS occurred in group B, while four patients developed the syndrome in group A. The implantation rate was slightly but not significantly lower in group B (χ 2 ⍧ 1.03). These results suggest that elective cryopreservation of all zygotes might prevent the risk of OHSS in patients undergoing IVF treatment. In contrast to what has been reported by other authors, our results show that the elective cryopreservation of zygotes does not affect pregnancy and live birth rates.

Research paper thumbnail of Rescue of implantation potential in embryos with poor prognosis by assisted zona hatching

Human Reproduction, 1998

The effect of the assisted zona hatching (AZH) procedure was investigated on 135 cycles with a po... more The effect of the assisted zona hatching (AZH) procedure was investigated on 135 cycles with a poor prognosis of pregnancy due to: (i) maternal age ജ 38 years (45 cycles); (ii) three or more failed in-vitro fertilization (IVF) attempts (70 cycles), and (iii) patients possessing both inclusion criteria (20 cycles). The control groups (113 cycles) included patients possessing the same characteristics (42, 53 and 18 cycles respectively) and who did not undergo the AZH procedure. A total of 505 embryos was treated with AZH before transfer, resulting in: 14, 25 and 6 clinical pregnancies. The percentage of clinical pregnancies per cycle was significantly higher than controls for the first (31 vs 10% in control 1, P Ͻ 0.05) and second groups (36 vs 17% in control 2, P Ͻ 0.05). No significant difference in percentage of clinical pregnancies was found for the third group (30 vs 6%). Similarly, higher rates of implantation were obtained (11.5, 15 and 11%) compared to the respective controls (4%, P Ͻ 0.02; 6.3%, P Ͻ 0.01; and 1.5%). The rate of miscarriage in the AZH groups was similar to that obtained in the controls (22 vs 21%). Finally, the morphological analysis of the embryos transferred revealed that the poor prognosis condition is associated to a significantly slower rate of development and a higher rate of fragmentation. The present results indicate that AZH procedure improves pregnancy and implantation rates in patients with a poor prognosis of pregnancy by facilitating the hatching process in embryos which would otherwise be trapped inside the zona pellucida.

Research paper thumbnail of Frequency of aneuploidy in sperm from patients with extremely severe male factor infertility

Human Reproduction, 2005

BACKGROUND: A protocol for the chromosomal analysis of sperm samples with a severely reduced numb... more BACKGROUND: A protocol for the chromosomal analysis of sperm samples with a severely reduced number of sperm cells was designed. METHODS: A severe male factor condition was the main cause of infertility for 38 couples: 27 were oligoasthenoteratospermic (OAT) and 11 with non-obstructive azoospermia underwent testicular sperm extraction (TESE). A two-round fluorescence in situ hybridization (FISH) protocol was performed with probes specific for the chromosomes X, Y, 13, 15, 16, 17, 18, 21 and 22. The recording of the position of each sperm cell at the microscope allowed diagnosis of each spermatozoon for the nine tested chromosomes. RESULTS: A mean number of 122 6 78.5 sperm were diagnosed per patient with an incidence of total abnormalities corresponding to 13.4%. x 2-tests for the observed frequencies and goodness-of-fit test were highly significant in all cases. A significantly higher proportion of total aneuploidy was detected in 79% of the tested samples compared to the normal population. Testicular sperm were significantly more prone to aneuploidy than ejaculated sperm. CONCLUSIONS: The designed FISH protocol for the analysis of severe OAT and TESE sperm samples is reliable, implying that the studied sample is representative of the original population. In view of the high incidence of aneuploidy in most severe OAT and TESE sperm, the FISH analysis of pathological sperm samples can be routinely performed in order to estimate the chances of the paternal contribution to aneuploidy in the resulting embryos.

Research paper thumbnail of Reply of the Authors

Fertility and Sterility, 2005

Research paper thumbnail of Exogenous luteinizing hormone in controlled ovarian hyperstimulation for assisted reproduction techniques

Fertility and Sterility, 2004

To investigate the role of exogenous LH in controlled ovarian hyperstimulation for assisted repro... more To investigate the role of exogenous LH in controlled ovarian hyperstimulation for assisted reproductive technologies. Design: Prospective randomized study. Setting: SISMER fertility unit. Patient(s): Women showing a hyporesponsiveness to FSH under GnRH agoinst down-regulation were randomized into three groups: group A (n ϭ 54) received an increased dosage of FSH; group B (n ϭ 54) was administered recombinant LH in addition to the increased dose of FSH; group C (n ϭ 22) was given additional FSH and LH using hMG as a combined drug. Fifty-four age-matched women with no need to increase the FSH dose were included as a control group (D). Intervention(s): None. Main Outcome Measure(s): Implantation and live birth rate per started cycles. Result(s): In group B, the pregnancy and implantation rates were statistically higher when compared with groups A and C and did not differ from the control group for normal response. The live birth rate was similar in groups B and D but was half as high in groups A and C. Conclusion(s): Hyporesponsiveness to FSH could be related to iatrogenic LH deficiency that, in turn, could affect oocyte competence. Addition of a small amount of recombinant LH is able to rescue oocyte competence to produce viable embryos.

