Current and future methods for embryo selection: on a quest for reliable strategies to reduce time to pregnancy.

INTRODUCTION: The aim of this study was to analyze the usefulness of the principal embryological strategies to reduce time to pregnancy. EVIDENCE ACQUISITION: A systematic search of publications in the PubMed/MEDLINE, Embase and Scopus databases from inception to present including "IVF," "blastocyst," "embryo colture," "competent embryo," "time to pregnancy," "aneuploid," "euploid," "vitrification," "preimplantation genetic," "IVF strategies" and "embryo selection" alone or in combinations has been done. EVIDENCE SYNTHESIS: We have selected 230 articles and 9 of them have been included in this mini-review. CONCLUSIONS: Several embryological strategies aimed to select the most competent embryo and reduce time to pregnancy have been proposed, even if few publications on this specific topic are available. preimplantation genetic testing (PGT-A) represents the unique method able to assess the embryonic chromosomal status, but this does not mean that PGT-A is a reliable strategy to reduce time to pregnancy. There is no consensus on a specific method to reduce time to pregnancy, nevertheless this final goal could be probably reached through a harmonious combination of procedures. Thus, a reliable strategy to reduce time to pregnancy could be achieved when embryo culture, embryo cryopreservation and PGT-A are perfectly integrated and appropriately offered to selected patients.

Nitric oxide as an early marker of human embryo metabolic cleavage in ART using fresh or thawed oocytes.

OBJECTIVE: To study a possible role of nitric oxide (NO) as a marker of development in the early phases of human embryo cleavage during assisted reproduction. STUDY DESIGN: 179 women having ART were included. 123 women used fresh oocytes and 56 oocyte thawing cycles in the Center of Reproductive Medicine, Department of Obstetrics and Gynecology, Arcispedale S. Maria Nuova, between July 2005 and June 2006; 57 patients had IVF and 122 patients had ICSI. NO concentrations in IVF or ICSI embryo culture media were assessed by monitoring levels of NO stable oxidation products (nitrites/nitrates). Analysis of embryo quality was performed. Student's t-test or Mann-Whitney and logistic regression model tests were applied to the data. RESULTS: In patients using fresh oocytes, there were greater NO production in embryos derived from ICSI than from IVF after 52 h of culture (38.64 micromol/L vs 11.2 micromol/L, p<0.05). No correlation with embryo quality was observed. Embryos derived from fresh oocytes produce more NO than embryos from thawed oocytes both after 48 and 52 h of culture (16.12 micromol/L vs 6.83 micromol/L and 25.93 micromol/L vs 2.98 micromol/L respectively, p<0.05). CONCLUSION(S): NO in embryo culture media is not a metabolic cleavage marker or a marker of embryo quality in ART. However, it could be an important parameter in the investigation of metabolism in frozen/thawed oocytes.

Limited importance of pre-embryo pronuclear morphology (zygote score) in assisted reproduction outcome in the absence of embryo cryopreservation.

OBJECTIVE: To investigate the hypothesis that Z-score criteria represent a reliable predictor of implantation rate and pregnancy outcome in in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI) cycles, excluding the possibility of embryo selection before the embryo transfer. DESIGN: Retrospective clinical study. SETTING: Centre of Reproductive Medicine, Department of Obstetrics and Gynecology, Arcispedale S. Maria Nuova (ASMN), Reggio Emilia, Italy. PATIENT(S): We analyzed 393 pregnancies obtained by IVF or ICSI cycles. INTERVENTION(S): Morphologic evaluations of Z-score in pre-embryos obtained from IVF or ICSI cycles. MAIN OUTCOME MEASURE(S): Evaluations of Z-scores, implantation rate, and clinical pregnancy outcome. RESULT(S): We did not find any statistically significant correlation between the Z-score of 1032 embryos transferred in 393 embryo transfers and the implantation rate or the pregnancy outcome. In particular, the best Z-score identified (Z1, 7.2%) did not seem to correlate with embryo implantation rate or pregnancy outcomes any better than those with worse scores (Z2, 6.9% and Z3, 85.9%). CONCLUSION(S): Our results seem to confirm that Z-score alone cannot be considered a better tool than standard morphologic criteria for identifying, controlling, or selecting embryos with a better chance of successful ongoing pregnancy.

Outcome of 518 salvage oocyte-cryopreservation cycles performed as a routine procedure in an in vitro fertilization program.

