Adolfo Allegra

Is there a uniform basal endometrial gene expression profile during the implantation window in women who became pregnant in a subsequent ICSI cycle

BACKGROUND To understand which genes are really involved in the implantation process, we planned to study the gene basal expression profile during the window of implantation (WOI) of patients who became pregnant in a subsequent ICSI cycle. METHODS Women attending their first ICSI cycle at ANDROS Day Surgery for severe male factor infertility were included in the study. An endometrial biopsy was performed during the WOI, in one of the last two cycles before the ICSI cycle. Forty-seven selected gene profiles were analyzed using Low Density Array technology. Only biopsies from women who subsequently became pregnant were evaluated, to exclude any bias regarding embryo viability and embryo transfer difficulties. RESULTS Fifteen patients were included in the analysis as they became pregnant after ICSI procedure. Four of 47 selected genes were excluded from the analysis. Of the 43 genes analyzed, only 6 genes (VEGFA, PLA2G2A, ALPL, LIF, NNMT and STC1) showed a statistically uniform expression among patients who subsequently became pregnant. For all the other genes analyzed there were considerable differences in their expression levels amongst women who subsequently became pregnant. CONCLUSIONS Our data suggest that very few genes, which change their expression level during the WOI, show a quantitative homogeneous expression in endometrially-receptive patients. In conclusion, in this study only six genes showed a homogeneous expression, and are probably involved in embryo implantation mechanisms.

A randomized controlled trial investigating the use of a predictive nomogram for the selection of the FSH starting dose in IVF/ICSI cycles.

The number of oocytes retrieved is a relevant intermediate outcome in women undergoing IVF/intracytoplasmic sperm injection (ICSI). This trial compared the efficiency of the selection of the FSH starting dose according to a nomogram based on multiple biomarkers (age, day 3 FSH, anti-Müllerian hormone) versus an age-based strategy. The primary outcome measure was the proportion of women with an optimal number of retrieved oocytes defined as 8-14. At their first IVF/ICSI cycle, 191 patients underwent a long gonadotrophin-releasing hormone agonist protocol and were randomized to receive a starting dose of recombinant (human) FSH, based on their age (150 IU if ≤35 years, 225 IU if >35 years) or based on the nomogram. Optimal response was observed in 58/92 patients (63%) in the nomogram group and in 42/99 (42%) in the control group (+21%, 95% CI = 0.07 to 0.35, P = 0.0037). No significant differences were found in the clinical pregnancy rate or the number of embryos cryopreserved per patient. The study showed that the FSH starting dose selected according to ovarian reserve is associated with an increase in the proportion of patients with an optimal response: large trials are recommended to investigate any possible effect on the live-birth rate.

Gene expression of stem cells at different stages of ontological human development

OBJECTIVES To compare multipotent mesenchymal stem cells (MSCs) obtained from chorionic villi (CV), amniotic fluid (AF) and placenta, with regard to their phenotype and gene expression, in order to understand if MSCs derived from different extra-embryonic tissues, at different stages of human ontological development, present distinct stemness characteristics. STUDY DESIGN MSCs obtained from 30 samples of CV, 30 of AF and 10 placentas (obtained from elective caesarean sections) were compared. MSCs at second confluence cultures were characterized by immunophenotypic analysis with flow cytometry using FACS CANTO II. The expression of the genes Oct-4 (Octamer-binding transcription factor 4, also known as POU5F1), Sox-2 (SRY box-containing factor 2), Nanog, Rex-1 (Zfp-42) and Pax-6 (Paired Box Protein-6), was analyzed. Real-time quantitative PCR was performed by ABI Prism 7700, after RNA isolation and retro-transcription in cDNA. Statistical analysis was performed using non-parametric test Kruskal-Wallis (XLSTAT 2011) and confirmed by REST software, to estimate fold changes between samples. Each gene was defined differentially expressed if p-value was <0.05. RESULTS Cells from all samples were negative for haematopoietic antigens CD45, CD34, CD117 and CD33 and positive for the typical MSCs antigens CD13, CD73 and CD90. Nevertheless, MSCs from AF and placentas showed different fluorescence intensity, reflecting the heterogeneity of these tissues. The gene expression of OCT-4, SOX-2, NANOG was not significantly different among the three groups. In AF, REX-1 and PAX-6 showed a higher expression in comparison to CV. CONCLUSIONS MSCs of different extra-embryonic tissues showed no differences in immunophenotype when collected from second confluence cultures. The expression of OCT-4, NANOG and SOX-2 was not significantly different, demonstrating that all fetal sources are suitable for obtaining MSCs. These results open new possibilities for the clinical use of MSCs derived from easily accessible sources, in order to develop new protocols for clinical and experimental research.

