Paolo Emanuele Levi-Setti

Insemination of HIV-negative women with processed semen of HIV-positive partners

Many HIV-discordant couples want to have children so much that they are willing to abandon condom-protected sexual intercourse irrespective of the risks. Previous testing in our laboratory showed that gradient centrifugation followed by a swim-up procedure effectively removed HIV-1-infected cells from the semen of HIV-seropositive men. 85 HIV-discordant couples were screened for fertility; 29 women were found suitable for a timed insemination course with the processed semen of their HIV-seropositive partner. None of the inseminated women seroconverted, and 17 pregnancies were achieved in 15 women. All 10 babies born to these mothers remain HIV seronegative. The findings may help in the counselling of such couples and also give them hope of having healthy babies.

Sperm protein 17 is expressed in the sperm fibrous sheath

BackgroundSperm protein 17 (Sp17) is a highly conserved mammalian protein characterized in rabbit, mouse, monkey, baboon, macaque, human testis and spermatozoa. mRNA encoding Sp17 has been detected in a range of murine and human somatic tissues. It was also recognized in two myeloma cell lines and in neoplastic cells from patients with multiple myeloma and ovarian carcinoma. These data all indicate that Sp17 is widely distributed in humans, expressed not only in germinal cells and in a variety of somatic tissues, but also in neoplastic cells of unrelated origin.MethodsSp17 expression was analyzed by immunocytochemistry and transmission electron microscopy on spermatozoa.ResultsHere, we demonstrate the ultrastructural localization of human Sp17 throughout the spermatozoa flagellar fibrous sheath, and its presence in spermatozoa during in vitro states from their ejaculation to the oocyte fertilization.ConclusionThese findings suggest a possible role of Sp17 in regulating sperm maturation, capacitation, acrosomal reaction and interactions with the oocyte zona pellucida during the fertilization process. Further, the high degree of sequence conservation throughout its N-terminal half, and the presence of an A-kinase anchoring protein (AKAP)-binding motif within this region, suggest that Sp17 might play a regulatory role in a protein kinase A-independent AKAP complex in both germinal and somatic cells.

Experimental contamination assessment of a novel closed ultravitrification device

OBJECTIVE To evaluate the safety of a new ultravitrification closed device. DESIGN Ultravitrification research. SETTING Private assisted reproduction center. ANIMAL(S) Microorganisms (bacteria). INTERVENTION(S) A styrofoam container holding 1,000 mL of liquid nitrogen (LN2) was contaminated with Pseudomonas aeruginosa and Escherichia coli. Forty closed devices (Ultravit) and 20 open devices (Cryotop) loading approximately 0.5 μL of antibiotic-free medium were plunged into this contaminated LN2 for 5-10 seconds and then inoculated into selective culture dishes. Colony-forming units were analyzed and counted after an overnight incubation at 37°C. MAIN OUTCOME MEASURE(S) Detection of micro-organisms in different devices after incubation. RESULT(S) There was no contamination in any of the closed devices, whereas in 45% of open devices these bacteria were present. CONCLUSION(S) With this study we demonstrated, in an experimental model using contaminated LN2, that this new closed device is a safe system that does not allow cell contact with LN2, avoiding cell contamination.

Human oocyte ultravitrification with a low concentration of cryoprotectants by ultrafast cooling: a new protocol

OBJECTIVE To evaluate the efficacy of a new ultravitrification technique with a low concentration of cryoprotectants. DESIGN Ultravitrification research. SETTING Private assisted reproduction center. PATIENT(S) Oocytes donated voluntarily with the aim of research. INTERVENTION(S) Ultravitrification with different protocols of 100 mature oocytes and 100 immature oocytes divided in four groups to determine which is the adequate cryoprotectant concentration and the appropriate cooling solution. MAIN OUTCOME MEASURE(S) Human oocytes survival rate with low concentration of cryoprotectants by ultravitrification technique. RESULT(S) We obtained higher survival rates with slush nitrogen than with liquid nitrogen (92% vs. 56%) and better results with 2 M of cryoprotectants than with 1.5 M (92% vs. 60%). The best protocol was 2 M PrOH + 0.5 M sucrose + slush nitrogen with a mature oocytes survival rate of 92% (23 of 25) and immature of 88% (22 of 25). CONCLUSION(S) This ultravitrification technique is a new option to preserve human oocytes that avoids the use of a high cryoprotectant concentration while obtaining a high survival rate with a concentration of cryoprotectants typical of slow freezing.

Ejaculate Oxidative Stress Is Related with Sperm DNA Fragmentation and Round Cells

Oxidative stress (OS) plays an essential role in male infertility aetiology by affecting sperm quality, function, and also the integrity of sperm DNA. The assessment of oxidative stress in semen may be an important tool to improve the evaluation of sperm reproductive capacity. The purpose of this study was the evaluation of any possible relation between the unbalance of oxidative stress caused by superoxide anion in the ejaculate with the presence of sperm DNA fragmentation and high concentration of round cells. 56 semen samples from males from couples suffering from infertility were evaluated according to World Health Organisation (WHO) 2010 guidelines. Oxidative stress levels from N1 (low) to N4 (high) were assessed in ejaculates using oxiSperm; DFI (sperm DNA fragmentation index) as assessed by the SCSA (Sperm Chromatin Structure Assay) was used for evaluation of sperm chromatin integrity. Our data show that high oxidative stress (N3-N4 levels) correlated positively with a DFI ≥ 30% (P = 0.0379) and round cells ≥1.500.000/mL (P = 0.0084). In conclusion, OS increases sperm DNA damage. Thus evaluation of semen OS extent of sperm DNA damage in infertile man could be useful to develop new therapeutic strategies and improve success of assisted reproduction techniques (ART).

