Francesco Capodanno

FSHR polymorphism p.N680S mediates different responses to FSH in vitro

The single nucleotide polymorphism p.N680S of the follicle-stimulating hormone (FSH) receptor (FSHR) is a discrete marker of ovarian response but previous in vitro studies failed to demonstrate differences in the response to FSH between N and S carrier cells. Here we demonstrate that p.N680S mediates different kinetics of the response to FSH in vitro. Intracellular cAMP production is faster in p.N680S N than in S homozygous human granulosa cells (45 versus 90 min to achieve the plateau, respectively; Mann-Whitneys U-test; p < 0.005; n = 4). Reflecting the cAMP kinetics, phospho-ERK1/2 and -CREB activation, AREG and STARD1 gene expressions and progesterone production were qualitatively and quantitatively different in N versus S homozygous cells. Finally, the blockade of ERK pathway by U0126 abolishes the genotype-mediated different effects on gene expression and progesterone production (Mann-Whitneys U-test; p ≥ 0.005; n = 3).

Pronuclear morphology evaluation in in vitro fertilization (IVF) / intracytoplasmic sperm injection (ICSI) cycles: a retrospective clinical review

BackgroundThe assessment of the embryo quality is crucial to maintain an high pregnancy rate and to reduce the risk of multiple pregnancy. The evaluation of the pronuclear and nucleolar characteristics of human zygote have been proposed as an indicator of embryo development and chromosomal complement. The aim of the current study was to assess the role of pronuclear morphology evaluation in vitro fertilization (IVF) / intracytoplasmic sperm injection (ICSI) cycles.MethodsRetrospective clinical analysis on 755 non-elective transfers of only one embryo (ET). Embryo assessment was performed in days 1 and 2. Clinical and biological data were recorded and analyzed according to embryo and/or pronuclear morphology.ResultsBoth pronuclear and embryo morphology were significantly related to clinical pregnancy and live-birth rates. No significant difference in clinical pregnancy and live-birth rates was detected when the pronuclear and embryo morphology assessments were combined. Embryo morphology and maternal age were the only independent predictors of favorable outcome by logistic regression analysis.ConclusionsPronuclear evaluation is effective to select the best zygotes if ET is performed at day 1, whereas it did not improve the clinical outcomes when combined with embryo morphology evaluation in day 2.

Protein microarrays on midtrimester amniotic fluids: a novel approach for the diagnosis of early intrauterine inflammation related to preterm delivery

Novel technologies that allow simultaneous assessment of multiple biomarkers provide new and promising diagnostic/prognostic approaches. By protein microarrays, here we analyzed amniotic fluids (AF) from 50 women with preterm delivery (PTD) and 50 control women, who delivered at term. In detail, cytokines, chemokines, matrix metalloproteinases and antigen-specific antibodies were assessed. The AF analysis showed significant differences between women with preterm and term delivery in the levels of IL-1α, IL-1β, IL-4, IL-6, IL-8, MCP-1, IFN-γ and anti-HSV2 IgG. No significant differences were observed in the levels of TNF-α, MMP-2, MMP-9 and specific IgG for seven vertically transmitted pathogens. In conclusion, we demonstrated the feasibility of protein microarrays in the diagnosis of early intrauterine inflammation. The significant association between the increased levels of certain cytokines and preterm delivery argues on their relevance as early pathogenetic markers for identification of risk patients.

Pronuclear morphology evaluation for fresh in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI) cycles: a systematic review

The current systematic review was aimed to assess the effectiveness of the zygote morphology evaluation in fresh in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI) cycles. All available studies reporting on zygote morphology and clinical and/or biological outcomes were analyzed. Forty studies were included in the final analysis. Fourteen different zygote scoring systems were employed. Zygote morphology correlated significantly with embryo quality and cleavage, blastocyst stage, embryonic chromosome status, in a high proportion of the studies which assessed the specific outcome [15/25 (60%), 15/20 (75%), 7/8 (87.5%), 6/6 (100%), respectively]. On the other hand, only a reduced proportion of papers showed a statistically significant relationship between implantation, pregnancy and delivery/live-birth rates and zygote morphology score [12/23 (52.2%), 12/25 (48%), 1/4 (25%), respectively]. In conclusion, our findings demonstrate the lack of conclusive data on the clinical efficacy of the zygote morphology evaluation in fresh IVF/ICSI cycles, even if biological results showing a good relationship with embryo viability suggest a role in cycles in which the transfer/freezing is performed at day 1.

