Giuseppe Ricci

Coagulation and fibrinolysis changes in normal pregnancy : Increased levels of procoagulants and reduced levels of inhibitors during pregnancy induce a hypercoagulable state, combined with a reactive fibrinolysis

OBJECTIVE To establish the physiologic changes in the coagulation and fibrinolytic systems during normal pregnancy and puerperium. STUDY DESIGN One hundred and seventeen normal pregnant women were investigated in a longitudinal study involving five measurements: blood samples were collected at 10, 20, 30, 36 weeks and on the second day puerperium and were assayed for prothrombin time (PT expressed in INR), activated partial thromboplastin time (PTT), fibrinogen (FBG), antithrombin III activity (AT III), protein C activity (PC), protein S activity (PS), prothrombin fragments 1+2 (F1+2), type 1 plasminogen activator inhibitor activity (PAI) and tissue-plasminogen activator antigen (t-PA). Student t-test, One Way Analysis of Variance (ANOVA) and Bonferroni test were used for statistical analysis. P<0.05 (two tails) was assumed to indicate a significant difference. RESULTS Fibrinogen concentrations were always increased with respect to controls (P<0.001), while protein S was always decreased, with values averaging 60% of those of controls from the 10th week of pregnancy onwards (P<0.001). Variance analysis showed a statistically significant increase with gestational age for procoagulant factors (INR: P<0.001; FBG: P<0.001), a reduction for anticoagulants (PC: P<0.0001; PS: P<0.0001), and a rise for F1+2 (P<0.0001). With regard to fibrinolysis, there was an increase both for t-PA (P<0.0001) and PAI-1 (P<0.0001) during pregnancy. The t-PA values were always comprised in the normal range. PAI-1 were increased with respect to control values starting from 31st week. The most significant variations in the procoagulants (expressed by PT and FBG) were recorded up to the 20th week (P<0.001); from the 30th week onwards, they remained stable until after the delivery. The same was true for protein S levels (P<0.001), except that the difference between the 10th and the 20th weeks was not statistically significant. The level of F1+2 gradually increased throughout pregnancy (P<0.001), and then fell in the puerperium (P<0.001). CONCLUSIONS The parameters showing the greatest variation during pregnancy were PT, FBG, PS, F1+2 and PAI-1. The existence of a hypercoagulable state in pregnancy was suggested by the increased levels of F1+2.

Semen preparation methods and sperm apoptosis: swim-up versus gradient-density centrifugation technique.

OBJECTIVE To compare the effects of density-gradient centrifugation and swim-up on sperm apoptosis by using a multiparameter flow cytometric method. DESIGN Autocontrolled split-sample study. SETTING Tertiary infertility center. PATIENT(S) Sixty-two male partners of couples undergoing infertility investigations. INTERVENTION(S) Each sample was analyzed both before and after semen preparation by optical microscopy and by flow cytometry. MAIN OUTCOME MEASURE(S) Percentage of viable, apoptotic, and necrotic sperm and recovery rate of total motile, progressive motile, and viable sperm before and after the two sperm preparation methods. RESULT(S) Compared with the original semen, the mean percentages of apoptotic and necrotic sperm were significantly lower after both sperm preparation methods. The mean percentage of viable sperm was significantly higher after swim-up compared with gradient centrifugation. The recovery rates of total motile, progressive motile, and viable sperm were significantly higher using gradient centrifugation compared with swim-up. The viable sperm percentage and the progressive sperm motility were significant predictors for negative difference between the two methods in terms of viable sperm percentage after preparation. CONCLUSION(S) Both sperm preparation methods allow obtaining a sperm population with a low percentage of apoptotic sperm. Therefore, the risk of using apoptotic sperm for clinical treatment seems to be rather low. The choice of method will depend on whether IVF/ICSI or intrauterine insemination is to be performed.

Factor V Leiden and prothrombin gene G20210A mutation and in vitro fertilization: prospective cohort study

BACKGROUND The influence of thrombophilia on fertility and on IVF outcome is very controversial. The objectives of this study were: (i) to compare the prevalence of Factor V Leiden (FVL) and prothrombin gene G20210A mutation (PGM) in women undergoing IVF to women with spontaneous pregnancy; (ii) to compare the IVF outcomes and the risk of complications in FVL and PGM carrier to non-carrier women. METHODS From March 2005 to December 2009, a total of 510 women requiring IVF were recruited in a prospective cohort study. A separate population of 490 nulliparous women who conceived naturally was also evaluated as fertile controls. All women were tested for the presence of FVL and PGM. RESULTS The prevalence of thrombophilic mutations was the same among women requiring IVF (6.9%) and women with spontaneous pregnancy (6.9%). A total of 480 patients underwent 1105 IVF cycles. There were 30 women carriers (86 IVF cycles) and 450 non-carriers for thrombophilic mutations (1019 IVF cycles). No significant differences in the mean number of oocytes retrieved and the number of good quality embryos transferred were found between the mutation carrier and non-mutation carrier women; likewise the reproductive outcome and the IVF complications were not statistically different between the two groups. The cumulative live birth rate after six IVF cycles was similar in the mutation carrier and non-mutation carrier women. For the mutation carrier women, the optimistic estimate of cumulative live birth rate after six IVF cycles was 60.8% and the conservative estimate was 50.0%. Corresponding rates for the non-mutation carrier women were 56.8 and 36.2%, respectively. CONCLUSIONS The results of this study suggest that FVL and PGM presence in asymptomatic women and in the absence of other risk factors do not influence IVF outcome, or represent risk factors for ovarian hyperstimulation syndrome (OHSS), or favour thrombosis after IVF. Screening for FVL and PGM does not appear to be justified to identify the patients at the risk for IVF failure, and/or for OHSS, and/or for thrombotic complications.