Research paper thumbnail of Embryo morphology and development are dependent on the chromosomal complement

Fertility and Sterility, 2007

A total of 662 patients with a poor prognosis for pregnancy underwent 916 cycles of preimplantati... more A total of 662 patients with a poor prognosis for pregnancy underwent 916 cycles of preimplantation genetic diagnosis for aneuploidy. Intervention(s): The chromosomes XY, 13, 15, 16, 18, 21, and 22 were analyzed in blastomeres biopsied from day 3 embryos. Main Outcome Measure(s): Embryo morphology, chromosomal status of the analyzed blastomeres, and spreading and reanalysis of nontransferred embryos. Result(s): The incidence of chromosomal abnormalities was significantly higher in arrested or slow-cleaving embryos, and in embryos cleaving too fast, compared to embryos with eight cells at 62 hours after insemination. The presence of an uneven number of blastomeres or fragments scattered in the perivitelline space was associated with an increased incidence of chromosomal abnormalities. Conclusion(s): A correlation between embryo development and chromosomal complement makes the incidence of chromosomal abnormalities significantly higher in embryos dividing according to a time frame and a symmetry plan which are different from what are expected. The type of fragmentation is also related to chromosomal status, which explains why the extrusion of fragments might severely affect embryo viability.

Research paper thumbnail of A Study to Sustain the Hypothesis of the Multiple Genesis of Oligoasthenoteratospermia in Human Idiopathic Infertile Males

Biology of Reproduction, 2008

Interdependence between sperm concentration, motility, morphology, and the percentage of aneuploi... more Interdependence between sperm concentration, motility, morphology, and the percentage of aneuploid sperm was explored to test whether oligoasthenoteratospermia (OAT) may have a multiple origin in idiopathic infertile males. A total of 174 men (age, 35.8 6 4.3 yr) with idiopathic infertility were studied. Seven patients had nonobstructive azoospermia, 55 had severe OAT, 30 had OAT, 27 had isolated alterations of motility, 45 had alterations of morphology and of motility, and 10 had isolated alterations of morphology. The sperm morphology was assessed with strict criteria. The percentage of aneuploid sperm was assessed with fluorescent in situ hybridization for chromosomes X, Y, 13, 15, 16, 17, 18, 21, and 22. Relationships between sperm features, and the relationship between sperm features and aneuploidies were analyzed with multivariate regression analysis. Statistical analysis did not find any significant relationship between the percentage of typical forms and sperm concentration or between morphology and motility. On the other hand, a positive and significant relationship was found between sperm concentration and motility. The percentage of aneuploid sperm was inversely and significantly related to the percentage of typical forms but not to motility and concentration. Sperm morphology is an independent characteristic with respect to concentration and motility, whereas it showed a significant inverse relationship with respect to the percentage of aneuploid sperm. This means that idiopathic OAT may occur by means of at least two independent pathways, the first affecting concentration and/or motility and the second affecting morphology.

Research paper thumbnail of Trends over 15 years in ART in Europe: an analysis of 6 million cycles†

Human Reproduction Open, 2017

Study question Was the European IVF Monitoring (EIM) Consortium, established in 1999 by ESHRE, ab... more Study question Was the European IVF Monitoring (EIM) Consortium, established in 1999 by ESHRE, able to monitor the trend over time of ART in Europe? Summary answer The initial aims of the EIM programme (to collect and publish regional European data on census and trends on ART utilization, effectiveness, safety and quality) have been achieved. What is already known ART data in Europe have been collected and reported annually in Human Reproduction. Study design, size, duration A retrospective data analysis and summary of the first 15 years of ART activity in Europe (1997–2011) was carried out, using the key figures from the annual ESHRE reports and focusing on how the practice of ART has evolved over the years. Participants/materials, setting, method A total of 5 919 320 ART cycles are reported, including IVF, ICSI, frozen embryo relacment and egg donation, resulting in the birth of more than 1 million infants. A total of 1 548 967 IUIs are also reported, including husband/partner’s s...

