Stefania Luppi

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.

Comparative analysis of the seminal plasma proteomes of oligoasthenozoospermic and normozoospermic men

A comparative proteomic study of oligoasthenozoospermic and normozoospermic seminal plasmas was conducted to establish differences in protein expression. Oligoasthenozoospermia (when semen presents with a low concentration and reduced motility of spermatozoa) is common in male infertility. Two-dimensional protein maps from seminal plasma samples from 10 men with normozoospermia and 10 men with idiopathic oligoasthenozoospermia were obtained by isoelectric focusing followed by sodium dodecyl-sulphate polyacrylamide electrophoresis. Map images were analysed using dedicated software involving normalization, spot-to-spot volume comparison and statistical treatment of the results to establish the significance of differences between normal and oligoasthenozoospermic samples. Six out of 1028 spots showed over 1.5-fold relative intensity differences (P < 0.05, analysis of variance). Four proteins were identified by nano liquid chromatography-electrospray ionization-mass spectrometry/mass spectrometry of their tryptic peptides and database searches. Two proteins were more than three-fold under-expressed in oligoasthenozoospermia, namely epididymal secretory protein E1 and galectin-3-binding protein; the other (lipocalin-1 and a prolactin-inducible protein form) were over-expressed. The identity and differential expression of epididymal secretory protein E1 was verified by Western-blotting. The statistically significant differential expression of these four proteins in oligoasthenozoospermia compared with normozoospermia provides a molecular basis for further investigations into the pathogenic mechanisms underlying idiopathic oligoasthenozoospermia.

Association between the JC Polyomavirus Infection and Male Infertility

In recent years the incidence of male infertility has increased. Many risk factors have been taken into consideration, including viral infections. Investigations into viral agents and male infertility have mainly been focused on human papillomaviruses, while no reports have been published on polyomaviruses and male infertility. The aim of this study was to verify whether JC virus and BK virus are associated with male infertility. Matched semen and urine samples from 106 infertile males and 100 fertile males, as controls, were analyzed. Specific PCR analyses were carried out to detect and quantify large T (Tag) coding sequences of JCV and BKV. DNA sequencing, carried out in Tag JCV-positive samples, was addressed to viral protein 1 (VP1) coding sequences. The prevalence of JCV Tag sequences in semen and urine samples from infertile males was 34% (72/212), whereas the BKV prevalence was 0.94% (2/212). Specifically, JCV Tag sequences were detected in 24.5% (26/106) of semen and 43.4% (46/106) of urine samples from infertile men. In semen and urine samples from controls the prevalence was 11% and 28%, respectively. A statistically significant difference (p<0.05) in JCV prevalence was disclosed in semen and urine samples of cases vs. controls. A higher JC viral DNA load was detected in samples from infertile males than in controls. In samples from infertile males the JC virus type 2 strain, subtype 2b, was more prevalent than ubiquitous type 1. JCV type 2 strain infection has been found to be associated with male infertility. These data suggest that the JC virus should be taken into consideration as an infectious agent which is responsible for male infertility.

The application of scanning near field optical imaging to the study of human sperm morphology

BackgroundThe morphology of spermatozoa is a fundamental aspect to consider in fertilization, sperm pathology, assisted reproduction and contraception. Head, neck, midpiece, principal and terminal part of flagellum are the main sperm components to investigate for identifying morphological features and related anomalies. Recently, scanning near-field optical microscopy (SNOM), which belongs to the wide family of nanoscopic techniques, has opened up new routes for the investigation of biological systems. SNOM is the only technique able to provide simultaneously highly resolved topography and optical images with a resolution beyond the diffraction limit, typical of conventional optical microscopy. This offers the advantage to obtain complementary information about cell surface and cytoplasmatic structures.ResultsIn this work human spermatozoa both healthy and with morphological anomalies are analyzed by SNOM, to demonstrate the potentiality of such approach in the visualization of sperm morphological details. The combination of SNOM topography with optical (reflection and transmission) images enables to examine typical topographic features of spermatozoa together with underlying cytoplasmic structures. Indeed the head shape and inner components as acrosome and nucleus, and the organization of mitochondria in the midpiece region are observed. Analogously for principal tract of the tail, the ridges and the columns are detected in the SNOM topography, while their internal arrangement can be observed in the corresponding SNOM optical transmission images, without requiring specific staining procedures or invasive protocols.ConclusionsSuch findings demonstrate that SNOM represents a versatile and powerful tool to describe topographical and inner structural details of spermatozoa simultaneously. This analysis could be helpful for better characterizing several morphological anomalies, often related to sperm infertility, which cannot be examined by conventional techniques all together.

Pitfalls and promises in FTIR spectromicroscopy analyses to monitor iron-mediated DNA damage in sperm

Many drugs, chemicals, and environmental factors can impair sperm functionality by inducing DNA damage, one of the important causes of reduced fertility potential. The use of vibrational spectromicroscopy represents a promising approach for monitoring DNA integrity in sperm, although some limitations exist, depending from the experimental conditions. Here, we report that when using FTIR spectromicroscopy to reveal oxidative stress mediated by Fentons reaction on hydrated sperm samples, DNA damage interpretation is partially compromised by unexpected cell surface precipitates. The precipitates give a broad band in the 1150-1000cm(-1) infrared region, which partially covers one of the signatures of DNA (phosphate stretching bands), and are detected as iron and oxygen containing material when using XRF spectroscopy. On the other hand, the analyses further support the potential of FTIR spectromicroscopy to reveal cellular oxidative damage events such as lipid peroxidation, protein misfolding and aggregations, as well as DNA strain breaks.

