Low molecular weight heparin -induced miRNA changes in peripheral blood mononuclear cells in pregnancies with unexplained recurrent pregnancy loss.

Unexplained recurrent pregnancy loss (uRPL) is a clinical condition for which there is a lack of evidenced-based therapies. However, in clinical practice, low molecular weight heparin (LMWH) has been widely used as an empirical therapy since immune effects have been hypothesized in modulating immune tolerance at the fetal-maternal interface. Epigenetic mechanisms are involved in establishing of immune tolerance, at fetal-maternal interface. To investigate potential induced immune-epigenetic changes at maternal periphery level, which could reflect the maternal-fetal interface condition, seems to open up new therapeutical strategies, since microRNAs circulating in maternal plasma and in peripheral blood mononuclear cells (PBMCs) may be specific and sensitive immunological markers/predictors of adverse pregnancy outcomes such as RPL. Our aim in this pilot study is to evaluate potential LMWH effects on genes regulating immunological response key mechanisms related to maternal-fetal tolerance processes, by studying circulating miRNAs in maternal peripheral blood. We tested a panel of selected miRNAs on three groups: 18 healthy pregnant women, 20 pregnant women affected by uRPL, 18 pregnant women affected by uRPL, treated with LMWH. The majority of differentially expressed miRNAs (miR 374a-5p, 19a-3p, 30e-5p, 128-3p, 155-5p and 200c-3p) were found to be modulated by LMWH, which seems to have a positive function in RPL patients, by bringing patients' values back to those comparable to the control ones. Selected microRNA panels would appear to be an effective clinical tool for uRPL diagnosis and management. LMWH-modified miRNA expression levels could be targets for immunotherapy, as LMWH would appear to restore physiological miRNA levels, which are dysregulated in uRPL.

Machine Learning (ML) based-method applied in recurrent pregnancy loss (RPL) patients diagnostic work-up: a potential innovation in common clinical practice.

RPL is a very debated condition, in which many issues concerning definition, etiological factors to investigate or therapies to apply are still controversial. ML could help clinicians to reach an objectiveness in RPL classification and access to care. Our aim was to stratify RPL patients in different risk classes by applying an ML algorithm, through a diagnostic work-up to validate it for the appropriate prognosis and potential therapeutic approach. 734 patients were enrolled and divided into 4 risk classes, according to the numbers of miscarriages. ML method, called Support Vector Machine (SVM), was used to analyze data. Using the whole set of 43 features and the set of the most informative 18 features we obtained comparable results: respectively 81.86 ± 0.35% and 81.71 ± 0.37% Unbalanced Accuracy. Applying the same method, introducing the only features recommended by ESHRE, a correct classification was obtained only in 58.52 ± 0.58%. ML approach could provide a Support Decision System tool to stratify RPL patients and address them objectively to the proper clinical management.

Uterine blood flow indices, antinuclear autoantibodies and unexplained recurrent miscarriage.

OBJECTIVE: To study the correlation between 2D and 3D uterine flow indexes and the presence or the absence of antinuclear antibodies (ANA) in women with unexplained recurrent miscarriage (uRM). METHODS: Fifty-two subjects (26 uRM and 26 control women) underwent 2D Doppler measurement of pulsatility index and resistance index of the uterine arteries in both the follicular and midluteal phase of the cycle. Additionally, 3D ultrasonography determination of vascularisation index, flow index, and vascularisation flow index was carried out with the aid of the VOCAL technique. Serum assay for the presence of ANA was performed in all women. RESULTS: Pulsatility index of ANA+ uRM women was higher than that of ANA- uRM women and control ANA+ and ANAwomen, both in the follicular and in the midluteal phase of the cycle. Vascularisation index in ANA- uRM women was significantly higher than that in ANA+ control women. Flow index in uRM ANA+ women was significantly lower than that of each of the other groups. CONCLUSION: ANA might be involved in uRM by determining an impairment in uterine blood flow hemodynamic, particularly in uterine blood flow intensity and uterine artery impedance.

Plasminogen activator inhibitor-1, factor V, factor II and methylenetetrahydrofolate reductase polymorphisms in women with recurrent miscarriage.

