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The serodiscordant couples, where the male is HIV-positive, are treated in fertility clinics, using the sperm washing technique by gradient centrifugation. This protocol cannot be carried out in oligo-azoospermic patients, where spermatozoa retrieval from the epididymis and testis must be performed. We developed a single sperm washing technique, where the spermatozoa, after the retrieval, are washed with the aid of a micromanipulator, to obtain virus decontamination and then used for the intracytoplasmic sperm injection (ICSI). The experiment was performed by using sperm samples containing three different viral loads. After one hour of incubation, spermatozoa were taken one by one from the HIV loaded drop and washed in four different microdrops. Before each passage into the next washing drop, the pipette was emptied in a first waste drop and then loaded with new washing medium from a second separate loading drop. After transferring of 10 spermatozoa in these four successive drops, the washing medium and the virus-loaded drops were tested for the HIV RNA presence by the nested RT-PCR technique. The presence of the virus was detected in the waste drop of all three viral loads. The four washing microdrops were each time negative for the presence of HIV-1 RNA, tested by the nested RT-PCR technique. The results show that by rinsing the spermatozoa four times, we are able to diminish the viral load to an undetectable level. Our data demonstrate that single sperm washing can be performed in the cases of extreme male sterility in HIV-positive men. From now on the couples, where the male is oligoazoospermic and HIV positive, could be included in our ICSI program, respecting the usual viral safety level of the ART techniques for the embryo.

Objective To determine gonosomal and autosomal aneuploidy rate in sperm and preimplantation embryos from nonmosaic 47,XYY males. Design Sperm and blastomere analysis by fluorescence in situ hybridization. Setting Fertility clinic, academic hospital. Patient(s) Two 47,XYY men undergoing preimplantation genetic diagnosis (PGD) and eight 46,XY males distributed in two control groups (fertile and infertile). Intervention(s) Sperm-sample collection for fluorescence in situ hybridization and PGD. Main Outcome Measure(s) Aneuploidy frequencies for chromosomes X, Y, 13, 16, 18, 21, and 22 in sperm and embryos. Result(s) Patients with 47,XYY presented global sperm gonosomal and autosomal aneuploidy frequency of 37.23%–37.80%, with XY disomy being the most frequent abnormality (16.70%–19.01%). This aneuploidy rate was statistically significantly different from that found in both 46,XY infertile controls (1.07%) and 46,XY fertile controls (1.04%). In total, 47 preimplantation embryos were analyzed, of which 32 were classified as normal (68%) and 15 as aneuploid (32%). Among the abnormal embryos, 9 presented gonosomal abnormalities, and 6, autosomal abnormalities. Conclusion(s) High rate of gonosomal and autosomal aneuploidy was observed in sperm and preimplantation embryos from nonmosaic 47,XYY males. The offspring of this category of patients may be at higher risk of chromosomal abnormalities, and therefore PGD can be suggested to these patients.

BACKGROUND The existence of a real benefit of blastocyst transfer is still a matter of debate. The aim of this study was to compare, in a prospective randomized trial, the outcome of day 2 and day 5 transfer of human embryos cultured in an 'in-house' sequential medium. METHODS A total of 193 cycles from 171 patients with less than four previous IVF cycles

The aim of the present study was to verify whether culturing testicular tissue, to obtain a higher percentage of motile spermatozoa and a better post-thaw recovery rate, affected the ratio between single/double-stranded sperm DNA and, consequently, DNA sensitivity to damage. Testicular biopsy samples from men with obstructive and secretory azoospermia, candidates for assisted reproductive treatment, were cultured for 72 h. The percentage of motile spermatozoa and the single/double stranded DNA ratio were assessed on the day of retrieval (day 0) and again on day 3. The single/double stranded DNA ratio was measured by the acridine orange (AO) staining method. Spermatozoa were classified as green (double-stranded chromatin) or red fluorescing (single-stranded chromatin). In obstructive azoospermia, median motility was 22% (range 10-44%) on day 0 and 50% (range 38-63%) on day 3 (P < 0.01). The median percentage of red stained spermatozoa was 53.5% (range 0.1-88%) on day 0 and 20% (range 2.7-99.9%) on day 3 (P < 0.05). No changes were observed in secretory azoospermia. The culture procedure from obstructive azoospermia not only increased the post-thaw recovery rate, as previously observed, but also reduced the portion of spermatozoa containing single-stranded DNA, thereby increasing the availability of double-stranded DNA spermatozoa for ICSI use.

