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Since the end of February 2020, SARS-CoV-2 dramatically spread in Italy. To ensure that most of National Health System (NHS) resources were employed to control the pandemic, non-urgent medical procedures (including IVF) were suspended in March 2020. Here, we aimed at assessing the impact of the restrictive measures on Italian IVF activity.In May 2020, the Italian ART Register launched an online survey (multiple choices and open answers) across ART centers (89.0% response rate; N = 170/191) to investigate how they were facing the emergency and estimate the reduction in their activity. In February 2022, the official data of the whole 2020 were published and retrospectively analyzed. The ART cycles conducted in Italy in 2020 (67,928 by 57,423 patients) were then compared to those conducted in 2019 (82,476 by 67,633 patients). The estimates formulated through the survey were compared to the actual reduction.In 2020, 14,548 less IVF cycles were conducted with respect to 2019 (- 17.6% reduction). This led to 2539 fewer live births (- 19.8%) than 2019. If the reduction unveiled by the survey launched in May 2020 (i.e., - 35%) would have persisted throughout 2020, a significantly larger impact was expected (4200 less newborns). Instead, the activity was gradually recovered, and it compensated the months of greatest emergency, thus fulfilling the most optimistic scenario.Italy suffers from the lowest birth rate in Europe, and COVID-19 impact on IVF-derived live births testified how key ART is for Italian demographics. The government should support access to these treatments with dedicated actions.

Purpose Infertility is increasing worldwide, and many couples seek IVF. Clinical management and laboratory work are fundamental in the IVF journey. Therefore, the definition of reliable key performance indicators (KPIs) based on clinical and laboratory parameters, is essential for internal quality control (IQC). Laboratory performance indicators have been identified and a first attempt to also determine clinical ones has been recently published. However, more detailed indicators are required. Methods An Italian group of experts in Reproductive Medicine from both public and private clinics on behalf of SIFES-MR and SIERR was established to define IVF indicators to monitor clinical performance. Results The working group built a consensus on a list of KPIs, performance indicators (PIs) and recommendation indicators (RIs). When deemed necessary, the reference population was stratified by woman age, response to ovarian stimulation and adoption of preimplantation genetic testing for aneuploidies (PGT-A). Each indicator was scored with a value from 1 to 5 and a weighted average formula – considering all the suggested parameters—was defined. This formula generates a center performance score, indicating low, average, good, or excellent performance. Conclusion This study is intended to provide KPIs, PIs and RIs that encompass several essential aspects of a modern IVF clinic, including quality control and constant monitoring of clinical and embryological features. These indicators could be used to assess the quality of each center with the aim of improving efficacy and efficiency in IVF.

Infertility may have a very strong emotional impact on individuals, requiring adequate support, but few studies on patients' demands toward psychological support have been conducted. This study aims to explore the emotions related to the infertility and to the Assisted Reproductive Technology (ART) procedure for which patients consider useful a psychological support. A total of 324 women completed a sociodemographic and clinical questionnaire and an open-ended questionnaire on emotional needs for psychological support. The written texts were explored by the Linguistic Inquiry and Word Count (LIWC) programme and linguistic characteristics were related to sociodemographic and anamnestic variables. Specific linguistic features were connected to several individual characteristics. More specifically, differences in linguistic processes emerged comparing women with an age over or under 40 years, women undergoing their first attempts versus more attempts, women undergoing ART with or without gamete donation, and women undergoing ART for male or unknown causes, as well as those undergoing ART for female or both partners’ problems. These differences seem to confirm that older age, more attempts, gamete donation, and ART for unknown or male causes are risk factors that may worsen women's psychological well-being. This study contributes to increase the knowledge about the emotional needs of patients undergoing an ART procedure to develop specific psychological intervention programs.

Infertility has an impact on the psychophysical health of individuals and couples, and its treatment through an assisted reproductive technology (ART) is a very exacting experience. The aim of this study was to explore the characteristics of psychological counselling services in Italian ART centres through a specially designed questionnaire administered to the physicians in charge of the centres.The questionnaire online was sent to 306 ART authorized centres. It consisted of 26 questions. A total of 15 were single-selection questions, 5 were multiple-choice and the remaining 6 were open-ended.113 (37%) ART centres responded to the questionnaire. All the ART centres offered psychological counselling, but only in 47% of them the psychologist is a permanent staff member. In 69% of the centres, 10 to 20% of couples use the psychological counselling service.Although the Italian Guidelines under Law 40/2004 stress the need to provide couples with psychological support and counselling and the literature highlights the efficacy of such interventions, the results of our study show that psychological counselling services are not yet fully operational in Italian ART centres or integrated into their day-to-day medical practice.In conclusion, the results show that psychological services in Italian ART centres are not yet fully operational and integrated in the ART procedure. All of this highlights the importance of further investigations with the aim to shared information to use to establish common protocols for psychological interventions in ART centres.