Research paper thumbnail of Recombinant Luteinizing Hormone supplementation in poor responders undergoing IVF: a systematic review and meta-analysis

Gynecological Endocrinology, 2013

Research paper thumbnail of Signal Transduction of hCG Induces Decidualization and Uterine Receptivity

Journal of Fertility Biomarkers, 2017

All independent experimental data on epithelial and glandular cells lines of human endometrium su... more All independent experimental data on epithelial and glandular cells lines of human endometrium support the evidence for a rapid production of eicosanoids from the LH/hCG receptors when exposed to the hCG hormone. Prostaglandins rapidly act on the surrounding endometrial stromal cells throughout the adenylyl cyclase enzyme leading to very large amounts of cAMP and angiogenic factors (VEGF) production. The cAMP is the most important intracellular second messenger and along with progesterone accomplishes the full process of decidualization and acquisition of receptivity after estrogenic priming of the endometrium. The status of uterine receptivity lasts few days only and timing for successful embryo-signal transduction system activation by the endometrium is probably short. In absence of in vivo embryonic signals it is impossible to predict, on individual bases, how the intensity of all the complex interlinked molecular changes of decidualization might ever be in case of exposure to native hCG. In other terms, amount of prostaglandins and cAMP produced in response to variably glycosylated hCG are all, in vivo, not measurable variables and should be viewed as a "wave" of biochemical chain reactions. Embryonic hCG is secreted in form of multiple isomers having an unpredictable variable level of glycosylation and control of this variable remains elusive. During cycles of ovarian stimulation many drugs (FSH, LH, HCG) interact with different G-protein coupled receptors (GPCRs) making it possible to alter the prostaglandins-mediated decidualization process ready to be elicited only by hCG of pregnancy. Since the molecules (cAMP and progesterone) controlling endometrial stromal cells differentiation into decidual cells are critical for successful implantation and placenta formation, the evidence of fast eicosanoids production associated with endometrial LH/hCG receptors exposure to hCG and the potential by human endometrium to produce, in response, very large amounts of cAMP has biological and clinical relevance.

Research paper thumbnail of 20 years of the European IVF-monitoring Consortium registry: what have we learned? A comparison with registries from two other regions

Human Reproduction, 2020

STUDY QUESTION How has the performance of the European regional register of the European IVF-moni... more STUDY QUESTION How has the performance of the European regional register of the European IVF-monitoring Consortium (EIM)/European Society of Human Reproduction and Embryology (ESHRE) evolved from 1997 to 2016, as compared to the register of the Centres for Disease Control and Prevention (CDC) of the USA and the Australia and New Zealand Assisted Reproduction Database (ANZARD)? SUMMARY ANSWER It was found that coherent and analogous changes are recorded in the three regional registers over time, with a different intensity and pace, that new technologies are taken up with considerable delay and that incidental complications and adverse events are only recorded sporadically. WHAT IS KNOWN ALREADY European data on ART have been collected since 1997 by EIM. Data collection on ART in Europe is particularly difficult due to its fragmented political and legal landscape. In 1997, approximately 78.1% of all known institutions offering ART services in 23 European countries submitted data and i...

Research paper thumbnail of DEBATE The Bologna

criteria for the definition of poor ovarian responders: is there a need for revision?

Research paper thumbnail of Luca Gianaroli1'3, M.Cristina Magli1

Reducing the time of sperm-oocyte interaction in human in-vitro fertilization improves the implan... more Reducing the time of sperm-oocyte interaction in human in-vitro fertilization improves the implantation rate

Research paper thumbnail of Sperm chromosome abnormalities in patients with normal karyotype and in translocation carriers: clinical relevance for assisted reproductive technology