Towards an ideal source of mesenchymal stem cell isolation for possible therapeutic application in regenerative medicine

BACKGROUND The possibility of obtaining mesenchymal stem cells (MSCs) from fetal tissue such as amniotic fluid, chorionic villi and placenta is well-known and a comparison between MSCs originating in different sources such as fetal tissue and those from bone marrow in terms of yield and function is a topical issue. The mesenchymal stem cells isolated from bone marrow are well-characterized. Unfortunately the low quantitative yield during isolation is a major problem. For this reason, other tissue sources for MSCs are of paramount importance. CONCLUSION In this review, starting from a description of the molecular and cellular biology of MSCs, we describe alternative sources of isolation other than bone marrow. Finally, we describe the potential therapeutic application of these cells.

The Fertility Quality of Life Questionnaire (FertiQoL) Relational subscale: psychometric properties and discriminant validity across gender

STUDY QUESTION Is the Fertility Quality of Life Questionnaire (FertiQoL)-Relational Scale a valid measure to assess the relational domain regarding quality of life in women and men undergoing infertility treatment? SUMMARY ANSWER The FertiQoL-Relational scale (FertiQoL-REL) showed good psychometric properties and captured core aspects of couple relationships. WHAT IS KNOWN ALREADY FertiQoL has become a gold standard for the assessment of infertility-related quality of life in patients undergoing assisted reproduction treatment (ART). Despite its growing importance, no previous studies have examined the convergent validity of the FertiQoL-REL and its discriminant validity across gender. STUDY DESIGN, SIZE, DURATION Baseline cross-sectional data as part of a longitudinal study of infertile couples undergoing an ART between February 2013 and January 2015. PARTICIPANTS/MATERIALS, SETTING, METHODS Five hundred and eighty-nine patients (301 females and 288 males), prior to starting an ART in a private clinic, filled in the Fertility Quality of Life Questionnaire (FertiQoL) and several measures of the marital relationship (Dyadic Adjustment Scale, Marital Commitment Inventory and ENRICH Marital Satisfaction Scale) and infertility-related distress (Fertility Problem Inventory). MAIN RESULTS AND THE ROLE OF CHANCE Confirmatory factor analysis showed that the FertiQoL four-factor solution provided a good fit for the observed data. Reliability of the FertiQoL-REL was higher for women than men. Significant correlations between the FertiQoL-REL scores and all the other measures of marital relationship were found for both women and men. FertiQoL-REL scores did not differ significantly in women and men. The FertiQoL-REL was able to differentiate subjects as regards the Dyadic Adjustment Scale and ENRICH Marital Satisfaction Scale threshold. LIMITATIONS, REASONS FOR CAUTION Findings are limited because the data were obtained from only one Italian private clinic. WIDER IMPLICATIONS OF THE FINDINGS FertiQoL-REL threshold scores are useful for identifying those patients undergoing ART who are more likely to report poor or good relationship quality. Clinicians should tailor their counselling strategies to the positive qualities in a couples relationship, so as to reinforce the overall quality of life, especially among women, and to support patients in tackling the psychological burden, so that they can either continue treatment or choose discontinuation. STUDY FUNDING/COMPETING INTERESTS This research was supported by funds provided by Centro Andros S.r.l., Palermo, Italy. The authors declare no financial or commercial conflicts of interest in this study. TRIAL REGISTRATION NUMBER Not necessary.

Assessing infertility-related stress: the factor structure of the Fertility Problem Inventory in Italian couples undergoing infertility treatment

Abstract The factor structure of the Fertility Problem Inventory (FPI) and its invariance across gender were examined in Italian couples undergoing infertility treatment. About 1000 subjects (both partners of 500 couples) completed two questionnaires prior to commencing infertility treatment at a private Clinic in Palermo, Italy. Confirmatory Factor Analysis demonstrated that the original factor structure of the FPI was partially confirmed. Two correlated factors (Infertility Life Domains and Importance of Parenthood) were obtained via a post hoc Exploratory Factor Analysis. Finally, the invariance of this factor structure across gender was confirmed. The study supported the relevance of two interrelated factors specific to infertility stress which could help clinicians to focus on the core infertility-related stress domains of infertile couples.

The pellet swim-up is the best technique for sperm preparation during in vitro fertilization procedures

PurposeThe aim of this study was to investigate the most suitable sperm preparation technique to apply in order to obtain a spermatozoon population with minimal DNA damage during in vitro fertilization procedures. We compared four preparation techniques: direct swim-up (DSU), pellet swim-up (PSU), density gradient (DG), and density gradient followed by swim-up (DG-SU), evaluating the effects of each technique on the DNA damage rate, evaluated by DNA fragmentation index of the spermatozoa obtained.MethodsIn this observational study, 98 semen samples from couples undergoing IVF/ICSI cycles were included. Data were collected between April and November 2014 at the ANDROS Day Surgery Clinic, Palermo, Italy.Result(s)The percentages of DNA fragmentation were 18.30 ± 10.8 in raw samples, 6.6 ± 5.7 after DSU, 4.2 ± 3.8 after PSU, 12.9 ± 9.9 after DG, and 3.7 ± 4.0 after DG-SU respectively. Compared to the raw evaluation, all the preparation techniques significantly decreased the total rate of the DNA fragmentation (DSU Z = −8.60, P < 0.008; PSU Z = −8.54, P < 0.008; DG Z = −6.42, P < 0.008, and DG-SU Z = −8.60, P < 0.008, respectively). Comparing them, spermatozoa with intact DNA after PSU and DG-SU were significantly higher than after DSU (Z = −7.12, P < 0.008; Z = −6.59, P < 0.008, respectively) and after DG (Z = −8.41, P < 0.008; Z = −8.60, P < 0.008, respectively). The difference between PSU and DG-SU was not significant (Z = −2.21, P = 0.03).Conclusion(s)There are, above all, two techniques of sperm preparation which allow for the recovery of spermatozoa with the lowest DNA fragmentation rate. Furthermore, given low costs and reduced time, we believe that PSU is the best option in the treatment of semen samples during IVF/ICSI.