Supplementation of culture media with amino acids has been shown to benefit preimplantation embryo development in several species. This randomized study analysed the in-vitro development of human embryos obtained after IVF in the presence or absence of a combination of amino acids from the 2- to 4-cell stage to the blastocyst stage. A total of 129 human embryos was randomly distributed between three serum-free chemically defined sequential media: (i) glucose-free Earle's balanced salt solution (EBSS) with glutamine (Gln) prior to morula stage, supplemented with glucose for blastocyst formation; (ii) glucose-free EBSS with glutamine and non-essential amino acids (AA) for cleavage stage development, and supplemented with all 20 AA for blastocyst formation (Earle's+AA); and (iii) a sequential commercial medium containing amino acids (K-SCIM). Embryos were individually cultured for successive periods of 24 h. On day 6 of development, blastocysts were differentially labelled and the numbers of trophectoderm and inner cell mass cells, mitoses and dead cells were examined. Blastocyst development was similar for the three sequential media. The mixture of AA significantly increased total blastocyst cell numbers from 61.8 +/- 4.2 with Earle's+Gln to 99.3 +/- 8.4 with Earle's+AA and 100.2 +/- 9.4 with K-SCIM (P = 0.005). This increase was present in both the trophectoderm and inner cell mass lineages (P < 0.02). Furthermore, the dead cell index was significantly lower with Earle's+AA (P = 0.047).

Mosaicism in 4-8-cell human embryos analysed by fluorescence in situ hybridisation (FISH) has been widely reported, but few studies have addressed the incidence of mosaicism in more advanced embryonic stages. In the present study we analysed spare human embryos in a case of preimplantation genetic diagnosis (PGD) for increased risk of aneuploidy because of an infertile 47,XYY man. After replacement of two embryos typed as 1818XX at PGD, six spare embryos (not frozen because of their low quality) were re-analysed on Day 5 for PGD confirmation. Out of five embryos typed as 1818XY at PGD, four were diploid mosaic (DM) and one was normal in all cells. The sixth embryo, typed as 18XYY/1818181818X at PGD, was a DM. In spite of the bias of our small series of morphologically low-quality embryos, the surprisingly high proportion of mosaics (which confirms previous findings) questions the validity of PGD, but supports the strategy of transferring only the embryos where two blastomeres gave normal and concordant results at PGD. More data are required to understand the clinical significance of early diploid mosaicism (and its impact on implantation rate) and to determine whether some diploid mosaic embryos might be considered safe for transfer.

The aim of the study was to analyse the toxicity, the osmolar and cryoprotective activity of ethylene glycol (ETG) in terms of survival rate (SR), cleavage rate (CR) and expanded blastocysts percentage (EBP) of mouse embryos. Early mouse embryos and blastocysts were slowly cooled with ETG, 1,2-propanediol (PROH) or glycerol, and thawed. The Van t'Hoff curve for 1.5 mol/l ETG showed recovery of initial volume within 4 min. No differences were observed in CR and EBP of ETG-exposed compared with non-exposed mouse zygotes. The SR of zygotes frozen with PROH was significantly better than with ETG (92% and 60% respectively; P < 0.01), and a significantly better EBP was achieved for blastocysts frozen with glycerol compared with ETG (75% and 50% respectively; P < 0.05). For 4-cell stage embryos, no differences were observed in SR and EBP between ETG and PROH. Higher EBP was observed for 4-cell stage embryos (53%) frozen with ETG compared with pronucleate stage (19%) and blastocysts (48%). Low toxicity, good SR and EBP were observed for mouse embryos frozen with ETG, the best results being obtained at the 4-cell stage. At other embryonic stages, PROH and glycerol respectively seemed to provide better results.

Objective:Our objective was to analyze the outcome of cryopreserved embryos obtained after intracytoplasmic sperm injection (ICSI) and in vitro fertilization (IVF) in terms of survival rate, implantation rate (IR), total and clinical pregnancy rate (PR) in a retrospective, comparative study. Methods:Three hundred seventy-five IVF and 463 ICSI surnumerary cleaved embryos, frozen on Day 2 with 1,2-propanediol, were thawed. Results:Thirty-two percent of the thawed IVF embryos survived and 11 pregnancies (8 clinical) were obtained from 68 transfers (16.1%). Fourty-seven percent of the ICSI embryos survived, with 19 pregnancies (18 clinical) from 116 transfers (16.4%). The IR was 8.5% (8/94) in IVF cycles and 10.8% (20/185) in ICSI cycles. Conclusions:A significantly better survival rate of ICSI embryos was observed but with no difference in PR, preclinical, and clinical abortion rate, or IR.