Medical Treatments for Endometriosis-Associated Pelvic Pain

The main sequelae of endometriosis are represented by infertility and chronic pelvic pain. Chronic pelvic pain causes disability and distress with a very high economic impact. In the last decades, an impressive amount of pharmacological agents have been tested for the treatment of endometriosis-associated pelvic pain. However, only a few of these have been introduced into clinical practice. Following the results of the controlled studies available, to date, the first-line treatment for endometriosis associated pain is still represented by oral contraceptives used continuously. Progestins represent an acceptable alternative. In women with rectovaginal lesions or colorectal endometriosis, norethisterone acetate at low dosage should be preferred. GnRH analogues may be used as second-line treatment, but significant side effects should be taken into account. Nonsteroidal anti-inflammatory drugs are widely used, but there is inconclusive evidence for their efficacy in relieving endometriosis-associated pelvic pain. Other agents such as GnRH antagonist, aromatase inhibitors, immunomodulators, selective progesterone receptor modulators, and histone deacetylase inhibitors seem to be very promising, but there is not enough evidence to support their introduction into routine clinical practice. Some other agents, such as peroxisome proliferator activated receptors-γ ligands, antiangiogenic agents, and melatonin have been proven to be efficacious in animal studies, but they have not yet been tested in clinical studies.

Immediate postpartum perineal examination as a predictor of puerperal pelvic floor dysfunction.

OBJECTIVE To determine the relation between postpartum perineal trauma and the development of puerperal pelvic floor dysfunctions. METHODS A prospective study was conducted on 218 primiparae immediately after vaginal delivery. Women were divided in three groups according to perineal trauma: group A (n = 171) intact perineum or superficial tear, group B (n = 39) perineal muscle tears, group C (n = 8) anal sphincter tears with or without disruption of the rectal mucosa. Two months later, each woman was questioned about urogynecologic symptoms and examined by digital test, vaginal perineometry, and uroflowmetric stop test score. RESULTS No significant difference was found among the groups with regard to the incidence of stress incontinence, frequency/urgency, and urge incontinence, whereas anal incontinence was found more commonly in group C (group C versus group A: P =.003, odds ratio 18.78). No significant difference was found for digital test, perineometry, and uroflowmetric stop test. CONCLUSION Immediate postpartum perineal examination is not a good predictor of stress incontinence and pelvic floor weakness but could identify women at risk for anal dysfunctions: intact perineum does not exclude the appearance of symptoms related to perineal trauma after vaginal delivery.

Leukocytospermia and sperm preparation - a flow cytometric study

BackgroundLeukocytes represent the predominant source of reactive oxygen species both in seminal plasma and in sperm suspensions and have been demonstrated to negatively influence sperm function and fertilization rate in assisted reproduction procedures. Peroxidase test is the standard method recommended by WHO to detect semen leukocytes but it may be inaccurate. The aims of this study were (i) to compare the efficiency of swim-up and density-gradient centrifugation techniques in removing seminal leukocytes, (ii) to examine the effect of leukocytes on sperm preparation, and (iii) to compare flow cytometry and peroxidase test in determining leukocyte concentration in semen using a multiparameter flow cytometric method.MethodsSemen samples from 126 male partners of couples undergoing infertility investigations were analyzed for leukocytospermia using standard optical microscopy and flow cytometry. Sixty-nine out of 126 samples were also processed using simultaneously the swim-up and density-gradient centrifugation techniques. A multiparameter flow cytometric analysis to assess simultaneously sperm concentration, sperm viability, sperm apoptosis, and leukocyte concentration was carried out on neat and prepared sperm.ResultsBoth sperm preparation methods removed most seminal leukocytes. However, the concentration of leukocytes was significantly lower after swim-up compared to that after density-gradient centrifugation preparation. Leukocytes concentration, either initial or in prepared fractions, was not correlated with sperm parameters (optical microscopy and flow cytometry parameters) after semen processing. There was no correlation between leukocyte concentration in the ejaculate and sperm recovery rate, whereas a significant correlation was found between the concentration of the residual leukocytes in prepared fractions and viable sperm recovery rate. Although the overall concordance between the flow cytometry and the optical microscopy was satisfactory, the sensitivity of peroxidase test for the detection of leukocytospermia resulted low.ConclusionSeminal leukocytes do not seem to influence sperm preparation results. However, for assisted conception, semen samples containing leukocytes should be processed using swim-up method. Although peroxidase-test is recommended by WHO as the standard method for determining semen leukocytes, it should not be used in clinical research study.