The present study investigated the association between genetic polymorphisms of selected thrombophilic factors with recurrent miscarriage (RM). The genetic polymorphisms for plasminogen activator inhibitor-1 4G/5G (PAI-1), Factor V Leiden (FVL), Factor II G20210A (FII) and methylenetetrahydrofolate reductase MTHFR C677T were determined in 186 RM women and 129 healthy women. In RM women, the frequency of heterozygosity for PAI-1 5G/4G (31%) was significantly higher than in controls (5G/4G: 22%) whereas no difference was found in the case of homozygosity 4G/4G and 5G/5G. The frequencies of genotype G/A for FVL and FII were significantly higher in RM women (FVL, 10%; FII, 8%) than in controls (FVL, 3%; FII, 2%). No difference was found in the case of MTHFR C677T. The polymorphisms of FVL and FII should be screened in RM women, whereas PAI-1 seems to be weakly associated with RM. The role of MTHFR C677T polymorphisms without hyperhomocysteinemia appears negligible.

The association of PTPN22 polymorphism with endometriosis: effect of genetic and clinical factors.

OBJECTIVE: To investigate the possible effect of clinical and genetic variables on the association between PTPN22 and endometriosis. METHODS: PTPN22, ACP1 and p53 codon 72 genetic polymorphisms and duration of previous pharmacological treatment were studied. The study sample consisted of 132 women hospitalized for endometriosis diagnosed by laparoscopic intervention and histologically confirmed: 359 healthy blood donors were studied as controls. PTPN22, ACP1 and p53 codon 72 genotypes were determined by DNA analysis. Discriminant statistical analysis, logistic regression analysis, chi square of independence, power test and linear correlation were performed using SPSS programs. RESULTS: A significant increase of PTPN22 *T allele in endometriosis is observed in women carrying ACP1*C allele, in women carrying p53 codon 72 *Pro allele and in women with prolonged pharmacological treatment. CONCLUSIONS: PTPN22 may not be a primary factor in the etiology of endometriosis but may cooperate with clinical and genetic factors influencing susceptibility and clinical course of disease. These new observations point to a multifactorial origin of endometriosis and help to explain the reported differences between human populations concerning the association between PTPN22 and endometriosis.

Diagnostic accuracy of liquid-based endometrial cytology in the evaluation of endometrial pathology in postmenopausal women.

OBJECTIVE: The aim of this study was to compare liquid-based endometrial cytology with hysteroscopy and endometrial biopsy regarding its diagnostic accuracy in a series of postmenopausal women with abnormal uterine bleeding (AUB) or asymptomatic women with thickened endometrium assessed by transvaginal ultrasound as a screening procedure. METHODS: Inclusion criteria were: menopausal status; the presence of AUB and/or thickened endometrium assessed by ultrasound (cut-off 4 mm); a normal Papanicolaou (Pap) smear; and no adnexal pathology at ultrasound. Exclusion criteria were: previous endometrial pathology; and previous operative hysteroscopy. Of 768 postmenopausal women referred to our general gynaecology clinics, 121 fulfilled the inclusion criteria and were recruited to the trial. Twenty-one refused to participate. Cytological sampling was carried out by brushing the uterine cavity using the Endoflower device with no cervical dilation and the vial was processed using a ThinPrep® 2000 automated slide processor. The slides were stained using a Pap method. RESULTS: In 98 cases with histological biopsies, endometrial cytology detected five cases of endometrial carcinoma, 10 of atypical hyperplasia and 47 of non-atypical hyperplasia; 36 cases were negative. In two cases cytology was inadequate because of uterine cervical stenosis. Taking atypical hyperplasia or worse as a positive test and outcome, the diagnostic accuracy of the endometrial cytology was 93.5%, with a sensitivity of 92% and specificity of 95%, a positive predictive value of 73% and a negative predictive value of 99%. All the carcinomas were detected by cytology. Only 42% of women with a positive diagnosis were symptomatic. The cytological sampling was well tolerated by all patients. No complication was registered. CONCLUSIONS: Liquid-based endometrial cytology can be considered an useful diagnostic method in the detection of endometrial pathology as a first-line approach, particularly if associated with transvaginal ultrasound.

Ak(1) genetic polymorphism and season of conception.