Effect of seminal leukocytes on in vitro fertilization and intracytoplasmic sperm injection outcomes

OBJECTIVE To investigate the influence of seminal leukocytes on conventional IVF and intracytoplasmic sperm injection (ICSI) outcomes, using a flow cytometry method. DESIGN Prospective study. SETTING Tertiary infertility center and research institute. PATIENT(S) One hundred sixty-four couples undergoing conventional IVF or ICSI. INTERVENTION(S) Seminal leukocytes were counted by flow cytometry. MAIN OUTCOME MEASURE(S) Correlation between seminal leukocytes concentration and reproductive outcomes in IVF and ICSI cycles. RESULT(S) The median number of oocytes retrieved, the fertilization and cleavage rate, the median number and grade of embryos transferred, the median number of good-quality embryos transferred, and the median percentage of good-quality embryos from total embryos transferred, in leukocytospermic and non-leukocytospermic patients were not statistically different after either IVF or ICSI. Similarly, there were no significant differences between the two groups for implantation rate and clinical pregnancy rate. Multivariate logistic regression analysis showed that the reproductive outcomes were not influenced by adjustment for female age, infertility diagnosis, number of previous attempts, treatment protocol (GnRH agonist or antagonist), assisted reproduction procedure (IVF or ICSI), and leukocytospermia. By profiling the proper Poisson regression models, no leukocytospermia cut-off value was able to identify the subjects at risk for oocyte fertilization or embryo cleavage failure. CONCLUSION(S) Using a flow cytometry method, we demonstrated that leukocytospermia does not significantly influence IVF or ICSI outcomes. The same results were obtained by using lower or higher cut-off values for leukocytospermia (from 0.2 to 2 × 10(6)/mL).

Comparative proteomic analysis of spermatozoa isolated by swim-up or density gradient centrifugation

BackgroundReports about the morphologic and functional characteristics of spermatozoa prepared by density gradient centrifugation (DC) or swim-up (SU) have produced discordant results. We have performed a proteomic comparison of cells prepared by DC and SU providing a molecular insight into the differences between these two methods of sperm cell isolation.MethodsProtein maps were obtained by 2-dimensional (2-D) separations consisting of isoelectrofocusing (IEF) from pI 3 to 11 followed by SDS-polyacrylamide gel electrophoresis. 2-D gels were stained with Sypro Ruby. Map images of DC and SU spermatozoa were compared using dedicated software. Intensities of a given spot were considered different between DC and SU when their group mean differed by >1.5-fold (p < 0.05, Anova).ResultsNo differences were observed for 853 spots, indicating a 98.7% similarity between DC and SU. Five spots were DC > SU and 1 was SU > DC. Proteins present in 3 of the differential spots could be identified. One DC > SU spot contained lactate dehydrogenase C and gamma-glutamylhydrolase, a second DC > SU spot contained fumarate hydratase and glyceraldehyde-3-phosphate dehydrogenase-2, and a SU > DC spot contained pyruvate kinase M1/M2.ConclusionsThe differences in protein levels found on comparison of DC with SU spermatozoa indicate possible dissimilarities in their glycolytic metabolism and DNA methylation and suggest that DC cells may have a better capacitation potential.

Ultrastructural Morphology of Sperm from Human Globozoospermia.

Globozoospermia is a rare disorder characterized by the presence of sperm with round head, lacking acrosome. Coiling tail around the nucleus has been reported since early human studies, but no specific significance has conferred it. By contrast, studies on animal models suggest that coiling tail around the nucleus could represent a crucial step of defective spermatogenesis, resulting in round-headed sperm. No observations, so far, support the transfer of this hypothesis to human globozoospermia. The purpose of this work was to compare ultrastructural morphology of human and mouse model globozoospermic sperm. Sperm have been investigated by using scanning and transmission electron microscopy. The images that we obtained show significant similarities to those described in GOPC knockout mice, an animal model of globozoospermia. By using this model as reference, we were able to identify the probable steps of the tail coiling process in human globozoospermia. Although we have no evidence that there is the same pathophysiology in man and knocked-out mouse, the similarities between these ultrastructural observations in human and those in the experimental model are very suggestive. This is the first demonstration of the existence of relevant morphological homologies between the tail coiling in animal model and human globozoospermia.

Thrombogenesis in Thrombophilic Pregnancy: Evaluation of Low-Molecular-Weight Heparin Prophylaxis

The aim of this study is to investigate thrombogenesis and the hypercoagulable changes in pregnant women affected by thrombophilia who received low-molecular-weight heparin (LWMH) prophylaxis. We included 21 pregnant women affected by thrombophilia treated with LWMH and 20 nontreated normal pregnant women as the control group. The sample group of thrombophilic pregnant women included different conditions (factor V Leiden mutation, protein C deficiency, protein S deficiency, antiphospholipid antibodies syndrome, and combined defects). Three blood samples were collected during pregnancy (i.e., at 16, 20, and 24 weeks) and tested for activated partial thromboplastin time and prothrombin fragment F1 + 2 (F1 + 2); anti-FXa activity was tested only in treated thrombophilic pregnant women. F1 + 2 levels progressively increased during pregnancy in both study groups. However, the F1 + 2 increase in women exposed to heparin prophylaxis was significantly lower than that in normal pregnant women in all 3 measurements carried out during gestation (p < 0.05); a statistically significant inverse correlation between F1 + 2 levels and anti-Xa activity (R = -0.8575, p < 0.05) was observed in treated women during pregnancy. Our findings suggest that F1 + 2 in addition to anti-Xa measurement could be used to adjust LWMH prophylaxis, at least in high-risk pregnant women.