Reproductive BioMedicine Online, 2020

Protein kinase C signals thromboxane induced increases in fibronectin synthesis and TGF.p bioacti... more Protein kinase C signals thromboxane induced increases in fibronectin synthesis and TGF.p bioactivity in mesangial cells. Previous studies have demonstrated that thromboxane (TX) stimulates matrix protein synthesis in mesangial cells (MC), and that this action is signalled by receptor mediated activation of protein kinase C (PKC). In the present study, we examined the hypothesis that activation of PKC by TX signals increases in transforming growth factor /3 (TGF-/3) bioactivity, which in turn induces enhanced matrix protein synthesis. In cultured rat MC, the TXAJ prostaglandin endoperoxide analogue U-46619, but not exogenous human platelet TGF-/31, activated PKC as reflected by enhanced in situ phosphorylation of MARCKS protein, an endogenous substrate of PKC. U-46619 and TGF-/31 stimulated fibronectin (Fn) synthesis in MC, as shown by [35S]methionine incorporation into immunoprecipitable Fn. Pan-specific rabbit anti-TGF-f3 antibody blocked the increases in Fn synthesis induced by exogenous TGF-/3 and those induced by U-46619 at 24 to 72 hours after addition. Anti-TGF-/3 antibody did not block the small increases in Fn synthesis observed six hours after addition of U-46619, suggesting that this acute response was not dependent on TGF-/3. Anti-TGF-13 antibody also failed to block activation of PKC by U-46619. U-46619 and 50 n of the PKC agonist phorbol dibutyrate (PDBu) significantly increased both the active fraction and total (latent plus active) TGF-/3 in MC culture media, as assayed with the mink lung epithelial cell bioassay system. PKC inhibition with bisindolylmaleimide GF 109203X (GFX) or down-regulation of PKC in MC by prior exposure to a high concentration (0.5 j.tM) of phorbol myristate acetate (PMA) blocked increases in TGF-/3 bioactivity induced by either U-46619 or PDBu. PKC down-regulation in MC also blocked increases in Fn synthesis induced by U-46619. By contrast, exogenous TGF-/3 stimulated Fn synthesis in both intact MC and in MC with down-regulated PKC. The findings indicate that activation of PKC by U-46619 signals an increase in TGF-j3 bioactivity, which in turn stimulates Fn synthesis in MC by processes not dependent on PKC. examined the possibility that TX activation of PKC is a signal to increase TGF-/3 bioactivity in MC, which in turn mediates the sustained increases in Fn synthesis observed in response to TX. Methods Rat MC culture

Research paper thumbnail of Cinnoxicam andl-Carnitine/Acetyl-l-Carnitine Treatment for Idiopathic and Varicocele-Associated Oligoasthenospermia

Journal of Andrology, 2004

The objective of this study was to detect a therapy for idiopathic and varicocele-associated olig... more The objective of this study was to detect a therapy for idiopathic and varicocele-associated oligoasthenospermia (OAT). Idiopathic and varicocele OAT patients were randomized into 3 groups. Each group was composed of varying degrees of left varicoceles (graded into 5 grades with echo-color Doppler) and of idiopathic OATs. Group 1 used a placebo, group 2 used oral L-carnitine (2 g/d) + acetyl-L-carnitine (1 g/d), group 3 used L-carnitine/acetyl-L-carnitine + 1 x 30-mg cinnoxicam suppository every 4 days. Drugs were administered for 6 months. The groups were composed as follows: group 1, 71 varicoceles and 47 idiopathic OATs; group 2, 62 varicoceles and 39 idiopathic OATs; group 3, 62 varicoceles and 44 idiopathic OATs. Sperm concentration, motility, and morphology before during and after treatments were assessed. Pregnancy rates and side effects were recorded. Group 1 did not have modified sperm patterns during treatment. Group 2 had significantly increased sperm patterns at 3 and 6 months into therapy in idiopathic patients and in patients with grades I, II, and III varicocele, but not in grades IV and V. Group 3 had significantly increased sperm parameters in all patients, with the exception of grade V varicocele. Group 3 sperm patterns proved significantly higher during therapy than group 2. All sperm patterns fell to baseline after therapy suspension. Minor side effects occurred. Pregnancy rates were 1.7% (group 1), 21.8% (group 2), and 38.0% (group 3) (P &amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;lt;.01). L-carnitine/acetyl-L-carnitine + cinnoxicam suppositories proved a reliable treatment for low-grade varicoceles and idiopathic OATs.

Research paper thumbnail of The Bologna criteria for the definition of poor ovarian responders: is there a need for revision?

Human reproduction (Oxford, England), 2014

The Bologna criteria were published by the European Society of Human Reproduction and Embryology ... more The Bologna criteria were published by the European Society of Human Reproduction and Embryology (ESHRE) in 2011 to help address the lack of a clear definition of poor ovarian responders. Since its publication, aspects of the criteria have been cited several times, whilst others have been criticized. In this debate, we re-examine the criteria (which address age, the number of oocytes retrieved and the results of ovarian reserve tests) following new evidence produced and we analyse the criticism received in an attempt to determine if revisions are needed.