Objective To analyze the impact of seropositivity with hepatitis C virus (HCV) on in vitro fertilization (IVF) outcomes. Design Retrospective, case-controlled study. Setting Fertility clinic of academic hospital. Patient(s) 42 IVF/intracytoplasmic sperm injection cycles in HCV-seropositive women and 84 matched control cycles. Intervention(s) IVF/intracytoplasmic sperm injection treatment for infertility. Main Outcome Measure(s) Ovarian response to stimulation, laboratory findings, and implantation and pregnancy rates. Result(s) Absence of ovarian response was statistically significantly higher for HCV-seropositive women compared with controls (10/42 vs 5/84 cycles, respectively). For cycles with oocyte retrieval, HCV-seropositive women required more gonadotropin units compared with controls. The maximum estradiol levels and number of collected oocytes were similar, but HCV-seropositive women had statistically significantly fewer embryos available compared with controls. Embryo morphologic features, number of transferred embryos, and rates of implantation and pregnancy were similar for HCV-seropositive women and controls. Conclusion(s) When compared with matched uninfected controls, HCV-seropositive women display a decreased ovarian response.

Is it necessary to analyze two blastomeres in preimplantation genetic diagnosis (PGD) by fluorescence in situ hybridization (FISH) or is one blastomere enough, as suggested by some teams? We analyzed the sensitivity (Se), specificity (Sp), positive predictive value (PPV), negative predictive value (NPV), false positives (FP), false negatives (FN), and the efficiency (Eff) of FISH performed on one (Group I) or two (Group II) blastomeres. Ninety embryos were analyzed (day 3), 19 blastocysts were replaced (day 5), 64 embryos were reanalyzed (day 5), (Group I = 23; Group II = 41). No differences were observed between the two groups for all of the parameters considered, but one false negative was observed in Group I. Furthermore, two embryos from Group II, which had a discordant diagnosis at PGD (one blastomere being normal and one abnormal), were read as abnormal after reanalysis. The accidental biopsy of the normal blastomere could have lead to the selection of these 2 embryos for transfer, causing a misdiagnosis rate of 4.8%. We conclude that embryo reanalysis is a useful tool to test the reliability of PGD in each laboratory: that PGD on two blastomeres is safer because the practice of PGD on one blastomere can result in a false-negative misdiagnosis.

Chromosomal mosaicism has been reported in in vitro-cultured embryos at early cleavage stages, as well as in morulae and blastocysts. We have assessed the incidence and pattern of mosaicism during in vitro development of human embryos from early-cleavage stages to morula and blastocyst. Fifty spare embryos were fixed for fluorescence in situ hybridization (FISH) analysis for chromosomes X, Y, 13, 18, and 21 on days 2 or 3 (4- to 10-cell stage) (n = 16), on day 4 (morula stage) (n = 14), on day 5 (pre-expanded blastocyst) (n = 5), and the expanded blastocyst stages (n = 15). Blocked embryos (no cleavage observed within the last 24 hr) were not included. A total of 2367 cells were analyzed. Four early-cleavage stage embryos were found uniformly diploid; all of the others were mosaic for the chromosomes analyzed (mean diploid nuclei 48.3% +/- 28.7). All of the embryos at more advanced developmental stages, except one fully normal morula, had mosaic chromosome constitutions, with an increase in the percentage of diploid cells in morulae, pre-expanded, and expanded blastocysts, respectively (mean diploid nuclei 78.6% +/- 11.7, 66.0% +/- 20.8, 79.6% +/- 12.8), in comparison with earlier stages. Hypotheses about the origin of mosaicism and embryo regulation mechanisms will be discussed.

The objective of this study was to optimize the use of testicular biopsies in 14 patients with obstructive azoospermia. Testicular specimens were retrieved from six patients (group I) and cultured at 32 and 37 degrees C for up to 20 days; changes in percentage motile spermatozoa were compared. In four men of group I, one portion of the specimen was frozen at retrieval, and changes in post-thaw motility after 24 h of culture at 37 degrees C were recorded. In the other eight patients (group II), testicular specimens were frozen at retrieval and after 72 h culture at 37 degrees C. Pre and post-freezing motility and post-thaw recovery rate were compared. No significant differences were observed until day 8 in the improvement of motility between 32 and 37 degrees C in-vitro culture. Maximum motility was reached, under both conditions, between 48 h and 72 h. Post-thaw 24 h culture at 37 degrees C of specimens frozen at retrieval did not improve motility; however, 72 h pre-freezing culture significantly improved initial motility (P: < 0.01), post-thaw motility (P: < 0.01) and post-thaw recovery rate (P: < 0. 001). The higher recovery rate of samples frozen 3 days after retrieval allows more economical use of the tissue that is available.

The straight line velocity of 500 individual spermatozoa was measured immediately before their direct injection into the cytoplasm of 500 metaphase II oocytes. The straight line velocity (VSL) did not have a normal distribution (P < 0.01) and ranged between 0 and 35 microm/s. The fertilization rate (84%) was significantly (P < 0.008) higher in the quartile of the sperm population with the highest VSL compared to the fertilization rate (68%) in the quartile with the lowest VSL. Embryo cleavage and embryo quality were not different in the quartiles of the sperm population used for injection.