Hereditary cancer syndromes are a heterogeneous group of genetic conditions that are associated with an increased risk of developing cancer during lifespan. In affected women, parenthood may be accompanied by concerns for the offspring, considering the common autosomal dominant inheritance. Moreover, fertility preservation to prevent the detrimental effects of cancer treatments differs compared to other clinical contexts. The necessity to preserve gametes is indeed predictable and expected to be common. For these reasons, we advocate a personalized and early fertility counseling. Carriers should be aware of the risk of transmission. The possibility to perform elective oocytes cryopreservation, either before (previvors) or after (survivors) cancer diagnosis should be discussed. Finally, they should be informed about the options of preimplantation genetic test (PGT) and oocytes donation. In conclusion, physicians engaged in oncofertility should personalize the counseling for women with hereditary cancer syndromes, being aware of their peculiar needs.

Purpose The risk of monozygotic twins (MZTs) is increased in couples undergoing assisted reproductive technology (ART) treatments. Several systematic reviews have investigated the possible determinants linked to ART, but results obtained have not been conclusive. The study aims to investigate whether the incidence of MZT differed among ART centers. Methods This is a multicenter retrospective cohort study using the Italian ART National Registry database and involving the centers reporting data from individual ART cycles from 2015 to 2019. To investigate the incidence of MZT, only single embryo transfer cycles were considered. Women who had sex-discordant deliveries were excluded. MZT rate was calculated as the number of multiple pregnancies (more than one gestational sac at first ultrasound) out of the total number of clinical pregnancies. A binomial distribution model was used to determine the 95% CI of the frequency of MZT. Results Eighteen centers were included, and they provided data on 10,433 pregnancies. The total number of MZT was 162, corresponding to an incidence of 1.5% (95% CI: 1.3-1.8%). The rate of MZT among centers varied between 0% (95% CI: 0.0-25.9%) and 3.2% (95% CI: 1.3-8.1%). All the 95% CIs included 1.5%, rejecting the hypothesis that the MZT rate may significantly differ among centers. Conclusions The rate of MZT did not significantly vary among ART centers. Local factors are unlikely to explain the increased rate of MZT in ART pregnancies.

Study question What are the characteristics of the psychological services for couples undergoing ART treatment in Italy? Summary answer In the 47% of ART clinics the psychologist is a stable member of the team whereas psychological services are used by 10–20% of couples. What is known already: ART treatments are complex and emotionally demanding for both men and women. During the procedure the couples often experience stress and several negative emotions. In Italy the Law 40/2004 establishes the guidelines for ART application. This law sustains the importance of offering psychological support to the people who undergo ART treatments at any stage of the medical procedure. However, in Italy there are no specific recommendations or protocols for psychological interventions in ART centres. Furthermore, there is little or no studies regarding the characteristics of the psychological services offered to those undergoing ART treatments. Study design, size, duration This observational study aims to explore the characteristics of psychological services in ART clinics. Therefore, the ART Italian National Register (National Health Institute) with the Department of Dynamic and Clinical Psychology and Health study (Sapienza University) proposed a national survay to the 306 Italian ART centres. The Medical Director of each centre was invited to participate by e-mail and to fill a questionnaire on an on-line platform. Data collection was between Jenuary to February 2020. Participants/materials, setting, methods The participation has been proposed to all the Italian ART centres (n = 306). 35 centres have been previously excluded because participating in the first phase of the study regarding the questionnaire construction. Around the 40% of the centres participated in the study (n = 113). The questionnaire contains 26 items with multiple response answers. It mainly explored: the presence of the psychologist in the ART centres, charachteristics of the intervention offered, percentage of couples using the psychological service. Main results and the role of chance In the 47% of ART centres the psychologist is a stable member of the team. The 38% of respondents reported that the psychologist works on call in the centre, the 18% reported that the psychologist is present in the centre 1–2 times a week whereas the 32% reported that the psychologist works in a private practice outside the centre. The reported percentages of couples using the psychological service are: 10–20% (69%), 20–30% (13%), 30–50% (5%), more than 50% (5%) and around the 100% (8%). The number of psychological sessions usually offered are: up to five (9%), four (13%), three (29%), two (27%), one (10%). Couples interventions represent the 73%, single patient intervention the 14%, whereas the 13% are group interventions. The 60% of respondent reported the absence of a protocol for psychological intervention in the ART centre whereas the 40% reported the presence of a protocol. In around the 50% of cases the psychological service represents an extra cost for the couple/individual. In the 60% of cases the psychologist is not involved in the team meeting, however the 87% of respondents reported that confronting with the psychologist on patients is percived as useful. Limitations, reasons for caution These results should be considered with cautions due to the rate of participation (around 40%). Furthermore, this is a national study and the charachteristics of psychological services offered in ART centres of different countries should be investigated. Wider implications of the findings: In conclusion, the results show that psychological services in Italian ART centers are not yet fully operational and integrated in the ART procedure. All of this highlights the importance of further investigations with the aim to shared information to use to establish common protocols for psychological interventions in ART centres. Trial registration number Not applicable