Research paper thumbnail of Mitochondrial DNA analysis and numerical chromosome condition in human oocytes and polar bodies

Molecular human reproduction, 2015

To investigate the mitochondrial DNA (mtDNA) segregation in human oocytes, the level of heteropla... more To investigate the mitochondrial DNA (mtDNA) segregation in human oocytes, the level of heteroplasmy in the three products of meioses, polar bodies (PBs) and corresponding oocytes, was assessed by studying the hypervariable region I (HVRI) of the D-loop region. The DNA from 122 PBs and 51 oocytes from 16 patients was amplified by whole genome amplification (WGA). An aliquot of the WGA product was used to assess aneuploidy, and another aliquot to study mtDNA. The HVRI was amplified and sequenced with an efficiency of 75.4 and 63%, respectively, in PBs, and of 100% in oocytes. The comparison with the mtDNA sequences from blood of the individual donors showed full correspondence of polymorphisms with the matching oocytes, whilst in PBs the degree of concordance dropped to 89.6%. Haplogroups were inferred for all 16 patients. Of the 89 diagnosed PBs from the 13 patients belonging to macrohaplogroup R, 23 were euploid and 66 aneuploid. The incidence of total anomalies was significantly l...

Research paper thumbnail of Effect of inner myometrium fibroid on reproductive outcome after IVF

Reproductive BioMedicine Online, 2005

To evaluate the influence of inner myometrium fibroids (myomas) on the outcome of IVF cycles, a r... more To evaluate the influence of inner myometrium fibroids (myomas) on the outcome of IVF cycles, a retrospective agematched controlled study was performed at SISMeR Reproductive Medicine Unit. The study group included 129 IVF/intracytoplasmic sperm injection cycles in 75 patients with one or more intramural and/or submucosal fibroids, while the control group consisted of 129 cycles in 127 patients without fibroids. The two groups were similar for mean oestradiol concentration at human chorionic gonadotrophin administration (1205.16 ± 874 versus 1395 ± 821 pg/ml), mean number of transferred embryos (2.02 ± 0.4 versus 2.14 ± 0.6) and clinical pregnancy rate (34.9 versus 41.1%). Conversely, the implantation rate was significantly lower in the study group (18.0%) than in the control group (26.5%; χ 2 = 4.81, P < 0.05), whereas the rate of spontaneous abortion demonstrated an opposite trend (40 versus 18.9%; χ 2 = 4.34, P < 0.05). Further research should be aimed at classifying fibroids on the basis of their location, especially when they are positioned in the junctional zone of the myometrium. Whether this classification will be superior in predicting the impact of fibroids on the reproductive outcome should be elaborated in a large multicentric study.

Research paper thumbnail of L- and T-Type Voltage-Gated Ca2+Channels in Human Granulosa Cells: Functional Characterization and Cholinergic Regulation

The Journal of Clinical Endocrinology & Metabolism, 2005

Using the whole-cell configuration of the patch-clamp technique, we have characterized two types ... more Using the whole-cell configuration of the patch-clamp technique, we have characterized two types of ionic currents through voltage-dependent Ca 2؉ channels in human granulosa cells. One is long-lasting, activates at approximately ؊20 mV, reaches the peak at approximately ؉20 mV, has an inactivation time constant of 132.5 ؎ 5.6 msec at 20 mV, and is sensitive to dihydropyridines. The other is transient, activates at approximately ؊40 mV, peaks at approximately ؊10 mV, has an inactivation time constant of 38.8 ؎ 1.8 msec at ؊10 mV, displays a voltage-dependent inactivation, and is sensitive to 100 M Ni 2؉ , but not to dihydropyridines. Biophysical and pharmacological properties of these currents indicate that they are gated through Land T-type calcium channels, respectively. The cholinergic receptor agonist carbachol (50 M) reduces the amplitude of the currents through both L-type (؊34.7 ؎ 6.4%; n ‫؍‬ 10) and T-type (؊52.6 ؎ 7.4%; n ‫؍‬ 8) channels, suggesting a possible role of these channels in the cholinergic regulation of human ovarian functions.

Research paper thumbnail of Positive outcome after preimplantation diagnosis of aneuploidy in human embryos *

Human Reproduction, 1999

Chromosomal abnormalities are responsible for a great deal of embryo wastage, which is reflected,... more Chromosomal abnormalities are responsible for a great deal of embryo wastage, which is reflected, at least partially, in decreased implantation and increased miscarriage in older women. To address this problem the transfer of only chromosomally normal embryos previously selected by preimplantation genetic diagnosis (PGD) has been proposed. We designed a multi-centre in-vitro fertilization (IVF) study to compare controls and a test group that underwent embryo biopsy and PGD for aneuploidy. Patients were matched retrospectively, but blindly, for average maternal age, number of previous IVF cycles, duration of stimulation, oestradiol concentrations on day ⍣1, and average mature follicles. All these parameters were similar in test and control groups. Only embryos classified as normal for those chromosomes were transferred after PGD. The results showed that the rates of fetal heart beat (FHB)/embryo transferred between the control and test groups were similar. However, spontaneous abortions, measured as FHB aborted/FHB detected, decreased after PGD (P < 0.05), and ongoing pregnancies and delivered babies increased (P < 0.05) in the PGD group of patients. Two conclusions were obtained: (i) PGD of aneuploidy reduced embryo loss after implantation; (ii) implantation rates were not significantly improved, but the proportion of ongoing and delivered babies was increased.

