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Purpose of Review: The purpose is to determine the efficiency and efficacy of oocyte cryopreservation by slow freezing versus vitrification, recent data collected from the Italian National Assisted Reproductive Technology Register during the period 2009-2014 will be presented and reviewed. The data on oocyte cryopreservation were also compared with the results obtained with embryo cryopreservation and relative IVF with fresh oocytes., Recent Findings: During the period 2009-2014 preservation of oocytes by vitrification had a significantly higher survival rate, implantation, and pregnancy rate than slow freezing; however, there are still large variations in success rates among centers in relation to the number of procedures performed., Summary: Vitrification has now become the method of choice for oocyte cryopreservation because of better results than slow freezing, but still requires a more standardized utilization. The transfer of fresh or cryopreserved embryo still shows a statistically significant better performance than transfers with embryos obtained with cryopreserved oocytes. Only in a few centers with much experience in cryopreservation are the results between transfers of frozen embryos or embryos obtained from oocyte cryopreservation comparable.

Purpose: Our aim was to evaluate the ultrastructure of human metaphase II oocytes subjected to slow freezing and fixed after thawing at different intervals during post-thaw rehydration., Methods: Samples were studied by light and transmission electron microscopy., Results: We found that vacuolization was present in all cryopreserved oocytes, reaching a maximum in the intermediate stage of rehydration. Mitochondria-smooth endoplasmic reticulum (M-SER) aggregates decreased following thawing, particularly in the first and intermediate stages of rehydration, whereas mitochondria-vesicle (MV) complexes augmented in the same stages. At the end of rehydration, vacuoles and MV complexes both diminished and M-SER aggregates increased again. Cortical granules (CGs) were scarce in all cryopreserved oocytes, gradually diminishing as rehydration progressed., Conclusions: This study also shows that such a membrane remodeling is mainly represented by a dynamic process of transition between M-SER aggregates and MV complexes, both able of transforming into each other. Vacuoles and CG membranes may take part in the membrane recycling mechanism., Competing Interests: Compliance with ethical standards All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards. Conflict of interest The authors declare that they have no conflict of interest.

A procedure based on ultra-high-pressure liquid chromatography tandem mass spectrometry has been developed for the determination of twenty three psychoactive drugs and metabolites in whole blood using dried blood spot (DBS). Chromatographic separation was achieved at ambient temperature using a reverse-phase column and a linear gradient elution with two solvents: 0.1% formic acid in acetonitrile and 5mM ammonium formate at pH 3. The mass spectrometer was operated in positive ion mode, using multiple reaction monitoring via positive electro-spray ionization. The method was linear from the limit of quantification (5ng/ml for all the analytes apart from 15ng/ml for Δ-9-tetrahydrocannabinol and metabolites) to 500ng/ml, and showed good correlation coefficients (r(2)=0.990) for all substances. Analytical recovery of analytes under investigation was always higher than 75% and intra-assay and inter-assay precision and accuracy always better than 15%. Using the validated method, ten DBS samples, collected at the hospital emergency department in cases of acute drug intoxication, were found positive to one or more psychoactive drugs. Our data support the potential of DBS sampling for non invasive monitoring of exposure/intoxication to psychoactive drugs., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Introduction: Gender disparity in different fields of addiction such as tobacco smoking, alcohol use, drugs of abuse consumption and doping practice has been investigated in Italian population., Methods: We used the surveys and studies carried out for the above reported issues in recent years as revised by the "National Observatory on Tobacco smoke, Drugs of abuse, Alcohol and Doping" at Istituto Superiore di Sanità., Results: Concerning tobacco habit, the trend of smoking women has been in constant decrease from a 19.7% in 2010 to a 16.9% in 2015, differently from men who passed from a 23.9% in 2010 to a 25.1% in 2015 with a slight increase in the habit. With respect to alcohol, in the last five years an increasing trend of consumption has been observed in 18-24 years old women, with 53% drinking women in the age range of 18-19 years overcoming the 50.4% general female population. Generally speaking, a one to four ratio can be underlined in the percentage of elderly women with a risky alcohol consumption with respect to men, while in case of adolescents and young adults gender disparity is not so pronounced. Drug abuse still remains a prevalent male phenomenon. However, an increase in cannabis users for both genders has been reported with a prevalence of "once in the life" around 20%, although more pronounced in females (+2.66 percentage points for females vs +0.93 percentage points in male). With respect to cocaine, the second most consumed drug, a reduction in consumption has been recently observed mainly in female population (-42.1%) than in men one (-27.5%). Finally, there are significant gender differences in doping attitude and/or in doping profiling. First of all, males seem to be more exposed to doping than females The prohibited substances most frequently used by females athletes are "Diuretics and Masking Agents" (38.3% positive female vs 14% males) compared to males athletes who use mostly anabolic agents (20.1% males vs 11.2% females)., Conclusions: Results presented for the different fields of addiction show that a gender disparity is apparent and that females are less prone in having an addiction behaviour, although the young generation seems to increase that tendency.