Study question Does fertilization rate (FR) affect cumulative success rates in assisted reproduction cycles? Summary answer These data indicate a positive association between FR with CLBR suggesting the predictive clinical relevance of this parameter and its adoption as Key Performance Indicator(KPI). What is known already Numerous studies have aimed at characterizing outcome predictors. Maternal age is historically and correctly recognized as the single most important factor impacting on the clinical outcome of ART. More recently ovarian response has also gained interest in this respect. However, the quest for novel, more comprehensive predictive factors is not over; new relevant evidence is starting to emerge. FR is a noteworthy parameter because expressing a fundamental aspect of both oocyte and sperm developmental competence. In fact it has been adopted as a key performance indicator of the IVF laboratory, to assess laboratory, operator, and gamete competence. Study design, size, duration Reported data concern a retrospective cohort study carried out between 2015 to 2017 involving 7,968 couples undergoing 9,394 complete ICSI cycles, i.e. whose all embryos were transferred or disposed.All women aged between 18-42 years were included.We excluded from analysis: surgical sperm retrieval cases, cycles resulting in neither fresh or frozen–thawed embryo transfers,cycles in which live birth were not achieved, but with remaining cryopreserved embryos,cycles of PGT, cycle with fertilization failure and standard IVF cycles. Participants/materials, setting, methods The cohort was groupped according to fertilization rate intervals based on recommendations of the Vienna Consensus (80% - Group 3). Harnessing the large size of the original dataset, further cycle stratifications were carried out based on female age (10 oocytes). Main results and the role of chance No significant difference in female age was observed between fertilization rate groups (p = 0.640). CLBR was progressively higher in relation fertilization rate in Groups 1, 2 and 3 (20.1%, 34.7%, 41.3%, P Limitations, reasons for caution The study design is retrospective and requires further refinement to control for factors that may impact clinical outcome. Wider implications of the findings These data indicate a positive association of FR with CLBR, thereby suggesting that fertilization, in addition to representing an assay for gamete quality and laboratory performance,has an independent clinical significance.Irrespective of the number of retrieved oocytes and female age, we observed that, rates of FR are positively associated with CLBR. Trial registration number None

Objective: To appraise the fertilization rate as a predictive factor for cumulative live birth rate (CLBR). Design: Multicenter retrospective cohort study. Setting: Ten in vitro fertilization clinics, whose data were collected and processed by the assisted reproductive technology (ART) Italian National Registry. Patient(s): 7,968 couples undergoing 9,394 complete intracytoplasmic sperm injection cycles. Intervention(s): None. Main Outcome Measure(s): The primary outcome measure was the CLBR in association with the fertilization rate intervals (80%—group 3). Further data stratification was performed on the basis of maternal age ( 10 oocytes). Result(s): The CLBR was progressively higher in relation to the fertilization rate in groups 1, 2, and 3 (20.1%, 34.7%, and 41.3%, respectively). The number of recovered oocytes, embryo number per cycle, and cumulative pregnancy rate followed the same trend. The decrease in CLBR with increasing maternal age was significantly correlated with the fertilization rate and CLBR in all 3 maternal age groups. Multivariate logistic regression analysis showed fertilization rate as a factor independently associated with CLBR. Conclusion(s): The present data indicated a positive association between the fertilization rate and the CLBR, suggesting the predictive clinical relevance of this parameter and its adoption as a key performance indicator.