Research paper thumbnail of Elective cryopreservation of all pronucleate embryos in women at risk of ovarian hyperstimulation syndrome: efficiency and safety

Human Reproduction, 1999

In a prospective randomized study, we analysed 125 patients at risk of ovarian hyperstimulation s... more In a prospective randomized study, we analysed 125 patients at risk of ovarian hyperstimulation syndrome (OHSS), selected in the period between January 1996 and July 1997. All the patients had blood oestradiol concentration ജ1500 pg/ml on the day of human chorionic gonadotrophin (HCG) administration and ജ15 oocytes were collected. The patients were matched in two groups: group A, control group (n ⍧ 67), had fresh embryo transfers; group B (n ⍧ 58) had cryopreservation of all obtained pronucleate embryos. Pregnancy, live birth rates and the incidence of OHSS were compared between the two groups. There were no significant differences in terms of pregnancies per patient (46.3 versus 48.3%) and live birth rates (38.8 versus 39.6%). No cases of OHSS occurred in group B, while four patients developed the syndrome in group A. The implantation rate was slightly but not significantly lower in group B (χ 2 ⍧ 1.03). These results suggest that elective cryopreservation of all zygotes might prevent the risk of OHSS in patients undergoing IVF treatment. In contrast to what has been reported by other authors, our results show that the elective cryopreservation of zygotes does not affect pregnancy and live birth rates.

Research paper thumbnail of Rescue of implantation potential in embryos with poor prognosis by assisted zona hatching

Human Reproduction, 1998

The effect of the assisted zona hatching (AZH) procedure was investigated on 135 cycles with a po... more The effect of the assisted zona hatching (AZH) procedure was investigated on 135 cycles with a poor prognosis of pregnancy due to: (i) maternal age ജ 38 years (45 cycles); (ii) three or more failed in-vitro fertilization (IVF) attempts (70 cycles), and (iii) patients possessing both inclusion criteria (20 cycles). The control groups (113 cycles) included patients possessing the same characteristics (42, 53 and 18 cycles respectively) and who did not undergo the AZH procedure. A total of 505 embryos was treated with AZH before transfer, resulting in: 14, 25 and 6 clinical pregnancies. The percentage of clinical pregnancies per cycle was significantly higher than controls for the first (31 vs 10% in control 1, P Ͻ 0.05) and second groups (36 vs 17% in control 2, P Ͻ 0.05). No significant difference in percentage of clinical pregnancies was found for the third group (30 vs 6%). Similarly, higher rates of implantation were obtained (11.5, 15 and 11%) compared to the respective controls (4%, P Ͻ 0.02; 6.3%, P Ͻ 0.01; and 1.5%). The rate of miscarriage in the AZH groups was similar to that obtained in the controls (22 vs 21%). Finally, the morphological analysis of the embryos transferred revealed that the poor prognosis condition is associated to a significantly slower rate of development and a higher rate of fragmentation. The present results indicate that AZH procedure improves pregnancy and implantation rates in patients with a poor prognosis of pregnancy by facilitating the hatching process in embryos which would otherwise be trapped inside the zona pellucida.

Research paper thumbnail of Frequency of aneuploidy in sperm from patients with extremely severe male factor infertility

Human Reproduction, 2005

BACKGROUND: A protocol for the chromosomal analysis of sperm samples with a severely reduced numb... more BACKGROUND: A protocol for the chromosomal analysis of sperm samples with a severely reduced number of sperm cells was designed. METHODS: A severe male factor condition was the main cause of infertility for 38 couples: 27 were oligoasthenoteratospermic (OAT) and 11 with non-obstructive azoospermia underwent testicular sperm extraction (TESE). A two-round fluorescence in situ hybridization (FISH) protocol was performed with probes specific for the chromosomes X, Y, 13, 15, 16, 17, 18, 21 and 22. The recording of the position of each sperm cell at the microscope allowed diagnosis of each spermatozoon for the nine tested chromosomes. RESULTS: A mean number of 122 6 78.5 sperm were diagnosed per patient with an incidence of total abnormalities corresponding to 13.4%. x 2-tests for the observed frequencies and goodness-of-fit test were highly significant in all cases. A significantly higher proportion of total aneuploidy was detected in 79% of the tested samples compared to the normal population. Testicular sperm were significantly more prone to aneuploidy than ejaculated sperm. CONCLUSIONS: The designed FISH protocol for the analysis of severe OAT and TESE sperm samples is reliable, implying that the studied sample is representative of the original population. In view of the high incidence of aneuploidy in most severe OAT and TESE sperm, the FISH analysis of pathological sperm samples can be routinely performed in order to estimate the chances of the paternal contribution to aneuploidy in the resulting embryos.

