Maria Matteo

Sex hormones modulate circulating antioxidant enzymes: Impact of estrogen therapy

Objective Ovarian senescence affects many tissues and produces a variety of symptoms and signs. We hypothesized that estrogens may also influence circulating redox balance by regulating activity of the cellular antioxidative enzyme system. We aimed to explore the impact of surgical estrogen deprivation and replacement (ERT) on the glutathione balance and antioxidant enzymes expression in fertile women. Study design Nineteen healthy premenopausal women who underwent total hysterectomy with bilateral salpingo-oophorectomy were evaluated at baseline, 30 days after surgery without ERT and 30 days after ERT. Redox balance was determined by measuring blood reduced (GSH) and oxidized (GSSG) glutathione, as well as the GSSG/GSH ratio. Antioxidant status was evaluated by measuring serum estrogen (E2) levels and mRNA expression of superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GSH-Px) and glutathione S-transferase (GST) in peripheral blood mononuclear cells. Results Serum E2 significantly lowered after surgery, and increased in 12 out of 19 patients after 30 days of ERT (Responders). In such patients, an increase in oxidative stress was observed after surgery that resolved after ERT. Oxidative stress was sustained by reduction in the mRNA expression of both SOD and GSH-Px, that recovered after 30 days of therapy in responders. CAT and GST mRNA expression were not modified by surgery and replacement therapy. Conclusions Menopause is associated with significant change in antioxidant gene expression that in turn affects circulating redox state. Estrogens replacement therapy is able to prevent and counteract such modifications by acting as regulators of key antioxidant gene expression. These findings suggest that antioxidant genes are, almost in part, under the control of sex hormones, and that pathophysiology of the difference in gender disease may depend on the redox biology.

Chronic Endometritis Due to Common Bacteria Is Prevalent in Women With Recurrent Miscarriage as Confirmed by Improved Pregnancy Outcome After Antibiotic Treatment

Recurrent miscarriage (RM) is defined as 3 or more miscarriages before 20 weeks’ pregnancy. In recent years, interest has been focused on chronic endometritis (CE), a subtle inflammation thought to be associated with RM. We aimed to evaluate the relationships between CE and RM. The records of 360 women with unexplained RM were retrospectively analyzed. Data from hysteroscopy, endometrial histology, endometrial culture, and polymerase chain reaction for chlamydia, performed before and after antibiotic treatment for CE, were analyzed. The occurrence of successful pregnancies within 1 year after treatment was also evaluated. Results showed that 208 (57.8%) women with RM showed CE at hysteroscopy; 190 (91.3%), positive at hysteroscopy, were also positive at histology, and 142 (68.3%) had positive cultures. Common bacteria were found in 110 (77.5%) patients. Mycoplasma and Ureaplasma were found in 36 (25.3%) patients and Chlamydia in 18 patients (12.7%). In 102 (71%) women, antibiogram-based antibiotic treatment normalized hysteroscopy, histology, and cultures (group 1); while in 40 (28.2%) patients, CE was still present at hysteroscopy (group 2). In 16 of the 66 patients positive at hysteroscopy, but not at cultures, the hysteroscopy becomes normal (group 3) after a Centers for Disease Control and Prevention-based therapy; while in 50 women, CE was still present (group 4). One year after treatment, group 1 showed a significantly higher number of pregnancies (78.4%) compared to group 2 (17.5%; P < .001) and group 4 (15.3%; P = .005). The CE is frequent in women with RM. Antibiotic treatment seems to be associated with an improved reproductive outcome.

ORIGINAL ARTICLE: Abnormal Pattern of Lymphocyte Subpopulations in the Endometrium of Infertile Women with Chronic Endometritis

Problem  Endometrial lymphocytes play a critical role in endometrial receptivity. This study aimed at evaluating the variations induced by chronic endometritis (CE) on endometrial lymphocyte subsets. We compared the results in infertile women diagnosed with CE with those in unexplained infertile women without any sign of CE.