To estimate the contribution of cryopreservation to the cumulative live birth rate (CLBR) after law modification in Italy in the era of vitrification and freeze-all.The Italian National Registry performed a cycle-based data collection. Nine Italian IVF clinics were involved incorporating a total of 10,260 fresh cycles performed between January 2015 and April 2016 resulting in 9273 oocyte retrievals and 3266 subsequent warming cycles from the same oocyte retrievals performed up to December 2016. Mean female age was 37 ± 4.3 years. Primary outcome measure was CLBR per oocyte retrieval. Confounding factors were tested in multivariate regression analysis, and the relative impact of cryopreservation to the CLBR in different patient categories was calculated.CLBR per oocyte retrieval was 32.6%, 26.5%, 18.7%, 13.0%, and 5.5% for women younger than 36, aged 36-39, 40-41, and older than 41 years, respectively. The total relative contribution of oocyte/embryo cryopreservation was 40.6% (95% CI 38.41-42.75). An association between maternal age, number of oocytes retrieved, fertilization rate, cryopreservation, and cumulative live birth was shown. When adjusted for confounders, a 2.3-fold increase was observed in the chance of live birth when cryopreservation was performed (OR 2.3; 95% CI 1.99-2.56). In high responder patients ( 15 oocytes retrieved) where freeze-all was applied in 67.6% of cycles to avoid the risk of hyper stimulation syndrome, the relative contribution of vitrification to the CLBR was 80.6%.Cryopreservation is essential in IVF and should always be available to patients to optimize success rates. Multicentric, cycle-based data analyses are crucial to provide infertile couples, clinicians, and regulatory bodies with accurate information on IVF effectiveness including fresh and cryopreserved cycles.

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.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.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.

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. Samples were studied by light and transmission electron microscopy. 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. 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.

Ethanol is the most widely used drug worldwide. Its consumption has been increasing, and it is reported even during childbearing. Prenatal exposure to ethanol can lead to irreversible damages of the fetus. Knowledge about this risk could prevent these damages. There is no information about knowledge of the Italian students on this issue.Therefore the aim of this study was to describe the awareness of the Italian students attending the last year of secondary school about risk of gestational alcohol drinking for the delivering mother and the fetus. An online multiple-choice anonymous survey for students was used and e-mailed to the all Italian secondary schools.The respondents were 9.921 and the obtained results evidenced that that young females are more informed than males, and students in Northern and Central Italy are better informed than those in the South, especially on general aspects. The most of respondents knows that alcohol consumption during pregnancy can damage the fetus. However, many youngsters failed to translate this belief into the practice.In conclusion, interventions are needed to enhance knowledge and prevent these damages, and health professionals, with nurses in the first row are entitled to provide education on this topic.

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. 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. 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). 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 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.

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.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.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).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.

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.

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 Human sperm cells are naturally birefringent when observed in a polarizing light due to the presence of longitudinally oriented protein filaments. The detection of birefringence in sperm heads reflects an organized and very compacted texture that indicates nuclear and acrosomal structural normality. Basically, three types of head birefringence have been observed: total, partial (localized in the post acrosomal region) and abnormal (absent or irregular). Data from electron microscopy has suggested a correlation between acrosome integrity and birefringence patterns. The aim of this study was to verify this hypothesis by direct analysis of the acrosome status in single spermatozoa. 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, 96% of spermatozoa with partial birefringence had a reacted acrosome, while 65% of totally birefringent heads had an intact acrosome. A large proportion of sperm cells with normal morphology showed total birefringence both in the presence (59%) or in the absence of motility (45%; P