Research paper thumbnail of Reply of the Authors

Fertility and Sterility, 2005

Research paper thumbnail of Exogenous luteinizing hormone in controlled ovarian hyperstimulation for assisted reproduction techniques

Fertility and Sterility, 2004

To investigate the role of exogenous LH in controlled ovarian hyperstimulation for assisted repro... more To investigate the role of exogenous LH in controlled ovarian hyperstimulation for assisted reproductive technologies. Design: Prospective randomized study. Setting: SISMER fertility unit. Patient(s): Women showing a hyporesponsiveness to FSH under GnRH agoinst down-regulation were randomized into three groups: group A (n ϭ 54) received an increased dosage of FSH; group B (n ϭ 54) was administered recombinant LH in addition to the increased dose of FSH; group C (n ϭ 22) was given additional FSH and LH using hMG as a combined drug. Fifty-four age-matched women with no need to increase the FSH dose were included as a control group (D). Intervention(s): None. Main Outcome Measure(s): Implantation and live birth rate per started cycles. Result(s): In group B, the pregnancy and implantation rates were statistically higher when compared with groups A and C and did not differ from the control group for normal response. The live birth rate was similar in groups B and D but was half as high in groups A and C. Conclusion(s): Hyporesponsiveness to FSH could be related to iatrogenic LH deficiency that, in turn, could affect oocyte competence. Addition of a small amount of recombinant LH is able to rescue oocyte competence to produce viable embryos.

Research paper thumbnail of Embryo morphology and development are dependent on the chromosomal complement

Fertility and Sterility, 2007

A total of 662 patients with a poor prognosis for pregnancy underwent 916 cycles of preimplantati... more A total of 662 patients with a poor prognosis for pregnancy underwent 916 cycles of preimplantation genetic diagnosis for aneuploidy. Intervention(s): The chromosomes XY, 13, 15, 16, 18, 21, and 22 were analyzed in blastomeres biopsied from day 3 embryos. Main Outcome Measure(s): Embryo morphology, chromosomal status of the analyzed blastomeres, and spreading and reanalysis of nontransferred embryos. Result(s): The incidence of chromosomal abnormalities was significantly higher in arrested or slow-cleaving embryos, and in embryos cleaving too fast, compared to embryos with eight cells at 62 hours after insemination. The presence of an uneven number of blastomeres or fragments scattered in the perivitelline space was associated with an increased incidence of chromosomal abnormalities. Conclusion(s): A correlation between embryo development and chromosomal complement makes the incidence of chromosomal abnormalities significantly higher in embryos dividing according to a time frame and a symmetry plan which are different from what are expected. The type of fragmentation is also related to chromosomal status, which explains why the extrusion of fragments might severely affect embryo viability.

Research paper thumbnail of A Study to Sustain the Hypothesis of the Multiple Genesis of Oligoasthenoteratospermia in Human Idiopathic Infertile Males

Biology of Reproduction, 2008

Interdependence between sperm concentration, motility, morphology, and the percentage of aneuploi... more Interdependence between sperm concentration, motility, morphology, and the percentage of aneuploid sperm was explored to test whether oligoasthenoteratospermia (OAT) may have a multiple origin in idiopathic infertile males. A total of 174 men (age, 35.8 6 4.3 yr) with idiopathic infertility were studied. Seven patients had nonobstructive azoospermia, 55 had severe OAT, 30 had OAT, 27 had isolated alterations of motility, 45 had alterations of morphology and of motility, and 10 had isolated alterations of morphology. The sperm morphology was assessed with strict criteria. The percentage of aneuploid sperm was assessed with fluorescent in situ hybridization for chromosomes X, Y, 13, 15, 16, 17, 18, 21, and 22. Relationships between sperm features, and the relationship between sperm features and aneuploidies were analyzed with multivariate regression analysis. Statistical analysis did not find any significant relationship between the percentage of typical forms and sperm concentration or between morphology and motility. On the other hand, a positive and significant relationship was found between sperm concentration and motility. The percentage of aneuploid sperm was inversely and significantly related to the percentage of typical forms but not to motility and concentration. Sperm morphology is an independent characteristic with respect to concentration and motility, whereas it showed a significant inverse relationship with respect to the percentage of aneuploid sperm. This means that idiopathic OAT may occur by means of at least two independent pathways, the first affecting concentration and/or motility and the second affecting morphology.