Endometrial expression of selected genes in patients achieving pregnancy spontaneously or after ICSI and patients failing at least two ICSI cycles.

The objective of this study was to identify the endometrial gene expression profile in receptive phase, which could represent a useful prognostic tool for selecting IVF patients. Endometrial expression of 47 selected genes biopsied during the window of implantation in natural cycles was compared between patients who achieved a successful pregnancy spontaneously or after subsequent intracytoplasmic sperm injection (ICSI) cycles and patients who did not achieve a pregnancy after at least two failed ICSI cycles. The comparative analysis showed significantly different levels of expression in 19 genes, five implicated in apoptosis (CASP8, FADD, CASP10, APAF1, ANXA4), three in immunity (LIF, SPP1, C4BPA), five in transcriptional activity (MSX1, HOXA10, MSX2, HOXA11, GATA2), two in lipid metabolism (LEPR, APOD) and four in oxidative metabolism (AOX1, ALDH1A3, GPX3, NNMT). The evidence for these genes being differently expressed could represent the starting point of identifying the ideal receptive endometrial gene expression profile, which could be used in the future as a prognostic tool for IVF patients. Gene expression analysis technology has opened new important perspectives on the study of the physiological processes of different tissues and organs. Specifically for the endometrium, it would be really interesting to find out an endometrial gene expression profile of receptive phase, which could be used in future as a useful prognostic tool for selecting IVF patients. To achieve this aim, the objective of the present paper was the comparison of endometrial expression in natural cycles of 47 selected genes between the biopsies of patients who achieved a successful pregnancy, either spontaneously or after subsequent ICSI cycles, and those of patients who did not achieve a pregnancy after at least two failed ICSI cycles. The comparative analysis showed a significant different expression in 19 genes: five implicated in programmed cell death, known as apoptosis (CASP8, FADD, CASP10, APAF1, ANXA4), three in immunity (LIF, SPP1, C4BPA), five in transcriptional activity (MSX1, HOXA10, MSX2, HOXA11, GATA2), two in lipid metabolism (LEPR, APOD) and four in oxidative metabolism (AOX1, ALDH1A3, GPX3, NNMT). The evidence of these genes being differently expressed could represent the starting point of identifying the ideal receptive endometrial gene expression profile which could be used in the future as a prognostic tool for IVF patients.

Population data of the 16 autosomal STRs loci of the Powerplex ESI 17 System in Sicilian Population (Italy)

Allele frequencies of 16 STR loci including the 5 new European Standard Set (ESS) loci [1] were calculated in a sample of 201 unrelated Sicilian individuals. Samples were collected from 42 different locations in Sicily. All samples were obtained from voluntary donors after informed consent. In order to make the samples anonymous, we were provided with a code before the analysis procedure. DNA was extracted from buccal swabs with Maxwell 16 Buccal Swab LEV DNA Purification Kit (Promega Corporation, USA) using the Maxwell 16 instrument (Promega) and quantified by Quantifluor dsDNA system (Promega) using Quantifluor-ST Fluorimeter (Promega). PCR amplification was performed by the Powerplex 17 ESI System (Promega) in a final volume of 12 ml in a 2720 Thermal Cycler (Applied Biosystems, Foster City, CA), to amplify 16 STR loci (D3S1358, D19S433, D2S1338, D16S539, D18S51, TH01, D21S11, vWA, D8S1179, FGA, SE33, D22S1045, D10S1248, D1S1656, D12S391, D2S441) and Amelogenin. PCR products were analysed by capillary electrophoresis in a ABI3130 Genetic Analyser (Applied Biosystems) using CC5 Internal Lane Standard 500 for Promega kits as size standard. Allele designations of the profiles were made by processing with Gene Mapper ID v3.2 using the sequenced ladder provided by

Identification of embryo-fetal cells in celomic fluid using morphological and short-tandem repeats analysis.

The main problem to wide acceptability of celocentesis as earlier prenatal diagnosis is contamination of the sample by maternal cells. The objective of this study was to investigate the cellular composition of celomic fluid for morphological discrimination between maternal and embryo–fetal cells.