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Background Transplantation of ovarian tissue is, at present, the only clinical option available to restore fertility using cryopreserved ovarian tissue. More than 30 transplantations of cryopreserved tissue have been reported, and six babies have been born, worldwide, following this procedure. Despite these encouraging results, it is essential to optimize the procedure by improving the follicular survival, confirming safety and developing alternatives. Here, we review the different factors affecting follicular survival and growth after grafting. Methods Relevant studies were identified by searching Pubmed up to January 2009 with English language limitation. The following key words were used: (ovarian tissue or whole ovary) AND (transplantation) AND (cryopreservation or pregnancy). Using the literature and personal experience, we examined relevant data on the different exogenous and clinical factors affecting follicular development after grafting. Results Clinical factors such as the patient's age and the transplantation sites influenced the lifespan of the graft. A heterotopic transplantation site is not optimal but offers some advantages and it may also promote the hormonal environment after a combined heterotopic and orthotopic transplantation. Exogenous factors such as antioxidants, growth factors or hormones were tested to improve follicular survival; however, their efficiency regarding further follicular development and fertility potential remains to be established. Conclusion Additional evidence is required to define optimal conditions for ovarian tissue transplantation. Alternatives such as whole ovary or isolated follicles transplantations require further investigation but are likely to be successful in humans in the future.

Since 1999, the Erasme Hospital Fertility Clinic has carried a special programme for patients with HIV seropositivity. The philosophy of the programme is to give access to these patients in a secure environment to the same technological facilities available to any other patients. Many of these patients being native from sub-Saharan countries, they are often sickle cell disease (SCD) carriers, a common autosomal recessive disorder in these regions, and a severe affection in homozygotes. We hereby report, for the first time, the birth of a healthy sickle haemoglobin (HbS) heterozygous baby after preimplantation genetic diagnosis (PGD) for SCD in an HIV-serodiscordant couple of HbS mutation carriers with longstanding infertility. The prospective mother was 35 years old and HIV positive with an undetectable viral load under highly active antiretroviral therapy. One carrier embryo was transferred and resulted in the birth of a healthy HbS carrier baby girl. Despite stimulation difficulties, sometimes described in HIV patients, PGD represents an interesting additional technology, especially in populations where the coexistence of both diseases is frequent. PGD could even be preferred to prenatal diagnosis for couples of HbS carriers if the woman is HIV positive, as invasive prenatal samplings carry a risk of materno-foetal viral transmission.

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.

Objective To develop a method for same-day validation of processed semen in the setting of assisted reproductive techniques (ART) with patients who are seropositive for human immunodeficiency virus, type 1 (HIV-1). Design Laboratory experiments. Setting University hospital. Patient(s) Volunteers who are HIV-1 seronegative and seropositive. Intervention(s) Evaluation of the sensitivity of a reverse-transcriptase (RT)–nested polymerase chain reaction (PCR) in HIV-1 RNA–positive blood plasma, in artificially infected blood plasma and semen, and in 85 semen samples of 29 HIV-1–seropositive volunteers. Semen was submitted to gradient separation, followed by swim-up. Main Outcome Measure(s) Qualitative detection of HIV-1 RNA in blood plasma and in different parts of semen preparation by using RT-nested PCR, PCR inhibition control by dilution of samples, and an internal control. Result(s) The detection limit of our PCR was 20 HIV-1 RNA copies per milliliter. Among seropositive patients, RNA was detected in 25% of fresh semen, 36.5% of seminal plasma, 27.5% of gradient supernatants, and 7.1% of final preparations before the migration–sedimentation stage. Positive final preparations were observed in patients who had blood viral loads of ≥20,000 HIV-1 RNA copies per milliliter. Inhibition was present in 17.6% of seminal plasma and in 20% gradient supernatants and in 2 final preparations among 69 tested. Among 25 preparations tested after the migration-sedimentation stage, 2 were positive (1 patient; 70,000 HIV-1 RNA copies per milliliter). Conclusion(s) The RT-nested PCR detects low viral load and allows the validation of semen preparations of HIV-1–seropositive patients for ART on the day of sampling. For this purpose, the validation is performed on spermatozoa that are obtained after gradient separation before swim-up. Inhibition of the PCR must be controlled by using an internal control that is well-designed to explore the detection limit of the method.