The effect of the 2004 Italian law on outcomes of assisted reproduction technology in severe male factor infertility

The Italian law regulating assisted reproductive technologies that came into force in 2004 restricts the number of fertilized oocytes per cycle to three, obliges the subsequent transfer of all resulting embryos and prohibits the freezing of surplus embryos. This study evaluates the impact of the law on severe oligozoospermic, cryptozoospermic, obstructive azoospermic and non-obstructive azoospermic patients. Intracytoplasmic sperm injection outcomes of 1066 cycles performed in the 4years before the passing of the law were compared with 804 cycles performed in the 4years after the law came to pass. Globally, analysis of clinical and obstetric outcomes showed a significant decrease in terms of pregnancy and delivery rates per cycle (17.8% versus 10.9% and 14.2% versus 8.5%, respectively) and per embryo transfer (18.8% versus 13.8% and 15.0% versus 10.7%, respectively), and a significant drop in multiple deliveries (35.1% versus 17.6%) in the post-law period. Cryptozoospermic and azoospermic couples were affected by the Italian law more than severe oligozoospermic couples. The results showed that the Italian law limits the efficiency of assisted reproduction treatment in couples with severe male factor. It is hoped that the Italian assisted reproductive technologies law is altered as soon as possible, allowing the insemination of more than three oocytes.

The effect of the 2004 Italian legislation on perinatal outcomes following assisted reproduction technology.

Abstract Objective: To assess the perinatal outcomes of the first three years under the 2004 Italian reproductive legislation obligating transfer of all embryos resulting from insemination of ≤3 oocytes. Study design: We compared the perinatal results of clinical assisted reproductive technology (ART) pregnancies during the three years following the new Italian legislation with the previous three years. Results: There were 583 and 571 clinical pregnancies during the respective periods. Before the law, the overall embryonic and fetal loss rates were significantly higher resulting in a significantly lower rate of live born infants and significantly fewer clinical pregnancies with at least one live born infant. Quadruplet and quintuplet pregnancies were entirely eliminated following the 2004 law but the neonatal mortality rate was not different between the two study periods. Conclusion: The 2004 Italian infertility legislation led to improved quantitative and qualitative outcomes of ART.

Detection of follicular fluid and serum antibodies by protein microarrays in women undergoing in vitro fertilization treatment.

A protein microarray serological assay was used to assess the antibody profile of 102 women subjected to in vitro fertilization treatment. The studies were conducted on pairs of serum and follicular fluid samples, collected from each woman on the same day at the time of oocyte recovery. The samples, stored as frozen aliquotes, were assessed by both microarray and ELISA. Follicular fluids and sera were screened to detect the presence of specific IgG and IgM antibodies against seven vertically transmitted pathogens. The IgG reactivity of follicular fluids closely mirrored that of serum in all the patients and for all the antigens, with an agreement of more than 85%. IgM antibodies were undetectable in follicular fluids. The antibody patterns were subsequently related to the biological and clinical outcomes of in vitro fertilization cycles. The results showed that varicella zoster virus (VZV) IgG positive women and cytomegalovirus (CMV) IgG negative women had on average a higher number of inseminated, good quality oocytes compared to VZV IgG negative and CMV IgG positive women. In addition, the rate of successful embryo transfers was significantly higher in Toxoplasma gondii IgG negative women than in their positive counterparts. Overall, the microarray was proven to be a suitable tool for detecting analytes in follicular fluids, therefore supporting its application in a wide spectrum of investigations.

NADPH oxidase-4 and MATER expressions in granulosa cells: Relationships with ovarian aging

AIMS Relevant roles in follicular development and ovulation are played by maternal antigen that embryos require (MATER), product of a maternal effect gene, and by reactive oxygen species (ROS), indispensable for the induction of ovulatory genes. At the moment, the relationship between these two biological systems and their involvement in the ovarian aging have not been still clarified. The aim of the current experimental study was to analyse the age-related changes of the MATER and NOX proteins. MATERIALS AND METHODS MATER and ROS homeostasis was studied in granulosa cells (GCs) and cumulus cells (CCs) of infertile patients who undergone oocyte retrieval for in vitro fertilization cycles using Western blot and confocal immunofluorescence analysis. Samples were obtained from subjects with age≥40years (cases) and with age≤37years (controls). KEY FINDINGS The expression pattern of MATER and NOX observed in GCs was not different from that observed in CCs. High levels of both proteins were detected in the control samples. A significant lower expression of both MATER and NOX4 was observed in the case versus control samples. SIGNIFICANCE The expression of MATER and NOX4 proteins are closely related to the follicular development and ovulation with particular regard for ovarian aging.

Nitric oxide as an early marker of human embryo metabolic cleavage in ART using fresh or thawed oocytes.