Purpose: This study is a retrospective collection of aggregated data from all the Italian ART centers reporting to the Italian National Register from cycles started between January 2005 and December 2013., Methods: Data from both slow freezing (SF) and vitrification (V) were assessed for the period 2007-2013, while during the years 2005-2006 cryopreservation was exclusively performed by SF., Results: In the study period, a total of 2,526,024 oocytes were retrieved (from 378,543 retrievals), of which 1,346,061 (53.3 %) were inseminated in fresh cycles and 214,481 (8.5 %) were cryopreserved. Cryopreserved oocytes were used in 24,173 cycles yielding 19,453 transfer cycles (80.5 % of the thawing/warming cycles) and 3043 clinical pregnancies (15.6 % per transfer). A significant difference in implantation (8.7 vs 12.9 % OR 1.30 CI 1.20-1.40) and pregnancy rates per transfer (12.2 vs 14.9 % OR 1.34 CI 1.23-1.46) was found between SF and V. Complete outcome data was available for 2708 pregnancies (89.8 %), leading to 1882 deliveries and 2152 live births. Neonatal major congenital anomalies were 0.9 % (20/2152)., Conclusions: A wide variation in pregnancy rates were found among different centers and lower rates were reported in donor cycles and in centers with more experience.

Objective: To compare mature human oocytes cryopreservation with slow freezing (SF) and vitrification (VT) in infertile couples., Design: Retrospective study of national Italian data submitted during the period 2007-2011., Setting: National ART registry., Patient(s): Infertile patients with supernumerary oocytes., Intervention(s): Thawing or warming of cryopreserved oocytes and ICSI., Main Outcome Measure(s): oocyte survival, fertilization, implantation and clinical pregnancy rate between SF and VT., Result(s): A total of 14,328 cycles with 11,599 transfers, 1,850 pregnancies, 1,168 deliveries and 1,342 babies born were analyzed from 146 reporting centers (range of cycles 1-1,255 per center). The SF oocytes' survival rate was lower than in VT (51.1% vs. 63.1%). Fertilization rate was significantly higher in SF than in VT (SF 71.6% vs. VT 70.1%). VT showed a significantly higher pregnancy rate, both per started cycle (14.4% vs. 12.0%) and per transfer (18.0% vs. 14.8%), and implantation rate (9.5% vs. 8.1%) than SF. However, the range and median pregnancy rate per started cycle were, respectively, 0%-50% and 7.7% in SF and 0%-100% and 6.7% in VT., Conclusion(s): VT showed a statistically significant higher performance than SF. As with other ART procedures, the results are not homogeneous among clinics and protocols, but the confirm the clinical value of oocyte cryopreservation in infertile patients., (Copyright © 2014 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.)

Platelet-derived growth factor (PDGF) and its receptors (PDGFRs) play a key role in the regulation of the embryonic and postnatal development of male gonads. PDGF deficiency is associated with severe spermatogenic impairment. ACP1 is a phosphoprotein tyrosine phosphatase that is able to dephosphorylate PDGFR, decreasing its activity as growth factor. The enzyme is polymorphic and shows strong differences in enzymatic activity among genotypes. At the Outpatient Department for Infertility, University of Rome La Sapienza, we investigated the effect of high-activity ACP1 genotype on spermatic parameters in 105 subjects referred to for varicocele. ACP1 genotype was determined by DNA analysis. In ACP1 *B/*C genotype, which shows the highest enzymatic activity, spermatic concentration is significantly lower and atypical spermatozoa are significantly more frequent as compared to other ACP1 genotypes. It is concluded that subjects carrying *B/*C genotype who represent about 10% of the population have a severe impairment of spermatic parameters in the presence of varicocele., (© 2012 Blackwell Verlag GmbH.)

Objective: To analyze the fetal and neonatal outcomes of pregnancies achieved with fresh and/or frozen oocytes in the same group of patients., Design: Observational study and comparative analysis., Setting: Research unit of an academic medical center., Patient(s): A group of 855 women with cryopreserved oocytes and their resulting 954 assisted reproductive technology clinical pregnancies were enrolled and followed up during the same time period and in the same clinical setting; the outcomes of 197 pregnancies from frozen/thawed oocytes were compared with 757 obtained from fresh sibling oocyte cycles., Intervention(s): None., Main Outcome Measure(s): Pregnancies were followed until delivery, and neonatal data (up to 28 days after delivery) were collected., Result(s): No significant differences were found between the use of fresh and frozen oocytes in the rates of therapeutic abortions for fetal anomaly (1.5% vs. 0.8%) and ectopic pregnancies (3.6% vs. 2.9%), but a significantly higher rate of spontaneous abortions at ≤ 12 weeks (17.6% vs. 26.9%) was observed in the frozen/thawed oocytes group. No statistical differences were found in major anomalies at birth (2.8% vs. 4.6%). Despite no difference in gestational age at delivery, the mean birth weights were significantly lower with fresh oocyte pregnancies, both in singleton (2,725 ± 727 g) and twins (2,128 ± 555 g), than with frozen-thawed oocytes (3,231 ± 615 g and 2,418 ± 492 g, respectively). However, the analysis of the 63 patients who obtained pregnancies both in fresh and thawed cycles (138 pregnancies) showed no differences in the abortion rate and in the mean birth weight., Conclusion(s): These results provide strong support to the notion that fetal and perinatal complications and congenital anomalies do not differ between pregnancies from frozen-thawed and fresh oocytes. The significantly lower mean birth weight observed with pregnancies from fresh oocytes supports similar observations reported for pregnancies from embryo cryopreservation and requires further prospective studies., (Copyright © 2013 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.)