Reduced percentage of natural killer cells associated with impaired cytokine network in the secretory endometrium of infertile women with polycystic ovary syndrome

OBJECTIVE To evaluate lymphocyte subset distribution in the secretory endometrium from infertile patients with polycystic ovary syndrome (PCOS), and the expression of the cytokines known to play a role in determining the endometrial lymphocyte pattern. DESIGN Experimental clinical study. SETTING Outpatient clinic in a university hospital. PATIENT(S) Twenty-eight patients with PCOS (PCOS group) and 6 fertile patients (control group). INTERVENTION(S) On days 22-26 of a spontaneous cycle, subjects underwent endometrial biopsies. MAIN OUTCOMES MEASURE(S) In 19 of 28 patients with PCOS and 6 controls with a late secretory endometrium, the percentage and phenotype of lymphocyte subsets were analyzed by flow cytometry. In the late secretory endometrium of 11 patients with PCOS and 3 controls, the expression of interleukins 15 and 18 and of chemokine ligand 10 was also analysed by polymerase chain reaction. RESULT(S) In patients with PCOS the percentage of CD56+/CD16- and of CD56bright/CD16- cells was significantly lower (median [confidence interval]: 38% [31%-52.7%] vs. 63.7% [57.7%-69%] and 17.4% [8%-41.6%] vs. 52% [43%-60%], respectively), whereas the percentage of CD3+ was significantly higher (45% [33.3%-64%] vs. 26.1% [21%-32%]) as compared with controls. Accordingly, polymerase chain reaction analysis revealed a significantly lower expression of interleukins 15 and 18 and of chemokine ligand 10 in patients with PCOS than in controls. CONCLUSION(S) Results demonstrated an abnormal percentage of endometrial lymphocyte subsets, associated with an impaired cytokine network in patients with PCOS. This could explain the poor reproductive potential in these patients.

Fetal Sex Identification in Maternal Plasma by Means of Short Tandem Repeats on Chromosome X

Analysis of fetal DNA in maternal plasma has recently been introduced as a new method for noninvasive prenatal diagnosis. In the majority of cases, the Y chromosome–specific sequences are commonly used as a fetus‐specific marker with a high risk of false‐negative cases. We attempted to develop a sensitive and reliable X chromosome short tandem repeat (STR) multiplex PCR amplification system that is suitable for the amplification of short‐sized templates of free fetal DNA. Because of specific characteristics of fetal DNA in maternal plasma, cell‐free fetal DNA is smaller than corresponding maternal DNA, and so we selected 10 X‐STR loci in which the allele size was 250 bp. In addition, fetal sex was also investigated using the amelogenin gene in the same multiplex assay. Twenty‐six women were enrolled in the study. Eight of 26 total fetuses analyzed were male and 18 were female. In the whole sample, X‐STRs were informative with a mean of 4.84 ± 1.43. A mean of 2.67 ± 1.28 X‐STR markers per sample (range 1–5) of paternally inherited fetal alleles were detected in pregnant women carrying a female fetus. In all cases, blind determination of fetal sex by means of the identification of amelogenin and X‐STR markers was confirmed by fetal karyotyping. This study showed that this noninvasive technique is a reliable and accurate tool to investigate free fetal DNA in pregnancies within the first trimester and could be widely used in clinical research and diagnosis.

Preliminary evidence for high anti-PLAC1 antibody levels in infertile patients with repeated unexplained implantation failure