OBJECTIVE: The season of conception affects human reproduction, intrauterine growth, neonatal parameters, sex ratio, cognitive development and, in adult life, performance in many fields. Associations between polymorphic enzymes and season of conception have been also reported. In this study we searched for a possible association between season of conception and adenylate kinase locus 1 (Ak(1)). STUDY DESIGN: Two samples of 381 and 248 consecutively newborn infants from two Italian cities with different geographical positions and climatic conditions were considered. Three way contingency table analysis and Student t-test analysis were performed. RESULTS: Ak(1)2-1 phenotype is more frequent in males conceived in the summer-autumn period than in those conceived in winter-spring and this association depends on maternal Ak(1) phenotype (p=0.001). There is also an interaction between season of conception and Ak(1) phenotype concerning their effects on sex ratio and birth weight. CONCLUSION: The present data suggest a complex interaction involving seasonal cycles, maternal and foetal Ak(1) genotype and sex of foetus concerning their effects on intrauterine selection and neonatal parameters.

Isobaric gasless laparoscopy versus minilaparotomy in uterine myomectomy: a randomized trial.

BACKGROUND: Isobaric gasless laparoscopy and minilaparotomy have been used as more recent minimally invasive approaches to myomectomy. This randomized trial aimed to compare the surgical and immediate postoperative outcomes for myomectomy performed by isobaric gasless laparoscopy with those for minilaparotomy. METHODS: A total of 100 patients with symptomatic uterine myomas requiring myomectomy were randomly allocated to the gasless laparoscopy group or the minilaparotomy group. The randomization procedure was based on a computer-generated list. The primary outcome was a comparison of the discharge times between the two procedures. A power calculation verified that more than 26 patients for each group was necessary to detect a difference of more than 24 h in discharge time with an alpha error level of 5% and a beta error of 80%. Continuous outcome variables were analyzed using the Student's t-test. Discrete variables were analyzed with the chi-square test or Fisher's exact test. A p value less than 0.05 was considered statistically significant. RESULTS: The mean discharge time was longer for minilaparotomy than for gasless laparoscopy (98.4 +/- 1.4 vs 52.8 +/- 1.6 h; p < 0.001). Gasless laparoscopy resulted in shorter times for canalization (21.6 +/- 1.1 vs 32 +/- 1.3 h; p < 0.05) and surgery (79.5 +/- 25.1 vs 103.5 +/- 24.9 min; p < 0.001). The intraoperative blood loss was less with gasless laparoscopy (154.2 +/- 1.2 vs 188.6 +/- 1.3 ml; p < 0.001). No intraoperative complications occurred, and no case was returned to the theater in either group. No conversion to standard laparotomy was necessary. CONCLUSIONS: Isobaric gasless laparoscopy and minilaparotomy can be suitable options for uterine myomectomy. Several surgical and immediate postoperative outcomes were significantly better in the gasless laparoscopy group than in the minilaparotomy group. However, further controlled prospective studies are required to confirm the results.

Transmission ratio distortion in the spinal muscular atrophy locus: data from 314 prenatal tests.

BACKGROUND: Spinal muscular atrophy (SMA) is a recessive neurodegenerative disorder characterized by the loss of alpha-motor neurons in the spinal cord and subsequent death of motor neuron cells. SMA occurs with a frequency of 1 in 6,000 live births, with a carrier frequency of 1 in 40, and is a leading genetic cause of infant mortality. SMA is caused by loss or mutation of the telomeric survival motor neuron gene (SMN1), which is deleted in almost 94% of SMA patients OBJECTIVE: To analyze the transmission ratio at the SMA locus, examining the segregation of the SMN1-deleted alleles in 314 fetuses from carrier parents who requested prenatal testing for the disease. METHODS: Prenatal diagnosis of SMA in families at 25% risk of the disease has been performed on chorionic villous sampling specimens, through direct detection of the SMN1 gene mutation and linkage analysis using microsatellite markers from the 5q13 region. Analysis of the genotypic/allelic frequencies of the SMN1 gene was performed using the chi2 test, assuming a recessive mendelian inheritance. RESULTS: Of 314 fetuses analyzed, 95 were homozygous for the wild-type allele (30.3%), 154 were carriers (49.0%), and the remaining 65 were homozygous for the mutated allele (20.7%). Statistical analysis demonstrated that proportion of fetuses predicted with SMA is lower than 25% expected for a recessive disorder, resulting in a transmission rate of the SMN1-deleted allele deviant from the 50% expected in a random the segregation of a mendelian tract (p = 0.016) CONCLUSIONS: This is the first study to evaluate the genotypic frequencies at the spinal muscular atrophy (SMA) locus based on data derived from prenatal analysis, which are not subject to ascertainment bias. The analysis showed a transmission ratio distortion at the SMA locus in favor of the SMN1 wild-type alleles.