Aim: The aim of this study was to assess the quantitative and qualitative outcomes of assisted reproductive technologies in Italy, from 2005 to 2007, collected by the Italian Assisted Reproductive Technologies Register (IARTR)., Methods: We analyzed, retrospectively, 121,708 ART treatments performed by Italian assisted reproductive technologies (ART) centres from 2005 to 2007., Results: In the study period ART Italian centers were reporting data to the IARTR. Number of total cycles (all homologous) reported was 121,708 including 110,074 fresh (FRESH), 8682 frozen/thawed oocytes (FO) and 2952 frozen embryo (FER). Pregnancy rates per cycle with respect to FRESH, FO and FER treatments were 19.1%, 10.2% and 15%, respectively. Delivery rate per cycle with at least one live birth was 10.8%, 5.8% and 8.4%, respectively, per type of procedure. Twin and "triplet or more" birth rate per delivery was 21% and 2.8%; 12% and 0.4%; 14.9% and 0.8%, respectively. During the study period 15923 ART infants were born (0.95% of all newborn in Italy)., Conclusion: From 2005 to 2007 we registered a strong increase in ART procedures concomitantly with a qualitative improvement leading to increased pregnancy and live birth rates per cycles. Remarkably, pregnancy loss to follow-up decreased drastically during the three years study-period.

For the first time in Europe hair and urine testing have been applied to assess drugs of abuse consumption in couples undergoing assisted reproductive technology and the eventual association of toxic habits with other lifestyle, health status and sociodemographic factors was also investigated. Couples attending five assisted reproduction centers in Rome were invited to join the study. When they presented at the Centre for the visit, they were asked to answer a structured questionnaire concerning sociodemographic characteristics and lifestyle habits, and at the same time to provide hair and urine samples. Hair and urine testing for drugs of abuse, urinary profile of principal endogenous steroids involved in fertility process (testosterone, epitestosterone, androsterone, etiocholanolone and dehydroepiandrosterone) and of alcohol and tobacco smoke biomarkers were performed with validated methodologies. Of the 594 enrolled individuals (297 couples), 352 (164 couples and 24 single individuals from the couple) completed the questionnaire and gave both hair and urine samples, apart from 3 bald men, who only gave urine samples. Urine testing showed an overall 4.8% (17 individuals) positivity to drugs of abuse: 4.2% to cannabinoids, 1.4% to cocaine and 0.85% to both drugs. Results of 4cm segment hair samples testing matched those from urine samples. Thus, taking together, results of urine and hair testing confirmed repeated use of cannabis, cocaine and both drugs in 3.7, 0.85 and 0.57% examined individuals, respectively. Drug consumers were in a statistically higher percentage active smokers and alcohol drinkers, less prone to physical activity and with a trend towards higher weight than non consumers. Finally, repeated drug consumption was associated with significant lower concentration of urinary testosterone in males and of urinary dehydroepiandrosterone in females. The findings of the present study confirm the suitability of urine testing to disclose recent drugs of abuse consumption and of hair analysis to verify repeated consumption. Association between different toxic habits and sedentary lifestyle is also substantiated by the obtained results in our cohort of couples attending assisted reproduction centers., (Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.)

Birefringence in sperm heads reflects an organized and very compacted texture, indicating nuclear and acrosomal structural normality. This study performed a direct analysis of the acrosome integrity in single spermatozoa to verify whether a pattern of total or partial head birefringence reflected the acrosome status. The morphology in fresh samples was assessed according to World Health Organization criteria while the characteristics of birefringence were evaluated by polarized light. Acrosome integrity was evaluated by fluorescein isothiocyanate Pisum sativum agglutinin that binds selectively to the acrosome content. According to the results, a reacted acrosome was present in 96% of spermatozoa with partial birefringence and only in 35% of those with totally birefringent heads. A great proportion of sperm cells with normal morphology showed total birefringence both in the presence (59%) or in the absence of motility (45%; P < 0.01), while in morphologically abnormal spermatozoa the frequency of total birefringence was comparable to that of partial birefringence irrespective of motility (26% and 27%, respectively, in motile spermatozoa; 22% and 19%, respectively, in immotile spermatozoa). These data support a strong association between partial birefringence and reacted acrosome and show that the patterns of birefringence vary depending on sperm motility and morphology., (Copyright © 2012 Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved.)