OBJECTIVE To study a possible role of nitric oxide (NO) as a marker of development in the early phases of human embryo cleavage during assisted reproduction. STUDY DESIGN 179 women having ART were included. 123 women used fresh oocytes and 56 oocyte thawing cycles in the Center of Reproductive Medicine, Department of Obstetrics and Gynecology, Arcispedale S. Maria Nuova, between July 2005 and June 2006; 57 patients had IVF and 122 patients had ICSI. NO concentrations in IVF or ICSI embryo culture media were assessed by monitoring levels of NO stable oxidation products (nitrites/nitrates). Analysis of embryo quality was performed. Students t-test or Mann-Whitney and logistic regression model tests were applied to the data. RESULTS In patients using fresh oocytes, there were greater NO production in embryos derived from ICSI than from IVF after 52 h of culture (38.64 micromol/L vs 11.2 micromol/L, p<0.05). No correlation with embryo quality was observed. Embryos derived from fresh oocytes produce more NO than embryos from thawed oocytes both after 48 and 52 h of culture (16.12 micromol/L vs 6.83 micromol/L and 25.93 micromol/L vs 2.98 micromol/L respectively, p<0.05). CONCLUSION(S) NO in embryo culture media is not a metabolic cleavage marker or a marker of embryo quality in ART. However, it could be an important parameter in the investigation of metabolism in frozen/thawed oocytes.

Heterogeneous hCG and hMG commercial preparations result in different intracellular signalling but induce a similar long-term progesterone response in vitro

STUDY QUESTION Are four urinary hCG/menotropin (hMG) and one recombinant preparation characterized by different molecular features and do they mediate specific intracellular signaling and steroidogenesis? SUMMARY ANSWER hCG and hMG preparations have heterogeneous compositions and mediate preparation-specific cell signaling and early steroidogenesis, although similar progesterone plateau levels are achieved in 24 h-treated human primary granulosa cells in vitro. WHAT IS KNOWN ALREADY hCG is the pregnancy hormone marketed as a drug for ARTs to induce final oocyte maturation and ovulation, and to support FSH action. Several hCG formulations are commercially available, differing in source, purification methods and biochemical composition. STUDY DESIGN, SIZE, DURATION Commercial hCG preparations for ART or research purposes were compared in vitro. PARTICIPANTS/MATERIALS, SETTING, METHODS The different preparations were quantified by immunoassay with calibration against the hCG standard (Fifth IS; NIBSC 07/364). Immunoreactivity patterns, isoelectric points and oligosaccharide contents of hCGs were evaluated using reducing and non-reducing Western blotting, capillary isoelectric-focusing immunoassay and lectin-ELISA, respectively. Functional studies were performed in order to evaluate intracellular and total cAMP, progesterone production and β-arrestin 2 recruitment by ELISA and BRET, in both human primary granulosa lutein cells (hGLC) and luteinizing hormone (LH)/hCG receptor (LHCGR)-transfected HEK293 cells, stimulated by increasing hormone concentrations. Statistical analysis was performed using two-way ANOVA and Bonferroni post-test or Mann-Whitneys U-test as appropriate. MAIN RESULTS AND THE ROLE OF CHANCE Heterogeneous profiles were found among preparations, revealing specific molecular weight patterns (20-75 KDa range), isoelectric points (4.0-9.0 pI range) and lectin binding (P < 0.05; n = 7-10). These drug-specific compositions were linked to different potencies on cAMP production (EC50 1.0-400.0 ng/ml range) and β-arrestin 2 recruitment (EC50 0.03-2.0 μg/ml) in hGLC and transfected HEK293 cells (P < 0.05; n = 3-5). In hGLC, these differences were reflected by preparation-specific 8-h progesterone production although similar plateau levels of progesterone were acheived by 24-h treatment (P ≥ 0.05; n = 3). LARGE SCALE DATA N/A. LIMITATIONS, REASONS FOR CAUTION The biological activity of commercial hCG/hMG preparations is provided in International Units (IU) by in-vivo bioassay and calibration against an International Standard, although it is an unsuitable unit of measure for in-vitro studies. The re-calibration against recombinant hCG,quantified in grams, is based on the assumption that all of the isoforms and glycosylation variants have similar immunoreactivity. WIDER IMPLICATIONS OF THE FINDINGS hCG/hMG preparation-specific cell responses in vitro may be proposed to ART patients affected by peculiar ovarian response, such as that caused by polycystic ovary syndrome. Otherwise, all the preparations available for ART may provide a similar clinical outcome in healthy women. STUDY FUNDING AND COMPETING INTEREST(S) This study was supported by a grant of the Italian Ministry of Education, University and Research (PRIN 2015XCR88M). The authors have no conflict of interest.