OBJECTIVE: To document outcomes of oocyte cryopreservation performed as a routine procedure in an IVF program. DESIGN: Describing the rate of oocyte survival, embryo transfer (ET), implantation, and live births of IVF-intracytoplasmic sperm injection performed on thawed oocytes. SETTING: Reproductive medicine center in Italy. PATIENT(S): Women (n = 696) who failed to conceive after IVF-intracytoplasmic sperm injection with fresh oocytes. INTERVENTION(S): Surplus oocytes obtained during a failed cycle with fresh oocytes were frozen and then were thawed, micromanipulated, and transferred in a later cycle. MAIN OUTCOME MEASURE(S): Rates of oocyte survival, ETs, implantation, and live births were calculated in the entire cohort and in patients aged 38 years. RESULT(S): There were 29 pregnancies, for a total implantation rate of 6.3% (95% CI: 4.3, 9.0) per 456 ET cycles. The clinical-pregnancy rate was 19 (4.2%; 95% CI: 2.6, 6.4) of 456 ET cycles, with a take-home-baby rate of 7 (1.5%; 95% CI: 0.7, 3.0) of 456 ET cycles. CONCLUSION(S): Cryopreservation performed as a routine procedure for so-called salvaging of surplus oocytes is associated with poor implantation rates and with a probability of 1 live birth in 65 ET cycles.

Pregnancy loss and assisted reproduction: preliminary results after the law 40/2004 in Italy.

The new Italian law on assisted reproduction technology rules that no more than three oocytes can be fertilized at one time, and that all embryos obtained must be transferred to the maternal uterus simultaneously. The aim of the present study was to investigate the influence of the new law on spontaneous embryonic/fetal losses through comparison of data collected over an identical period of time: the first year of application of the new law compared with the same time period 1 year before (March 10, 2004 to March 9, 2005 versus March 10, 2003 to March 9, 2004). A total of 271 clinical pregnancies were analysed. In the post-law period, a significantly lower percentage of total spontaneous embryonic losses compared with the pre-law period, and a higher percentage of surviving embryos in singletons and twins was observed. In conclusion, the impact of the limitations imposed by the new legislation regulating assisted reproduction in Italy seems to exert positive effects on spontaneous embryonic loss both in singletons and multiple pregnancies. These findings are in contrast to international predictions on this issue and, in general, are counter-intuitive. This suggests that further investigations on a larger cohort of women are required to confirm these preliminary results.

Effect of the mode of assisted reproductive technology conception on obstetric outcomes for survivors of the vanishing twin syndrome.

Survivors of the "vanishing" twin syndrome, which occurred in dichorionic twins only, and singletons that began as singletons, had similar mean gestational duration and birth weights, as well as similar frequencies of maternal and neonatal complications. This similarity persisted when conventional IVF and IVF plus ICSI cases were separately evaluated.

Spontaneous embryonic loss rates in twin and singleton pregnancies after transfer of top- versus intermediate-quality embryos.

OBJECTIVE: To determine whether embryo quality is associated with early spontaneous loss rates in twin and singleton pregnancies after IVF/intracytoplasmic sperm injection (ICSI). DESIGN: Retrospective, single center analysis. SETTING: The Center of Reproductive Medicine, Arcispedale Santa Maria Nuova, Reggio Emilia, Italy. PATIENT(S): Women undergoing IVF/ICSI and two- or three-embryo transfer of intermediate- and top-quality embryos. INTERVENTION(S): First-trimester sonography at 6 to 7 weeks to determine number of embryos with positive heartbeat. Number of embryos lost was calculated from a second-trimester sonogram. MAIN OUTCOME MEASURE(S): Rates of total pregnancy loss, as related to embryo quality, initial number of embryos, maternal age <35 or > or =35 years, and IVF procedure. RESULTS: A total of 94 losses (23.1% of 407 pregnancies) were counted, with similar proportions in pregnancies after transfer of intermediate- or top-quality embryos. Neither the mode of IVF procedure nor the number of transferred embryos affected the loss rate. In contrast, the loss rate was significantly higher in older mothers after transfer of intermediate-quality embryos (odds ratio [OR 2.4], 95% confidence interval [CI] 1.1-5.5). Losses among singletons were significantly higher compared with losses among twins (OR 2.5, 95% CI 1.1-6.0), but this was observed in top-quality embryos only. CONCLUSION(S): Top-quality but not intermediate-quality ETs are associated with lower early spontaneous loss rates among twin pregnancies after IVF/ICSI.

Prevalence and determinants of genital shedding of herpes simplex virus among women attending Italian colposcopy clinics.