The application of the European Tissues and Cells Directive requires that a high standard of quality and safety be applied to all tissue establishments, including that of assisted reproductive centres. In the countries where the Directive has been implemented, changes in the ART clinic and laboratory procedures have been made. However, many requirements stipulated in the Directive are already in place in some European countries and are mandatory by the country specific laws, while some other requirements have been recently implemented. In this article the benefits and the potential risks of the Directive implementation on the efficacy, safety and cost of all the different assisted reproductive technologies performed, including cryopreservation techniques and storage policies have been widely analysed. The feasibility of the implementation of some of the specific requirements when considering the delicate issues of reproductive cells in contrast to other cells or tissues covered by the Directive has been evaluated., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Aim of the Study: To report the frequency of aberrant karyotype and mutated cystic fibrosis transmembrane conductance regulator (CFTR) gene, according to a careful application of Italian guidelines for genetic screening in infertile couple candidates for intrauterine insemination (IUI) and in vitro fertilization/intracytoplasmic sperm injection (IVF/ICSI)., Materials and Methods: Two thousand and sixteen consecutive infertile couple candidates for Assisted Reproduction Techniques (ART) were screened for karyotype and 616 couples for CFTR analysis., Results: Regarding karyotype analysis, 59 chromosomal abnormalities were diagnosed in candidates for IVF/ICSI: 27 mutations in women corresponding to a frequency equal to 1.53% (27/1762; 95% confidence interval [CI], 0.96-2.1%) and 32 mutations in men corresponding to a frequency equal to 1.82% (32/1762; 95% CI, 1.2-2.44%) for men. The frequency differs according to the sperm count. In couple candidates for IUI techniques, no genetic abnormalities were found in male patients and only one aberration in a female patient with a frequency of 0.41% (1/245 CI 0.01-0.81%). Regarding CFTR analysis, excluding the 5T variant, we obtained 20 mutations in couples undergoing IVF/ICSI and 8 mutations in IUI group., Conclusion: Couples undergoing IVF/ICSI show a higher prevalence of aberrant karyotypes than general population, whereas the frequency of a mutation of the CFTR gene is similar. On the other hand, couples undergoing IUI do not differ from the general population either for karyotype or for CFTR mutations.

Background: In May 2009, the Italian Constitutional Court banned most of the limitations of a restrictive law regulating assisted reproduction technology on the grounds that it limited a couple's right to have access to the best possible medical treatment and reduce any possible higher risk of complications. The aim of the study was to compare our results in fresh cycles before and after this change., Materials and Methods: We analysed retrospectively 3274 IVF cycles: 2248 before and 1026 after the law was modified., Results: There was no significant difference between the two groups in terms of age, basal FSH levels, years of infertility, the number of previous cycles or the number of oocytes retrieved but the number of oocytes used (2.7 ± 0.6 versus 4.6 ± 1.8; P = <0.001), the number of embryos obtained (2.0 ± 0.9 versus 3.3 ± 1.8; P = <0.001) and transferred (2.2 ± 0.7 versus 2.3 ± 0.7; P = <0.001) were all higher after the removal of the previous restrictions, as was the pregnancy rate per started cycle (23.49% versus 20.42%; P = 0.047). Before modification of the law, the pregnancies were single in 74.11% of the cases (versus 71.43% afterwards), twins in 23.44% (versus 26.89%; P = 0.318) and triplets in 2.46% (versus 1.68%; P = 0.594)., Conclusions: Our preliminary results after the removal of the previous legal restrictions show a higher pregnancy rate per started cycle (3.7% represents a 15% difference) and a positive (albeit non-significant) trend towards a reduction in the number of multiple pregnancies.

This paper reports on oocyte cryopreservation efficacy in Italy with respect to successful IVF from 2005 to 2007, presenting data from 193 centres collected by the Italian National Register. Post-thawing survival rates, number of transferred embryos, implantation rates and clinical pregnancy rates per transfer with respect to frozen/vitrified oocytes (FVO) were analysed. These numbers were compared with those obtained using frozen embryos or fresh oocytes. A total of 121,708 cycles were initiated, of which, 7.1% (8682) were FVO cycles and 2.4% (2952) were frozen embryo cycles. Of the 81,786 FVO, 52.5% (42,917) were thawed and 26.9% (22,005) inseminated. Of those inseminated, 68.0% (14,966) yielded good embryos. These numbers were significantly lower than those using fresh oocytes in which 77.9% (197,242; fresh oocytes versus FVO P<0.001) of inseminated oocytes generated good embryos. Implantation rate using FVO was 6.9%, which was significantly lower than that using fresh oocytes (13.5%; P<0.001) and frozen embryos (8.8%; P<0.001). Pregnancy rate per transfer using FVO was 12.5% and significantly lower than that using fresh oocytes (24.9%; P<0.001) or frozen embryos (16.4%; P<0.001). There were 505 deliveries after IVF with FVO and 582 babies., (Copyright © 2010 Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved.)

Purpose: To ascertain possible cell damage from cryopreservation, the ultrastructure of human oocytes cryopreserved by slow cooling was assessed., Materials and Methods: Cryopreservation was performed through two protocols with one-step or two-step propanediol. Fresh control oocytes were examined for comparison. Samples were processed for transmission electron microscopy analysis., Results: By light microscopy, both fresh and frozen-thawed oocytes appeared regularly rounded, with intact zona pellucida, and homogeneous cytoplasm. By electron microscopy observation, organelles were abundant and uniformly dispersed. Mitochondria-smooth endoplasmic reticulum associations appeared regular. However, both the amount and density of cortical granules appeared abnormally reduced in frozen-thawed samples. Slight to moderate vacuolization was also found in the ooplasm of oocytes of both frozen groups., Conclusions: Slow cooling ensures a good overall preservation of human oocytes. However, cytoplasmic vacuolization and cortical granule loss appears associated with cryopreservation, irrespective of the protocol used.