The urine stream interruption test and pelvic muscle function in the puerperium

Objectives: This study assesses the role of the uroflowmetric urine stream interruption test (UST) in the evaluation of postpartum pelvic floor muscle function. Method: Two months after vaginal delivery, 492 women who underwent a digital test, vaginal manometry, and a UST were divided into two groups: continent and incontinent. Variables were subjected to the Students t‐test and to Fishers exact test to verify the difference between the two groups. Results: Digital test and vaginal manometry results were higher in the continent group, but only the UST showed significantly different values (P=0.001). All test results of incontinent puerperae who underwent rehabilitation were significantly improved after treatment. Conclusions: UST is low cost, non‐invasive, and can give objective information about pelvic floor performance after a vaginal delivery. It can be used for both routine clinical use and an outcome measure for women who undergo rehabilitation treatment.

A simple method for fallopian tube sperm perfusion using a blocking device in the treatment of unexplained infertility

OBJECTIVE To evaluate the efficacy of fallopian sperm perfusion (FSP) using a new method similar to the FAST system in comparison with standard intrauterine insemination (IUI) in patients with unexplained infertility. DESIGN Prospective, randomized, controlled study. SETTING Assisted conception service in a University Hospital. PATIENT(S) Women with unexplained infertility undergoing controlled ovarian hyperstimulation (COH). INTERVENTION(S) After hCG administration, patients were randomized to either standard IUI or FSP. The women received the same treatment in the first and all subsequent cycles. A maximum of three cycles was performed. Intrauterine insemination was performed using a standard method, and fallopian sperm perfusion was performed using a commercial device for hysterosalpingography and tubal hydropertubation. MAIN OUTCOME MEASURE(S) Clinical and ongoing pregnancy rates. RESULT(S) A total of 132 cycles was completed: 66 IUI cycles and 66 FSP cycles. In the IUI group, there were 5 ongoing pregnancies, giving a pregnancy rate of 7.6 per cycle and 15.6% per patient; in the FSP group, 14 ongoing pregnancies occurred, giving a pregnancy rate of 21.2% per cycle and 42.4% per patient. The prevalence of multiple pregnancies, miscarriages and ectopic pregnancies was similar in the two insemination groups. Fallopian sperm perfusion was easy to perform, and no case of sperm reflux was observed. The procedure was well tolerated and no complications were observed. The costs were comparable with standard IUI. CONCLUSION(S) In the treatment of couples with unexplained infertility, the method for fallopian sperm perfusion described yields higher pregnancy rates than IUI, with no significant increase in costs or complications. However, these results need to be confirmed in larger studies before replacing IUI with FSP as standard practice.

Heparin's ‘potential to improve pregnancy rates and outcomes’ is not evidence-based

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Fas receptor is not present on ejaculated human sperm

BACKGROUND Apoptosis appears to have an essential role in the control of testis germ cell number and Fas expression has been reported in apoptotic spermatocytes and spermatids. We investigated if Fas (CD95) was present on ejaculated human sperm and any relationship between Fas on sperm and the apoptotic marker Syto16. METHODS Semen samples from 77 male partners of infertile couples were evaluated. Each sample was analysed both before and after semen preparation by conventional microscopical procedures and by flow cytometry (FC). A multiparameter FC analysis to assess simultaneously sperm concentration, sperm viability, sperm apoptosis, CD45 positive (leukocyte) and CD95 (Fas) positive cell concentration was carried out. A further 10 samples were studied by indirect immunofluorescence to confirm results. RESULTS The mean concentration of CD95 positive cells was very low (<1%), with no significant difference between normozoospermic and non-normozoospermic men. There was no correlation between apoptotic sperm and CD95 positive cell concentration. A linear correlation was found between CD95 positive cell and leukocyte (CD45 positive) concentration (r = 0.9946, P < 0.0001). CD95 mean fluorescence intensity of leukocytes was 10-fold greater than that of sperm and of isotypic control. Both incubation with activating anti-Fas antibody and betulinic acid induced apoptosis in leukocytes. Incubation with betulinic acid, but not with activating anti-Fas antibody, induced apoptosis in sperm. Pre-incubation with neutralizing anti-Fas antibody suppressed CD95 expression on leukocytes, whereas it did not change sperm CD95 peak fluorescence. CONCLUSIONS There is no detectable quantity of Fas on human ejaculated sperm.