OBJECTIVES: To determine the prevalence of herpes simplex virus (HSV) shedding among women attending Italian colposcopy clinics and describe their lifestyle, demographic characteristics, genital symptoms and signs. STUDY DESIGN: A cross-sectional study was performed to assess shedding of HSV among 4565 women requiring a gynecological consultation. An amplified enzyme immunoassay that detects an HSV type-common glycoprotein D was used to reveal HSV shedding in cervical specimens. Statistical analysis was performed using Chi-square test and Student's t test. RESULTS: A prevalence of 7.8% was found among colposcopy clinic patients. No significant differences regarding patients' average age, age at first sexual intercourse, contraceptive method used, and number of sexual partners in the previous year were found between subjects with and without viral shedding (P > 0.05). The detection of a concomitant genital infection with Trichomonas vaginalis as well as the report of previous episodes of genital herpes (GH) were significantly higher in the positive group (P < 0.01). Only 2.8% of the patients shedding HSV presented with vesicles and ulcers, with the majority of them being asymptomatic. CONCLUSION: This is the first Italian survey on genital herpes conducted among colposcopy clinic patients. Our data show that the prevalence of HSV shedding in this study population is high and confirms that the disease is often asymptomatic. The demographics and behavioural variables of women shedding HSV seem to differ from the ones assessed in high risk patients.

Spontaneous embryonic loss after in vitro fertilization with and without intracytoplasmic sperm injection.

OBJECTIVE: To determine whether pregnancies after IVF, with and without intracytoplasmic sperm injection (ICSI), have different early spontaneous loss rates. DESIGN: Retrospective analysis of IVF/ICSI dataset. SETTING: The Center of Reproductive Medicine, Arcispedale Santa Maria Nuova, Reggio Emilia, Italy. PATIENT(S): Women undergoing IVF with or without ICSI. INTERVENTION(S): First-trimester sonography at 6-7 weeks to count the number of embryos with positive heartbeat. The number of embryos lost was calculated from a second-trimester sonogram. MAIN OUTCOME MEASURE(S): Embryonic loss rates related to the initial number of embryos, maternal age <35 or > or =35 years, and IVF procedure. RESULT(S): In vitro fertilization and ICSI had similar embryonic loss rates (odds ratio [OR] 1.2, 95% confidence interval [CI] 0.9-1.7, and OR 1.3, 95% CI 0.9-1.8 for women aged <35 years and > or =35 years, respectively). Younger women had fewer losses after IVF (OR 0.7, 95% CI 0.5-0.9). Multiples had lower loss rates compared with singleton pregnancies. CONCLUSION(S): In vitro fertilization and ICSI have similar spontaneous embryonic loss rates. Factors other than the initial number of embryos, maternal age, and IVF technique, such as embryo quality or uterine environment, might be involved in the outcome of multiple pregnancies in assisted reproductive technology procedures.

Spontaneous embryonic loss following in vitro fertilization: incidence and effect on outcomes.

OBJECTIVES: The purpose of this study was to determine the early spontaneous loss rate in multiple pregnancies following assisted reproductive technology (ART). STUDY DESIGN: Analysis of pregnancies following ART as related to the initial number of embryos and maternal age was performed. RESULTS: At least one spontaneous loss was observed in 47.0% (95% CI 29.8-64.9) of quadruplets, 59.3% (95% CI 48.2-69.8) of triplets, and 35.2% (95% CI 29.6-41.2) of twins. The increased loss rates in mothers > or =35 years was related to early twin pregnancies, but not to early triplet and quadruplet gestations. We could not find an effect of the starting number of embryos on either gestational age or birth weight characteristics. CONCLUSION: Our observations did not identify a clear relation between early spontaneous loss of multiple gestations and either initial number of embryos or maternal age. Other factors might be involved in the outcome of ART multiple pregnancies.

Developmental outcomes at one and two years of children conceived by intracytoplasmic sperm injection.

BACKGROUND: Present opinions regarding developmental delay of children conceived with intracytoplasmic sperm injection (ICSI) are variable and without consensus. METHODS: We compared developmental outcome at 1 and 2 years of 50 children conceived by ICSI with 51 spontaneously conceived children. Assessments were performed using the Bayley Scales of Infant Development for motor and mental development and quality of behavior, and the CARE-Index for the quality of parent-child interaction. RESULTS: No difference was present in the motor and mental development of study and control groups, as assessed by the Bayley Scales. However, 1-year-old ICSI children had lower Behavioral Index scores than controls in the "Motor Quality" item (p = 0.04). Regarding interaction with parents, ICSI families had lower "father cooperation" CARE-Index scores (p = 0.02) and lower sensitivity scores (p = 0.007). No significant differences were present between study and control groups for the 2-year-old children. CONCLUSIONS: ICSI conceived children develop normally. When assessed by the Bayley Scales of Infant Development and the CARE-Index, the differences in quality of development and social interaction observed at 1 year of age had no negative effect on the overall level of development, and appeared to be related to developmental processes of the entire family unit rather than to ICSI-related biological effects. Observed differences dissipated with familial adaptation and were not observed at 2 years of age.