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 =

Objective To evaluate the efficacy of oocyte cryopreservation by a single slow-cooling protocol involving sucrose (0.2 mol/L) in the freezing solution. Design Observational comparison of the clinical outcome in fresh and frozen thawed cycles. Setting Public and private IVF centers. Patient(s) Infertile couples undergoing IVF treatment. Intervention(s) Use of a maximum three oocytes in fresh cycles, as established by local law, and cryopreservation and later use of surplus oocytes. Likewise fresh cycles, maximum three thawed oocytes were used per cycle. All thawed oocytes were microinjected. Main Outcome Measure(s) Embryologic and clinical parameters of fresh and thawed cycles. Result(s) Two thousand forty-six patients underwent 2,209 oocyte retrievals involving oocyte cryopreservation. Overall, the survival rate of thawed oocytes was 55.8%. In 940 thaw cycles, the mean numbers of inseminated oocytes and fertilization rates were significantly decreased vs. fresh cycles outcomes (2.6 ± 0.7 vs. 2.9 ± 0.2 and 72.5% vs. 78.3%, respectively), as were the rates of implantation (10.1% vs. 15.4%), pregnancy rates per transfer (17.0% vs. 27.9%), and pregnancy rates per cycle (13.7% vs. 26.2%). Differences in clinical outcome were found among centers. A pregnancy rate per thawing cycle above 14% was achieved by most clinics. Fifty-seven retrievals involving oocyte cryopreservation achieved a pregnancy after fresh embryo replacement. Implantation and pregnancy rates per embryo transfer and per thawing cycles were 17.5%, 28.6%, and 24.6%, respectively. Conclusion(s) Under the conditions tested, the clinical outcome of oocyte slow-cooling cryopreservation is reduced compared with fresh cycles. Nevertheless, in cases of inapplicability of embryo cryopreservation, oocyte cryopreservation should be offered to patients with surplus oocytes.

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.

To ascertain possible cell damage from cryopreservation, the ultrastructure of human oocytes cryopreserved by slow cooling was assessed.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.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.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.

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 μg/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 ≥ 0.99) were found in both UV-DAD and ESI-MS. Limits of quantifications ranged between 2.5 and 7.8 μg/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.

Resumen: Introducción: por primera vez en Europa, el «Proyecto Meconio» ha estimado la prevalencia del consumo de drogas ilegales por mujeres embarazadas y la consiguiente exposición fetal. Pacientes y método: entre octubre de 2002 y febrero de 2004, se incluyó en el estudio a 1.209 parejas madre-recién nacido del Hospital del Mar, Barcelona. Se recogieron datos sobre las características socioeconómicas y demográficas y los hábitos tóxicos durante el embarazo empleando un cuestionario. Se recogió el meconio neonatal antes de 24 horas después del nacimiento y se analizó empleando técnicas cromatográficas estandarizadas para detectar opiáceos, cocaína, cannabinoides y anfetaminas. Resultados: el análisis del meconio demostró una positividad para las drogas de abuso del 10,9% del total, con una prevalencia específica de exposición fetal a heroína, cocaína y cannabis del 4,7, el 2,6 y el 5,3%, respectivamente. El cuestionario estructurado detectó que el 0,3, el 1,2 y el 1,5% de las madres habían consumido heroína, cocaína y cannabis, y sólo una madre declaró el consumo de éxtasis, confirmado mediante el análisis del meconio. La etnia de los padres y la clase laboral no se asociaron con el consumo de drogas. Las madres consumidoras de drogas mostraron un número de abortos previos más elevado que las madres no consumidoras. Se encontró que el peso y la talla al nacimiento eran significativamente más bajos en los recién nacidos de las madres expuestas a cocaína sola o en combinación con otras drogas. Conclusiones: este estudio puede servir como aviso de un consumo de drogas durante el embarazo oculto y significativo. En este sentido, el análisis del meconio puede ser importante para identificar a recién nacidos en elevada sospecha de exposición intrauterina a drogas de abuso y proporciona la base para un tratamiento médico y un seguimiento social apropiados. Abstract: Introduction: For the first time in Europe, the «Meconium Project» 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.

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.

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.