OBJECTIVE Placenta-specific1 (PLAC1) is a trophoblast-specific gene encoding for a protein that is highly expressed in human placenta, on the surface of the syncytiotrophoblast. PLAC1 was found to elicit spontaneous antibody responses in cancer patients. We aimed to determine the levels of anti-PLAC1 antibodies in infertile women with a history of unexplained repeated implantation failure after IVF cycles as compared to fertile women. STUDY DESIGN An observational case-control clinical study. MAIN OUTCOME MEASURE(S) Two groups of patients were analysed in two different experimental settings: 21 infertile women and 81 control patients were enrolled in the first group, 16 infertile women and 67 fertile controls in the second group. Anti-PLAC1 antibody levels and ranking were analysed by ELISA test. RESULTS In both groups of infertile patients enrolled, optical densities (OD) from ELISA test ranked significantly higher than those of controls (0.27 ± 0.2 vs. 0.13 ± 0.1 respectively; p = 0.0009 in the first group), (0.62 ± 0.38 vs. 0.39 ± 0.35 respectively; p = 0.0044 in the second experiment). In the first group about one case in four (29%) had OD levels above the 95th percentile (0.337) for healthy controls (p = 0.005). In the second experiment 4 out of 16 cases (25%) had OD levels above the 95th percentile (0.878) for healthy controls (p = 0.023). CONCLUSIONS Anti-PLAC1 antibodies could represent a biomarker associated with infertility and with high probability of repeated implantation failure after ovarian stimulation and IVF-ET, greatly improving the diagnostic work up of infertile couples.

Markers of haemostasis and angiogenesis in placentae from gestational vascular complications: Impairment of mechanisms involved in maintaining intervillous blood flow

INTRODUCTION Preeclampsia (PE) and fetal growth restriction (FGR) are multifactorial diseases, whose pathogenesis is largely unknown. A significant relationship between haemostasis and angiogenesis in placentae from uneventful pregnancies was previously shown. MATERIALS AND METHODS RNA expression of haemostasis (TF, TFPI, TFPI-2, PAI-2, Anx V, TM) and angiogenesis (Ang-1, Ang-2, PlGF, VEGF) markers in placentae from PE (n=12), PE+FGR (n=17) and FGR (n=20) in respect of placentae from uncomplicated pregnancies (n=21) were investigated. RESULTS Placentae from complicated pregnancies showed a significant lower expression (p<or=0.05 Mann-Whitney U test) of TF, TFPI, TFPI-2, Anx V, PAI-2 than those from in uncomplicated ones. VEGF and PlGF were not different in the considered groups; Ang-1 and Ang-2 were significantly higher (p<or=0.05 Mann-Whitney U test) in the PE group. Correlations between factors involved in haemostasis and those involved in angiogenesis, observed in placentae from uneventful pregnancies are lacking in those from complicated ones. CONCLUSIONS Haemostasis factors are reduced in placentae from complicated pregnancies. The relationship between haemostasis and angiogenesis observed in uncomplicated pregnancies is impaired in PE and FGR.

Altered uterine contractility in women with chronic endometritis

OBJECTIVE To evaluate the alterations in endometrial waves (EW) originating from the contraction of the subendometrial myometrial layer in the periovulatory and midluteal phases in women diagnosed with chronic endometritis (CE). DESIGN Case-control study. SETTING University hospital. PATIENT(S) Forty-five women referred for hysteroscopy and diagnosed with CE. INTERVENTION(S) Three-minute recording of transvaginal ultrasound scanning on sagittal uterine plane at periovulatory (cycle days 11-14) and midluteal phase (cycle days 19-22). MAIN OUTCOME MEASURE(S) Direction and frequency of EW measured by transvaginal ultrasound scan. RESULT(S) The direction and frequency of EW were analyzed offline as accelerated (four to eight times normal speed) image sequences using video editing software, and the results were compared with 45 cycling women without CE. The EW pattern was significantly different when comparing the women with CE and controls at both the periovulatory and midluteal phases. During the periovulatory phase, we observed retrograde contractions in 26.7% versus 88%, anterograde in 24% versus 0, opposing in 22.7% versus 12%, not propagated in 13.3% versus 0, and absent in 13.3% versus 0, respectively, in the CE cases versus the control group. During the midluteal phase, we observed not propagated (41.3% vs. 61.3%), opposing (24% vs. 25.4%), absent (16.1% vs. 13.3%), anterograde (13.3% vs. 0), and retrograde (5.3% vs. 0), respectively, in the CE cases versus the control group. CONCLUSION(S) Women with CE show altered EW patterns in both the periovulatory and midluteal phases. Altered uterine contractility may aid in explaining the symptoms related to CE such as pain, abnormal uterine bleeding, infertility, and possibly endometriosis.