Drug abuse is a worldwide phenomenon with significant health and socioeconomic impact and it is of particular concern in women of reproductive age and in pregnant women. We aimed to investigate the prevalence of drug use by serum and hair testing in a cohort of pregnant women at 12th week gestation who decided voluntarily to interrupt their pregnancy and to investigate the relationship between drug exposure and induced abortions (IA), repeated IA and contraception. The study was conducted in an obstetrics clinic authorised to perform IA in Murcia, Spain during an 18 months period (2007-2009). Apart from serum and/or hair testing, the 142 women enrolled in the study completed a detailed questionnaire regarding drug, alcohol and tobacco use in the previous 3 months. Serum and hair samples were analyzed by gas chromatography mass spectrometry assays. Hair and serum samples showed a 30% overall positivity to drugs of abuse. Of these samples, 20.4, 14.1, 4.2 and 1.4% were positive for cannabinoids, cocaine, opiates, and MDMA, respectively, with polydrug use in 5.6% cases. In this cohort, a positive association was found between drug use and repeated IA. The results highlight the need for promoting pregnancy planning for young women in general, especially when consuming psychoactive substances as well as promote safe and accessible contraception in women of reproductive age. In women requesting IA, specific drug abuse counselling should be implemented., (Copyright 2009 Elsevier Ireland Ltd. All rights reserved.)

A simple high-performance liquid chromatography (HPLC) method with ultraviolet diode array (UV-DAD) and electrospray ionisation mass spectrometry (ESI-MS) detection has been developed for the determination of vardenafil, sildenafil, tadalafil, testosterone, procaine, lidocaine, prilocaine, and benzocaine in cosmetic creams sold as promising remedies for male erectile dysfunction and female genitals stimulation. The presence of these substances in commercial cosmetic samples is prohibited. Aliquots (1 g) of the cosmetic creams under investigation were diluted 1:100 in methanol, subjected to ultrasonic treatment, added with benzoic acid as internal standard, and analyzed by HPLC-DAD and HPLC-ESI-MS after a further 1:1000 dilution. The compounds were separated by reversed phase chromatography with water (0.02% trifluoroacetic acid) and acetonitrile gradient elution and detected by UV-DAD at 228, 255 and 290 nm and by ESI-MS positive ionisation mode. Benzoic acid was used as internal standard. Linearity was studied with UV-DAD detection from 2.5-7.8 to 250 microg/g range, depending on the different compounds and with ESI-MS in the 3.3-8.9 to 250 ng/g range. Good determination coefficients (r(2) > or = 0.99) were found in both UV-DAD and ESI-MS. Limits of quantifications ranged between 2.5 and 7.8 microg/g for HPLC-UV-DAD assay and between 3.3 and 8.9 ng/g for HPLC-ESI-MS assay depending on different analyzed substances. At three concentrations spanning the linear dynamic ranges of both UV-DAD and ESI-MS assay, mean recoveries were always higher than 90% for the different analytes and intra-assay and inter-assay precision always better than 15% and 12%. This method was successfully applied to the analysis of substances under investigations present in cosmetic creams, freely sold on the Internet web-sites.

We used hair testing to investigate the prevalence of unsuspected exposure to cocaine in a group of preschool children presenting to an urban pediatric emergency department without signs or symptoms suggestive of exposure. Hair samples were obtained from 90 children between 18 months and 5 years of age attending the emergency room of Hospital del Mar in Barcelona, Spain. In 85 cases, hair samples from the accompanying parent were also provided. The samples were analyzed for the presence of cocaine and benzoylecgonine by gas chromatography-mass spectrometry, which also determined opiates and amphetamines. Parental sociodemographics, possible drug history, and information on the child's features were recorded. Hair samples from 21 children (23.3%) were positive for cocaine (concentration range 0.3-5.96 ng/mg of hair) with 1 sample also positive for 3,4-methylenedioxymethamphetamine and another for opiates. In 88% of the positive cases, cocaine was also found in the hair of the accompanying parent (15 of 17 matched parent-child hair samples). Parental sociodemographics were associated neither with children's exposure to cocaine nor with somatometry of children at birth. However, the behavioral patterns with potential harmful effects for the child's health (eg, tobacco smoking, cannabis, benzodiazepines and/or antidepressants use, and shorter breast-feeding time) were significantly higher in the parents of exposed children. A statistically higher percentage of exposed children were in the lower weight percentile group compared with the nonexposed children. In the light of these results, we advocate general hair screening to disclose exposure to cocaine and other drugs of abuse in children from risky environments, which could provide the basis for specific social and health interventions.