Hormonal regulation of cytokine release by human fetal membranes at term gestation: effects of oxytocin, hydrocortisone and progesterone on tumour necrosis factor-alpha and transforming growth factor-beta 1 output.

Inflammatory cytokines can play an important role in the biomolecular processes leading to labour by regulating prostaglandin production in intrauterine tissues. In the setting of intrauterine infection, an increased production of these cytokines by placenta, decidua and fetal membranes occurs and is responsible for the onset and maintenance of preterm labour. However, the factors involved in the control of cytokine release by these tissues in normal pregnancy at term are still largely unknown. We investigated the possibility that the synthesis and release of tumour necrosis factor-alpha (TNF-alpha) and transforming growth factor-beta1 (TGF-beta1) by human fetal membranes at term gestation is regulated by several hormones potentially involved either in the maintenance of pregnancy or in the parturitional process. In the present study, the effects of hydrocortisone, progesterone and oxytocin on TNF-alpha and TGF-beta1 release by explants of fetal membranes at term gestation were evaluated. Reverse transcriptase-polymerase chain reaction (RT-PCR) was used to assess the effect of the above hormones on mRNA expression; TNF-alpha and TGF-beta1 release in culture medium was quantitifed by ELISA assays. Results show that both tissue mRNA expression for TNF-alpha and TNF-alpha release in culture medium were significantly increased by oxytocin, but not by hydrocortisone and progesterone. On the contrary, all the hormones tested increased both tissue TGF-beta1 mRNA expression and release in culture medium. These findings suggest that TNF-alpha and TGF-beta1 production by human fetal membranes in uncomplicated pregnancy at term is selectively modulated by oxytocin, hydrocortisone and progesterone.

Chromosomal abnormalities in fetuses with absent end-diastolic velocity in umbilical artery: analysis of risk factors for an abnormal karyotype.

OBJECTIVE: Our purpose was to evaluate the incidence and patterns of chromosomal abnormalities in fetuses with absent end-diastolic velocity in umbilical artery and to analyze maternal and fetal factors associated with abnormal karyotype. STUDY DESIGN: One hundred ninety-two fetuses of known karyotype with absent end-diastolic velocity in the umbilical artery at a gestational age > 20 weeks were considered. The following potential risk factors were analyzed in a multiple logistic regression model: maternal age, gravidity, parity, gestational age at diagnosis, presence of gestational hypertension and preeclampsia, presence of fetal malformations, different biometric measurements, head/abdominal circumference ratio, amniotic fluid volume, and several Doppler index values calculated from uterine arteries, fetal heart, and fetal peripheral arteries and veins. RESULTS: Sixteen cases had an abnormal karyotype. In two cases a triploidy was present, whereas the remaining 14 cases had autosomal aberrations. The risk factors statistically significantly and independently associated with the presence of an abnormal karyotype were maternal age > 35 years, gestational age at diagnosis < 27 weeks, presence of multiple malformations, and absence of gestational hypertension and preeclampsia. All the fetuses with an abnormal karyotype but one were correctly identified by at least one risk factor. CONCLUSIONS: An abnormal karyotype is present in 8.3% of fetuses with absent end-diastolic velocity in umbilical artery and is associated with maternal and fetal risk factors. The knowledge of these factors may be useful in the management of such fetuses.

Does measurement of amniotic fluid index detect changes in amniotic fluid volume after second-trimester amniocentesis?