Color-doppler velocimetry of uterine arteries in pregnant and nonpregnant patients during multiovulation induction for IVF

ObjectivesTo evaluate uterine artery resistance during multiovulation induction in relation to the implantation rate in patients attendingin vitro fertilization (IVF) cycles.PatientsMultiovulation induction for IVF was monitored by daily determination of the pulsatility index (PI) of the uterine arteries, obtained by a transvaginal probe (6.5 MHz) implemented with color-flow imaging. Doppler data were obtained from 5 days before hCG administration to the day of follicular aspiration. One IVF cycle was monitored in 70 patients. In 17 patients, 41 IVF cycles were monitored until a successful attempt occurred.ResultsIn the 70 patients studied during one IVF attempt, the PI of the uterine arteries significantly varied (P < 0.001) in the different phases of the cycle. In the 24 patients who conceived, a significantly lower PI (P < 0.03) was found throughout the cycle. This result was mainly due to a highly significant difference of PI values observed the day after hCG administration (P < 0.005). In the 17 patients who conceived after 1 to 4 negativein vitro fertilizations, no significant difference in PI was observed in the uterine artery resistance in cycles in which implantation was or was not successful.ConclusionsUterine artery resistance varies significantly during phases of the induction therapy. Uterine artery resistance is lower throughout the course of multiovulation induction in patients with higher pregnancy rates. The PI on the day after hCG administration was the best index of pregnancy rate. Low uterine artery resistance was present even in negative attempts in patients who eventually achieved a successful implantation. PI values ⩽3 can be considered a favorable prognostic factor for future IVF cycles.

Freeze/thaw stress induces organelle remodeling and membrane recycling in cryopreserved human mature oocytes

PurposeOur 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.MethodsSamples were studied by light and transmission electron microscopy.ResultsWe 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.ConclusionsThis 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.

The effect of parnaparin sodium on in vitro fertilization outcome: A prospective randomized controlled trial

INTRODUCTION In-vitro and in-vivo models suggest the influence of low-molecular weight heparin on conception in infertile women undergoing in vitro fertilization procedures (IVF). In this randomized controlled trial we assessed whether a low-molecular weight heparin (parnaparin) could affect IVF outcomes. MATERIALS AND METHODS 271cycles were analyzed in 247 women having a first or subsequent IVF cycle at Fertility Center of Humanitas Research Hospital. Patients, without severe thrombophilia and hormonal or active untreated autoimmune disorders, were randomly allocated (1:1) to receive for the whole cycle parnaparin, or routine hormonal therapy only. The primary endpoint was the clinical pregnancy rate and the secondary endpoints included implantation rate and live birth rate. RESULTS The clinical pregnancy and the live birth rate were similar in treated and controls (21.5% vs. 26.7%, p=0.389; 18.5% vs. 20.6%, p=0.757). The abortion rate was 10.3% vs 22.9%, p=0.319, respectively. The subgroups analysis, ≤35, 36-38, 39-40years, showed the following: comparable clinical pregnancy rate (22.5% vs 38.8%, p=0.124; 21.8% vs 17.3%, p=0.631; 19.4% vs 23.3%, p=0.762 respectively) and live birth rate (16.3% vs 32.7%, p=0.099; 20.0% vs 13.5%, p=0.443; 19.4% vs 13.3%, p=0.731 respectively) in treated vs controls. Sensitivity analyses on women with ≥3 previous attempts and first enrolment only, and subgroup analyses according to trial conclusion conditioning a small sample size with low statistical power. CONCLUSIONS Our study excludes positive effect of parnaparin, once a day for the whole cycle, on clinical pregnancy rate in infertile women undergoing in vitro fertilization techniques.

Evolution of human oocyte cryopreservation: slow freezing versus vitrification

Purpose of reviewThe 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 findingsDuring 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. SummaryVitrification 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.

A new, simple, automatic vitrification device: preliminary results with murine and bovine oocytes and embryos

PurposeThis paper reports the use of a novel automatic vitrification device (Sarah, Fertilesafe, Israel) for cryopreservation of oocytes and embryos.MethodsMice oocytes (n = 40) and embryos (8 cells, n = 35 and blastocysts, n = 165), bovine embryos (2PN, n = 35), and MII oocytes (n = 84) were vitrified using this automated device. A total of 42 (2 cells) mice embryos, 20 (2PN) bovine embryos, and 150 MII bovine oocytes were used as fresh controls and grown to blastocysts. Upon rewarming, all were assessed for viability, cleavage, blastocyst, and hatching rates.ResultsNinety-five % (38/40) of the mice MII oocytes regained isotonic volumes and all (100%) the surviving were viable. Rewarmed 8-cell mice embryos had 95% (33/35) blastulation rate and 80% (28/35) hatched. Rewarmed mice blastocysts had 97% survival rate (160/165) and 81% (135/165) hatched. Fresh control mice embryos had 100% (42/42) blastulation and 73% (21/42) hatching rates. Bovine embryos’ survival was 100% with 54% (19/35) cleavage and 9% (3/35) blastulation rate. Fresh control bovine embryos had 65% (13/20) cleavage and 20% (4/20) blastulation rate. Vitrified bovine oocytes had 100% survival (84/84), 73% (61/84) cleavage, and 7% (6/84) blastocysts’ rates; fresh control had 83% (125/150) cleavage and 11% (17/150) blastocysts’ rates.ConclusionThis novel automatic vitrification device is capable to produce high survival rates of oocytes and embryos. We anticipate that as the demand for vitrification of gametes, embryos, and reproductive tissues increases worldwide, the availability of an automated vitrification device will become indispensable for standardization, simplification, and reproducibility of the entire process.