Introduction: For the first time in Europe, the <> aimed to estimate the prevalence of drug use by pregnant women and the subsequent foetal exposure to illicit drugs., Patients and Method: Between October 2002 and February 2004, 1209 mother-infant dyads from the Hospital del Mar, Barcelona, Spain met eligibility criteria and agreed to participate in the study. Data on socio-economic and demographic characteristics and on drug habits during pregnancy were collected using a structured questionnaire. Neonatal meconium was collected within 24h after birth and analyzed by standardized chromatographic techniques for the presence of opiates, cocaine, cannabinoids and amphetamines., Results: Meconium analysis showed an overall 10.9% positivity for drugs of abuse, with a specific prevalence of heroin, cocaine and cannabis with foetal exposure of 4.7, 2.6 and 5.3%, respectively. Structured interviews also revealed that 0.3, 1.2 and 1.5% of mothers used heroin, cocaine and cannabis, respectively, while only one mother declared ecstasy consumption, confirmed by meconium analysis. Parental ethnicity and working class was not associated with drug use. Drug consuming mothers were shown to have a higher number of previous abortions when compared to non-consumer mothers, which was probably due to a lack of family planning. Significantly lower birth weight and length was found in newborns from mothers exposed to cocaine alone or in combination with other drugs., Conclusions: This study, although developed in a low socio-economic-status cohort, may serve as an eye opener for any hidden non-negligible drug consumption during pregnancy. In this sense, meconium analysis can be important to identify neonates with a high suspicion of exposure to drugs of abuse in utero, and provides the basis for appropriate treatment and adequate medical and social follow-up.

The metaphase II (MII) spindle of the human oocyte may be damaged by cryopreservation. High performance confocal microscopy was used to assess meiotic spindle and chromosome organization in oocytes after vitrification by the cryoleaf system. Three hours after retrieval, donor mature oocytes were fixed or vitrified. Vitrification was performed by equilibration in 7.5% ethylene glycol (EG) and 7.5% dimethylsulphoxide (DMSO), transfer to 15% EG, 15% DMSO and 0.5 mol/l sucrose, and loading onto cryoleaf strips. Tubulin staining was found in all survived vitrified-warmed oocytes, the majority (62.8%) of which displayed a bipolar spindle. A normal bipolar spindle configuration and equatorial chromosome alignment was observed only in a part of vitrified-warmed oocytes (32.6%). This frequency was significantly lower in comparison to fresh oocytes (59.1%). In another fraction of vitrified-warmed oocytes (30.2%), spindle bipolarity was associated to one or more non-aligned scattered chromosomes that often appeared tenuously associated with the lateral microtubules of the spindle. Furthermore, in cryopreserved oocytes with a bipolar spindle, a significantly increased pole-to-pole distance (14.9 +/- 2.3 microm) was found in comparison to the fresh control (12.4 +/- 2.6 microm) (P = 0.001). Therefore, under the conditions tested, vitrified-warmed oocytes maintain a MII spindle with a bipolar organization. However, chromosome alignment appears to be partly compromised.

Hyaluronan has recently been employed in the development of a commercial diagnostic kit for assessing sperm maturity, the so-called sperm-hyaluronan-binding assay (HBA). The aim of this study was to evaluate the usefulness of this test, in addition to routine semen analysis, to identify patients with poor reproductive prognosis in conventional IVF. Furthermore, the ability of hyaluronan to select spermatozoa with low DNA fragmentation was investigated. A total of 60 IVF patients were analysed with regard to reproductive outcome, sperm parameters, HBA score and sperm DNA fragmentation. The DNA fragmentation analysis was performed using the terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling assay on the total sperm population and on the hyaluronan-bound spermatozoa obtained from the same samples. No relationship between hyaluronan binding and fertilization, cleavage, good-quality embryos, implantation, clinical pregnancy, miscarriages and biochemical pregnancy rates was found. Otherwise, correlations between spermatozoa hyaluronan binding and morphology (P < 0.01) and a significant difference between DNA fragmentation of the total sperm population and DNA fragmentation of the hyaluronan-bound spermatozoa (P = 0.029) were found. The results underline the ability of hyaluronan to select spermatozoa with higher DNA integrity and morphology. Nevertheless, the clinical value of the HBA in the management of male infertility seems to be limited.

To date, several publications have focused their attention on a new method for observing spermatozoa called 'motile sperm organelle morphology examination' (MSOME), which enables the evaluation of the fine nuclear morphology of motile spermatozoa in real time at high magnification (>x6000). As a consequence, a new microinjection procedure called intracytoplasmic morphologically selected sperm injection (IMSI) has been developed. The aim of the present work is therefore to evaluate the efficacy of the IMSI technique in the light of the current literature, focusing attention on the potential clinical application of the selection of strictly morphologically normal spermatozoa in patients undergoing conventional intracytoplasmic sperm injection treatments. In addition, a brief analysis of preliminary data regarding the relationship between IMSI and assisted reproduction treatment outcome is presented.

This study describes and compares the possible effects of vitrification on the ultrastructural morphology of 20 human mature oocytes vitrified using two different supports, cryoleaf (n = 10) and cryoloop (n = 10). Fresh human mature oocytes (n = 15) were used as controls. Fresh and vitrified-warmed oocytes appeared rounded, with a homogeneous cytoplasm, an intact oolemma and a continuous zona pellucida. Sparse microvacuolization was only occasionally detected in fresh and vitrified-warmed oocytes, to the same extent. About 50% of the vitrified oocytes contained atypical, small and slender mitochondria-smooth endoplasmic reticulum aggregates, whereas a non-homogeneous microvillar pattern was observable in only 30% of the oocytes subjected to vitrification, regardless of the support utilized. Cortical granule content appeared generally reduced after vitrification, but cryoleaf-supported oocytes contained more cortical granules than cryoloop-supported oocytes (P < 0.05). Thus good overall preservation and virtual absence of cytoplasmic vacuolization seem to be the most relevant markers of quality in vitrified-warmed oocytes, using either support. In addition, cryoleaf-supported oocytes retained a higher number of cortical granules than cryoloop-supported oocytes. The variety of ultrastructural alterations recorded emphasizes the need for further studies aimed at assessing the actual tolerance of human oocytes to vitrification.