Background Recently described slow-cooling cryopreservation protocols involving elevated sucrose concentration have improved survival frequencies of human oocytes, potentially overcoming a major hurdle that has limited the adoption of oocyte storage. Because implantation rates of embryos from frozen oocytes remain generally low, it is still debated whether, irrespective of survival rates, this form of cryopreservation leads inevitably to the disruption or complete loss of the metaphase II (MII) spindle. Methods Human oocytes with an extruded polar body I (PBI) were cryopreserved using a slow-cooling method including 1.5 mol/l propane-1,2-diol (PrOH) and alternative sucrose concentrations (either 0.1 or 0.3 mol/l) in the freezing solution. Fresh control and frozen-thawed survived oocytes were analysed by confocal microscopy to evaluate MII spindle and chromosome organizations. Results Of the 104 oocytes included in the unfrozen group, 76 (73.1%) displayed normal bipolar spindles with equatorially aligned chromosomes. Spindle and chromatin organizations were significantly affected (50.8%) after cryopreservation involving lower sucrose concentration (61 oocytes), whereas these parameters were unchanged (69.7%) using the 0.3 mol/l sucrose protocol (152 oocytes). Conclusions Partial disruption of the MII spindle and associated chromosomes accompanies inadequate cryopreservation during slow cooling. However, protocols adopting higher sucrose concentration in the freezing solution promote the retention of an intact chromosome segregation apparatus comparable in incidence to freshly collected oocytes.

Assisted reproductive techniques (ART) include treatments where human gametes are handled outside the body to achieve a pregnancy. They include in vitro fertilization embryo transfer, intracytoplasmic sperm injection, gamete intrafallopian transfer, preimplantation genetic diagnosis oocyte and sperm donation, and surrogacy. Recently, assisted reproductive technologies have spread worldwide, leading to over 5 million babies born according to International Committee Monitoring Assisted Reproductive Technologies (ICMART) 2009. Europe is the world leader, with the number of cycles performed being >55% of all reported cycles. Many factors can influence the application and the availability of ART treatments in different countries. This chapter covers some of these factors considering data collection systems, different religious perspectives related to ART, the availability of the techniques, their application in different settings, and their principal outcomes.

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.

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.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.Seven teaching hospitals providing obstetrical care for mothers infected with HIV.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 count200 x 10(6)/l.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.

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.

The aim of this article is to clarify the different strategies used to induce controlled ovarian hyperstimulation in some groups of difficult-to-treat patients, namely poor-responder patients, polycystic ovarian patients and endometriosis patients. The success of in vitro fertilization depends on adequate follicle recruitment and the recovery of multiple good-quality oocytes by using controlled ovarian hyperstimulation. This is particularly difficult to achieve in patients with a high incidence of low ovarian response (stage III–IV endometriosis and premature ovarian failure patients) or an excessive ovarian response with a risk of ovarian hyperstimulation syndrome (polycystic ovary syndrome patients). Consequently, special methods are needed in these groups of patients in order to optimize in vitro fertilization results.

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).Two thousand and sixteen consecutive infertile couple candidates for Assisted Reproduction Techniques (ART) were screened for karyotype and 616 couples for CFTR analysis.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.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.

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.

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.

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.

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.

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.

BACKGROUND: We studied the ultrastructural characteristics of human mature oocytes frozen/thawed (F/T) using different concentrations of sucrose. Fresh human mature oocytes were used as controls. METHODS: The oocytes (n = 48) were fixed in 1.5% glutaraldehyde at sampling (n = 16) or after freeze/thawing performed using a slow cooling method with propane-1,2-diol 1.5 mol/l and sucrose at either 0.1 mol/l (n = 16) or 0.3 mol/l (n = 16) in the freezing solution. The oocytes were then processed for electron microscopy observations. RESULTS: Fresh and F/T oocytes belonging to both study groups were regularly rounded in sections, with a homogeneous cytoplasm and an intact zona pellucida (ZP). Organelles (mainly mitochondria-smooth endoplasmic reticulum aggregates and mitochondria-vesicle complexes) were abundant and uniformly dispersed in the ooplasm. The amount and density of cortical granules appeared to be abnormally reduced in some F/T samples, independently of the sucrose concentration in the freezing solution: this feature was frequently associated with an increased density of the inner ZP, possibly related to the occurrence of zona 'hardening'. Furthermore, slight to moderate microvacuolization was revealed in the ooplasm of some F/T oocytes, particularly in those treated with sucrose 0.3 mol/l. CONCLUSIONS: Freeze/thawing procedures are associated with ultrastructural alterations in specific oocyte microdomains, presumably linked to the reduced developmental potential of mature cryopreserved oocytes. Further work is needed to determine whether or not a high concentration of sucrose plays a role, at least in part, in producing the above alterations.

analyze slow freezing/rapid thawing or vitrification in one of the largest worldwide experience of oocyte cryopreservation.