The Italian STD 'Hot-Line'. Making people aware.

BACKGROUND: To increase the accessibility of qualified and anonymous information on sexually transmitted diseases (STDs) in Italy, a national telephone hotline was initiated in February 1997. GOAL: The goal was to determine the profile of callers to the national STD hotline, assess their concerns, identify their sources for the hotline telephone number, and to compare callers' disease interests with diagnoses made at STD clinics. STUDY DESIGN: The survey analyzed 3577 calls received from February 1997 to December 1999. Hotline operators addressed callers' questions, asked 6 short-answer questions, and recorded the data collected. RESULTS: The survey showed callers' sex distribution (49.8% women, 50.2% men), average age (women's mean+/-standard deviation [SD], 34.3 +/- 11.8 y; men's mean +/- SD, 36.2 +/- 12.2 y), level of education (66.4% had a secondary school degree), residence (47.9% northern part of Italy), risk category (44% reported being definitely infected by a STD), specific disease interest (30% were concerned about HIV), and source of hotline telephone number (67.3% learned the hotline telephone number from magazines or newspapers). Hotline callers' specific disease interests were compared with diagnoses made at STD clinics; the top 4 disease concerns of hotline callers were HIV, genital herpes, human papillomavirus/genital warts, and mycosis, whereas the top 4 diagnoses made at STD clinics were genital warts, nonspecific vaginitis, nonspecific urethritis, and genital herpes. CONCLUSION: The hotline seems to be an effective way to deliver information and to allay fears about STDs, although it needs to be promoted more widely, especially in central and southern Italy.

Correlations between concentrations of interleukin-12 and interleukin-13 and lymphocyte subsets in the follicular fluid of women with and without polycystic ovary syndrome.

OBJECTIVE: To investigate a possible correlation between interleukin-12 (IL-12) and IL-13 levels and lymphocyte subsets in the preovulatory follicles of patients with and without polycystic ovarian syndrome (PCOS). DESIGN: Controlled clinical study. SETTING: University hospital. PATIENT(S): Seventy-eight infertile women undergoing IVF-embryo transfer. INTERVENTION(S): The subjects underwent blood sampling, ovum retrieval, and embryo transfer. MAIN OUTCOME MEASURES: Follicular fluid levels of T, androstenedione (A); IL-12, IL-13, activated T cells, T helper, and T-suppressor lymphocytes. RESULT(S): The level of IL-12 detected in follicular fluid (FF) was significantly lower in patients with PCOS than in normally ovulating women (mean: 1.47 +/- 0.3 pg/mL vs. 2.25 +/- 0.7 pg/mL, respectively); in contrast, FF IL-13 concentrations were significantly higher in the patients with PCOS than in the normally ovulating women (mean: 32.5 +/- 3.7 pg/mL vs. 19.6 +/- 2.5 pg/mL, respectively), as was the total number of activated T lymphocytes (11.5% +/- 1.5% vs. 4.8% +/- 0.4%). A significant correlation was observed between FF activated T-cell concentrations and FF IL-12, IL-13, T, and A levels. No significant differences were observed when these data were compared with embryological parameters. CONCLUSION(S): The present study shows significant differences in the correlation between FF IL-12 and IL-13 levels and T lymphocyte numbers in the subset of patients with PCOS as compared to normally ovulating women.

Physiological levels of 1alpha, 25 dihydroxyvitamin D3 induce the monocytic commitment of CD34+ hematopoietic progenitors.

Although supraphysiological levels of 1alpha, 25 dihydroxyvitamin D3 (VD) have been demonstrated extensively to induce the monomacrophagic differentiation of leukemic myelo- and monoblasts, little is known about the role that physiological levels of this vitamin could play in the regulation of normal hematopoiesis. To clarify this issue, we adopted a liquid-culture model in which cord blood CD34+ hematopoietic progenitors, induced to differentiate in the presence of different combinations of cytokines, were exposed to VD at various concentrations and stimulation modalities. The data obtained show that physiological levels of VD promote a differentiation of CD34+ hematopoietic progenitors characterized by the induction of all the monomacrophagic immunophenotypic and morphological markers. This effect is not only exerted at the terminal maturation but also at the commitment level, as demonstrated by the decrease of highly undifferentiated CD34+CD38- hematopoietic stem cells, the down-regulation of CD34 antigen, and the increase of monocyte-committed progenitors. Molecular analysis suggests that the VD genomic signaling pathway underlies the described differentiation effects.