Over the last few years, there has been renewed interest and scientific debate concerning human oocyte cryopreservation. The aim of this study was to analyse the clinical data coming from our long experience of slow-freezing oocytes. Between 2001 and 2007, 1280 thawing cycles were carried out using oocytes previously frozen by means of a slow 1,2 propaniedol+sucrose protocol. A total of 7585 oocytes were thawed, of which 4409 survived and 3622 were microinjected; 144 clinical pregnancies were obtained. The number of thawing cycles increased from 19 in 2001 to 268 in 2007, and the number of thawed oocytes from 197 to 1652. Although the survival rate was significantly lower in the period 2002-2005 than in the period 2006-2007, pregnancy and implantation rates steadily improved from respectively 6.7% and 2.4% in 2001 to 15% and 8.2% in 2007. Our data demonstrate a clinically important improvement in oocyte crypreservation over the years in a Centres with proved experience, and can be offered as a standard of care not only before cancer treatment but also for couples refusing embryo crypreservation or in countries with very restrictive limitations on embryo or zygote freezing.

In recent years, the evaluation of in utero exposure to drugs of abuse has been achieved by testing biological matrices coming from the fetus or newborn (eg, meconium, fetal hair, cord blood, neonatal urine), the pregnant or nursing mother (eg, hair, blood, oral fluid, sweat, urine, breast milk), or from both the fetus and the mother (placenta, amniotic fluid). Overall, these matrices have the advantage of noninvasive collection (with the exception of amniotic fluid) and early detection of exposure from different gestational periods. Matrices such as amniotic fluid, meconium, fetal hair, and maternal hair provide a long historical record of prenatal exposure to certain drugs and can account for different periods of gestation: amniotic fluid from the early pregnancy, meconium for the second and third trimester of gestation, fetal hair for the third, and finally maternal hair (when long enough) for the whole pregnancy. Placenta may reveal the passage of a substance from the mother to the fetus. Cord blood and neonatal urine are useful for determining acute exposure to drugs of abuse in the period immediately previous to delivery. Drug detection in maternal blood, oral fluid, and sweat accounts only for acute consumption that occurred in the hours previous to collection and gives poor information concerning fetal exposure. Different immunoassays were used as screening methods for drug testing in the above-reported matrices or as unique analytical investigation tools when chromatographic techniques coupled to mass spectrometry were not commonly available. However, in the last decade, both liquid and gas chromatography-mass spectrometric methodologies have been routinely applied after appropriate extraction of drugs and their metabolites from these biological matrices.

A heavy smoking, lactating mother delivered a baby that exhibited spontaneous tremors, fluctuations of muscular rigidity, and opisthotonus at 48 hours of life. Although the symptoms did not disappear within the following days, they could be controlled by swaddling or wrapping the baby in a blanket. The absence of any other etiology generated a suspicion of prenatal exposure to heavy tobacco smoke and potential neonatal nicotine withdrawal syndrome. This diagnosis was supported by extremely high concentration of hair nicotine and cotinine in the infant's hair and in different segments of maternal hair. The presence of non-negligible amounts of nicotine and cotinine in breast milk confirmed that the mother did not quit smoking after delivery, despite her reports. The breast-fed newborn continued to have 3 to 4 crises of spontaneous tremors and alternant muscular rigidity per day for a month. More studies are needed to establish neonatal nicotine withdrawal.

Aims: To identify possible work related sources of psychosocial stress in order to develop pertinent questionnaire items for a cross-sectional health survey of 3000 Italian women flight attendants, and to solicit suggestions on improving survey participation., Methods: Qualitative study in which 26 current and former women flight attendants participated: three focus groups of 6-7 participants each and six in-depth individual interviews. The session themes included positive and negative aspects of the job; relationships with colleagues, superiors, and passengers; perception of occupational risk for serious diseases; compatibility of work and family; and experiences of work related sources of stress and their effect on health. A transcript based analysis of the focus groups and interviews was used to identify emerging themes related to risk factors for mental health problems., Results: The participants indicated that mental health was a major concern. Several work related risk factors possibly related to adverse outcomes, such as depression and anxiety, were highlighted. These included isolation and solitude, fears of being inadequate partners and mothers due to job demands, passenger relationships, and lack of protection by employers with respect to workplace exposures and violent passengers. The information gained was used to develop a mental health module for inclusion in the health survey questionnaire which included questions on history of severe depression or anxiety, suicidal ideation or attempt, substance abuse, workplace sexual harassment, social support, leisure time activities, relationship with a partner, and role as mother., Conclusions: Employing qualitative methods to identify work related sources of psychosocial stress enabled development of pertinent questionnaire items for a cross-sectional epidemiological study of women flight attendants. Follow up qualitative research may be necessary in order to put the cross-sectional study findings into context and to explore actions or strategies for preventing work related health problems evidenced from the survey.