Cervical pregnancy treated by uterine artery embolisation combined with office hysteroscopy.

OBJECTIVE To evaluate the safety and efficacy of uterine artery embolisation in conjunction with hysteroscopic resection of trophoblast in the conservative treatment of cervical ectopic pregnancies. STUDY DESIGN Five women diagnosed with cervical pregnancies at University Hospital of Foggia, Italy, between May 2009 and February 2012 underwent uterine artery embolisation followed by office hysteroscopic resection of trophoblast. Data on operating time, blood loss, blood transfusion, conversion to other techniques, complications related to surgery, change of serum β-hCG level, hospitalisation days and outcome of the women after discharge were collected. RESULTS The mean hysteroscopic operative time was 9.8 min, and the blood loss was negligible in all cases. Blood transfusion was not needed for any of the women. None of the women required conversion to other techniques. In all cases the operations were uneventful. The serum β-hCG level in all the cases declined to normal within 15 days of surgery. The total hospitalisation time was 4 days in all the cases. No vaginal bleeding or other side effects were observed throughout and after the treatment, all women recovered without complications. CONCLUSIONS Uterine artery embolisation with office hysteroscopic resection is an effective option in treatment of cervical ectopic pregnancy.

Five years (2004–2009) of a restrictive law-regulating ART in Italy significantly reduced delivery rate: analysis of 10 706 cycles

STUDY QUESTION Was the delivery rate of ART cycles negatively affected by the enactment of the Law 40/2004 by the Italian Parliament which imposed a long list of restrictions for ART procedures? SUMMARY ANSWER This large and extensive comparative analysis of ART outcomes prior to and after the introduction of the Law 40 revealed a significant reduction in pregnancy and delivery rates per cycle, independent of age or other clinical variables, once the law went into effect. WHAT IS KNOWN ALREADY Several studies have been published on the effect of Law 40/2004 on ART outcomes, some authors demonstrating a negative impact of the Law in relation to specific etiologies of infertility, other authors showing opposite conclusions. STUDY DESIGN, SIZE, DURATION Retrospective clinical study of 3808 patients treated prior to the enactment of the Law, September 1996-March 2004 (Group I) and 6898 treated during the Law, March 2004-May 2009 (Group II). PARTICIPANTS/MATERIALS, SETTING, METHODS A total of 10 706 ART cycles were analysed, 3808 performed before and 6898 after the application of the Law. An intention-to-treat statistical analysis was performed to detect pregnancy and delivery rates (pregnancies ≥ 24 weeks) per started cycle. A P value of <0.05 was considered statistically significant. We analysed different outcomes: differences in fertilization, pregnancy and delivery rate, multiple pregnancies and miscarriage rates between the two time periods. MAIN RESULTS AND THE ROLE OF CHANCE The delivery rate for started cycle was 20% before and 16.0% after the introduction of the Law representing a 25% reduction (P < 0.001). The multivariate analysis, corrected by female age of >38 years, duration of infertility, basal FSH level and number of retrieved oocytes, showed a 16% lower delivery rate (odds ratio: 0.84; confidence interval: 0.75-0.94). This statistical approach removed the risk that the observed effects were due to chance and confirmed unequivocally that the Law was an independent factor responsible for the reduced likelihood of a successful outcome. LIMITATIONS, REASONS FOR CAUTION This is a retrospective study. A prospective randomized study, with patients treated in the same time period and randomized to restrictions or not, would have minimized potential limitations due to differences in years of treatments. WIDER IMPLICATIONS OF THE FINDINGS Our findings based on the analysis of such a large number of cycles proved clearly and unequivocally that imposing restrictions on the practice of ART penalized patients. These data represent a relevant clinical contribution for countries still debating the enactment of restrictive limitations of ART.