The objective of this study was to establish whether variations of amniotic fluid volume induced by second-trimester amniocentesis could be detected by serial measurements of amniotic fluid index. A total of 130 singleton pregnancies undergoing second-trimester amniocentesis for genetic indications were considered. Amniotic fluid index was measured at three different time intervals: 30-60 min before amniocentesis, immediately after the procedure, and 60 min after the procedure. Serial measurements were obtained either by a single operator (n = 55) or by the three independent operators (n = 75). Significantly lower amniotic fluid index values were demonstrated immediately after amniocentesis when compared with the pre-amniocentesis and subsequent measurements in the study design with both the single and multiple operators. No statistically significant changes were found between the first amniotic fluid index measurements and those obtained 1 h after amniocentesis. These results suggest that second-trimester amniocentesis induces a temporary decrease of amniotic fluid volume detectable by serial amniotic fluid index measurements, no longer evident 1 h after the procedure.

Evaluation of pulsatility index nomograms based on fetal biometry in small for gestational age fetuses.

Reference limits for the PI from the umbilical, middle cerebral, and renal arteries were constructed using BPD, AC, FL, and transverse cerebellar diameter as independent variables and their efficacy tested in a population of SGA fetuses. Therefore, 153 normal fetuses and 90 SGA fetuses with established dates between 20 and 40 weeks of gestation were considered. Normal fetuses showed a linear negative relationship between the PI from all the vessels investigated and all the biometric parameters considered. Although the BPD related better with the PI from the umbilical artery (r = 0.646) and the renal artery (r = 0.765) and the transverse cerebellar diameter related better with middle cerebral artery PI values (r = 0.510), no evident differences in fitting were found among the variables tested. In SGA fetuses the nomograms on BPD, AC, and FL significantly underestimated PI values in all the vessels studied when compared to the nomograms based on gestational age, while a similar ability in identifying abnormal PI values was found for nomograms based on gestational age and transverse cerebellar diameter. These newly developed nomograms based on transverse cerebellar diameter may prove useful in the evaluation of Doppler indices of fetuses with uncertain gestational age.

Transverse cerebellar diameter in small-for-gestational-age fetuses: pregnancy dating is possible only when growth retardation is secondary to uteroplacental insufficiency.

The objective of this study was to establish whether measurement of the transverse cerebellar diameter to determine gestational age differs in small-for-gestational-age fetuses with normal or abnormal Doppler velocity waveforms. Our secondary objective was to compare the efficacy of measurement of transverse cerebellar diameter with that of femur length in pregnancy dating among small-for-gestational-age fetuses. A total of 107 small-for-gestational-age fetuses with established dates and free from structural and chromosomal abnormalities were considered for this study. According to the Doppler results, fetuses were divided into two groups: group A (n = 64), with normal Doppler values as expressed by a ratio of pulsatility indices between the umbilical artery and middle cerebral artery of 95th centile. Measurements of transverse cerebellar diameter and femur length were compared to previously established 95th centile prediction intervals. In both groups of small-for-gestational-age fetuses, the values for transverse cerebellar diameter were lower than in normally grown fetuses after normalization for gestational age. The difference was more evident in group A (p

Fetal cardiac and extracardiac flows preceding intrauterine death.

Extracardiac and cardiac flow velocity waveforms were recorded in a severely growth-retarded fetus 1 day and a few hours before fetal death. At the first scan, the typical Doppler patterns of a growth-retarded fetus were found, but the brain-sparing effect was lost at the last examination and a huge tricuspid insufficiency was demonstrated.

Analysis of factors influencing ventricular filling patterns in fetuses of type I diabetic mothers.

In an effort to determine the factors influencing the abnormal ventricular filling patterns of fetuses of type I diabetic mothers, Doppler flow velocity waveforms were recorded from fetal atrioventricular valves in 37 pregnancies complicated by type I diabetes immediately before an elective cesarean section. The ratio between the peak velocities during early passive ventricular filling and active atrial filling was calculated at the level of both atrioventricular valves and related to different factors including ventricular chamber wall thickness, heart rate, umbilical vein hematocrit and time to peak velocities values obtained at the outflow tract. Multiple stepwise regression demonstrated that the interventricular wall thickness, heart rate and hematocrit values significantly and independently affected the ratios between early and active ventricular filling from mitral and tricuspid valves. As a consequence all these factors should be taken into account in the interpretation of atrioventricular Doppler indices.