Vaginal isolates of Candida albicans from human immunodeficiency virus-positive (HIV+) and HIV- women with or without candidal vaginitis were examined for secretory aspartyl proteinase (Sap) production in vitro and in vivo and for the possible correlation of Sap production with pathology and antimycotic susceptibility in vitro. HIV+ women with candidal vaginitis were infected by strains of C. albicans showing significantly higher levels of Sap, a virulence enzyme, than strains isolated from HIV+, C. albicans carrier subjects and HIV- subjects with vaginitis. The greater production of Sap in vitro was paralleled by greater amounts of Sap in the vaginal fluids of infected subjects. In an estrogen-dependent, rat vaginitis model, a strain of C. albicans producing a high level of Sap that was isolated from an HIV+ woman with vaginitis was more pathogenic than a strain of C. albicans that was isolated primarily from an HIV-, Candida carrier. In the same model, pepstatin A, a strong Sap inhibitor, exerted a strong curative effect on experimental vaginitis. No correlation was found between Sap production and antimycotic susceptibility, as most of the isolates were fully susceptible to fluconazole, itraconazole, and other antimycotics, regardless of their source (subjects infected with strains producing high or low levels of Sap, subjects with vaginitis or carrier subjects, or subjects with or without HIV). Thus, high Sap production is associated with virulence of C. albicans but not with fungal resistance to fluconazole in HIV-infected subjects, and Sap is a potentially new therapeutic target in candidal vaginitis.

Epidemiological studies have demonstrated that cervical HPV infection and precancerous lesions of the cervix are more common in HIV-seropositive patients. However little is known about the natural history of these lesions in this population. In the present study cervical smears from 36 patients, 18 HIV-seropositive women and 18 matched controls were evaluated with the aim of quantifying morphological alterations and to evaluate DNA ploidy and HPV subtypes. Cervical lesions in HIV-seropositive patients were diploid in 50% of the cases compared to 25% in controls. The only HPVs identified by ISH were types 16/18 and no significant differences were observed in the control population. In contrast, cytological evidence of HPV infection and dysplastic changes was greatly increased in smears from HIV patients compared to HIV-seronegative women. Less than 5% of the cells showed HPV associated changes in controls while 10% to 30% of the cells were affected in HIV-patients. We suggest that the Papanicolaou test should be effective for detecting cervical disease and for a close follow-up of this population. Moreover, while additional studies with larger population groups and different population bases are needed, these findings are suggestive of the possible use of morphological criteria for the identification of HIV-seropositive subjects.

In view of the increasing heterosexual spread of HIV and reports of interventions which reduce the rate of mother to child transmission, a postal questionnaire survey was performed to determine the strategies for the management of pregnancy and delivery in HIV-infected women in 56 obstetric centres in 21 European countries. A response rate of 96% was achieved. Antenatal testing for HIV infection was routinely offered in 96% of centres. Pre- and post-test counselling was available in most centres and was mainly provided by the obstetrician; only 12 centres had trained counsellors. Compared to uninfected women, infected women made more antenatal visits in 36% of centres and had more screening tests in 30% of centres. Some delivery procedures (e.g. use of scalp electrodes) were less common in infected than in uninfected women. Nearly half the centres prescribe Zidovudine to infected women; most prescribed it specifically to reduce HIV vertical transmission. Disinfection of the birth canal and elective caesarean sections for infected women were routinely performed in 22% and 18% of centres, respectively. The survey revealed different approaches to the management of infected pregnant women both between and within countries, which may partly be due to limited knowledge of what constitutes optimal management, especially in relation to interventions to reduce vertical transmission. Randomised control trials are required to establish the most effective intervention.

Objective: To investigate the risks of post-operative complications in HIV-positive mothers who undergo a caesarean section (CS) because the delivery cannot be safely accomplished by the vaginal route or to protect the infant from viral infection., Design: In a multicentre study, we reviewed the incidence and type of post-operative complications in 156 HIV-positive women who underwent a CS. These results were compared with those observed in an equal number of HIV-uninfected women who matched for the indication requiring a caesarean delivery, the stage of labour, the integrity or rupture of membranes, and the use of antibiotic prophylaxis., Setting: Seven teaching hospitals providing obstetrical care for mothers infected with HIV., Results: We found that six HIV-infected mothers suffered a major complication (two cases of pneumonia, one pleural effusion, two severe anaemia and one sepsis) compared with only one HIV-negative woman who required blood transfusion after surgery. Minor complications like post-operative fever, endometritis, wound and urinary tract infections were significantly more frequent in HIV-positive women than controls. Multivariate analysis revealed that in HIV-infected women the only factor associated with a significant increase in the rate of complications was a CD4 lymphocyte count < 200 x 10(6)/l., Conclusions: The results of our study indicate that HIV-positive mothers are at an increased risk of post-operative complications when delivered by CS. The risk of post-operative complications is higher in HIV-infected women who are severely immunodepressed.