Research paper thumbnail of Trends over 15 years in ART in Europe: an analysis of 6 million cycles†

Human Reproduction Open, 2017

Study question Was the European IVF Monitoring (EIM) Consortium, established in 1999 by ESHRE, ab... more Study question Was the European IVF Monitoring (EIM) Consortium, established in 1999 by ESHRE, able to monitor the trend over time of ART in Europe? Summary answer The initial aims of the EIM programme (to collect and publish regional European data on census and trends on ART utilization, effectiveness, safety and quality) have been achieved. What is already known ART data in Europe have been collected and reported annually in Human Reproduction. Study design, size, duration A retrospective data analysis and summary of the first 15 years of ART activity in Europe (1997–2011) was carried out, using the key figures from the annual ESHRE reports and focusing on how the practice of ART has evolved over the years. Participants/materials, setting, method A total of 5 919 320 ART cycles are reported, including IVF, ICSI, frozen embryo relacment and egg donation, resulting in the birth of more than 1 million infants. A total of 1 548 967 IUIs are also reported, including husband/partner’s s...

Research paper thumbnail of Recombinant Luteinizing Hormone supplementation in poor responders undergoing IVF: a systematic review and meta-analysis

Gynecological Endocrinology, 2013

Research paper thumbnail of Signal Transduction of hCG Induces Decidualization and Uterine Receptivity

Journal of Fertility Biomarkers, 2017

All independent experimental data on epithelial and glandular cells lines of human endometrium su... more All independent experimental data on epithelial and glandular cells lines of human endometrium support the evidence for a rapid production of eicosanoids from the LH/hCG receptors when exposed to the hCG hormone. Prostaglandins rapidly act on the surrounding endometrial stromal cells throughout the adenylyl cyclase enzyme leading to very large amounts of cAMP and angiogenic factors (VEGF) production. The cAMP is the most important intracellular second messenger and along with progesterone accomplishes the full process of decidualization and acquisition of receptivity after estrogenic priming of the endometrium. The status of uterine receptivity lasts few days only and timing for successful embryo-signal transduction system activation by the endometrium is probably short. In absence of in vivo embryonic signals it is impossible to predict, on individual bases, how the intensity of all the complex interlinked molecular changes of decidualization might ever be in case of exposure to native hCG. In other terms, amount of prostaglandins and cAMP produced in response to variably glycosylated hCG are all, in vivo, not measurable variables and should be viewed as a "wave" of biochemical chain reactions. Embryonic hCG is secreted in form of multiple isomers having an unpredictable variable level of glycosylation and control of this variable remains elusive. During cycles of ovarian stimulation many drugs (FSH, LH, HCG) interact with different G-protein coupled receptors (GPCRs) making it possible to alter the prostaglandins-mediated decidualization process ready to be elicited only by hCG of pregnancy. Since the molecules (cAMP and progesterone) controlling endometrial stromal cells differentiation into decidual cells are critical for successful implantation and placenta formation, the evidence of fast eicosanoids production associated with endometrial LH/hCG receptors exposure to hCG and the potential by human endometrium to produce, in response, very large amounts of cAMP has biological and clinical relevance.

Research paper thumbnail of 20 years of the European IVF-monitoring Consortium registry: what have we learned? A comparison with registries from two other regions

Human Reproduction, 2020

STUDY QUESTION How has the performance of the European regional register of the European IVF-moni... more STUDY QUESTION How has the performance of the European regional register of the European IVF-monitoring Consortium (EIM)/European Society of Human Reproduction and Embryology (ESHRE) evolved from 1997 to 2016, as compared to the register of the Centres for Disease Control and Prevention (CDC) of the USA and the Australia and New Zealand Assisted Reproduction Database (ANZARD)? SUMMARY ANSWER It was found that coherent and analogous changes are recorded in the three regional registers over time, with a different intensity and pace, that new technologies are taken up with considerable delay and that incidental complications and adverse events are only recorded sporadically. WHAT IS KNOWN ALREADY European data on ART have been collected since 1997 by EIM. Data collection on ART in Europe is particularly difficult due to its fragmented political and legal landscape. In 1997, approximately 78.1% of all known institutions offering ART services in 23 European countries submitted data and i...