The embryo-maternal dialogue that starts very early in the life of the embryo is crucial for its own implantation. A disturbance in this dialogue is the major reason for which 60% of all pregnancies are terminated at the end of the periimplantation period. Many studies have been performed to improve the understanding of the molecular mechanisms involved in this dialogue. Both partners, the mother and the embryo, are equally involved in this exchange of signals. Much progress has been done in understanding the role of (i) chorionic gonadotrophin, (ii) growth factors and cytokines, and (iii) steroid hormones and other mediators, produced either by the embryo, by the mother, or by both, during the peri-implantation period. Today it is clear that their production dictates changes in the endometrium, in the immunological system of the mother and in embryo metabolism, that enable the embryo to implant. Knowledge of the molecular mechanisms involved in the embryo-maternal interaction are reviewed in this article.

Teams practising medically assisted reproduction techniques try to avoid viruses as much as possible. Attitudes towards chronic carriers of viruses are rapidly changing, especially for human immunodeficiency virus (HIV) patients. We focus our attention on the legitimacy of systematic screening before assisted reproductive techniques and the need for specialized approaches including an adapted laboratory for viral hazards as well as the need for a multidisciplinary team. Specificities of HIV, hepatitis C virus (HCV), hepatitis B virus (HBV) carriers and the hypothesis of a reduced fertility potential are discussed. Are male HIV carriers a new indication for assisted reproductive techniques in order to prevent virus transmission? It is largely proven that sperm gradient preparation techniques efficiently decrease viral loads and therefore have a protective effect on contamination risk during assisted reproductive techniques. Although a few thousand assisted reproductive technique cycles were performed in the world for this indication without contamination, it is still too early to demonstrate that this technology is fully safe. Two examples of contaminations during insemination are examined. Many questions remain unresolved, such as the lack of standardized techniques for semen preparation or virus detection or the relative merits of intrauterine insemination or ICSI to prevent HIV contamination during assisted reproductive techniques. The authors plead for well-structured, separate programmes of care linked to research objectives.

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

Purpose: To compare the frequency and the degree of mosaicism in human embryos from Robertsonian translocation (RT) t(13;14) carriers, with embryos from karyotypically normal IVF patients. Methods: FISH analysis of embryos from PGD cycles for RT t(13;14), with probes for chromosomes 13, 14, and 18 (Group I) and of embryos from karyotypically normal IVF patients with probes for chromosomes 13, 18, 21, X, and Y (Group II). Results: The incidence of abnormal mosaic embryos was significantly higher in group I (38/51) as compared with group II (6/45) (χ2: P < 0.01). Furthermore, in group I the percentage of diploid cells per embryo was lower for chromosome 13 and 14 in comparison with 18, while in group II no differences were observed between the five chromosomes analyzed. Conclusions: RT induces a high frequency of mosaicism specifically for the chromosomes implicated in the translocation; the analysis by FISH of two blastomeres is strongly recommended for these patients.

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.

Purpose:Our purpose was to compare the embryo culture performance of two types of petri dishes (Nunc and Falcon).

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.

Cryopreservation of ovarian tissue is now offered as an experimental procedure to preserve the fertility of young patients with a high risk for premature ovarian failure resulting from cancer therapy. This is the only available option to preserve the fertility of prepubertal patients treated with gonadotoxic chemotherapy. At present, thousands of patients all over the world have undergone this procedure with the hope of later restoring their fertility. Although the efficiency of the transplantation of cryopreserved ovarian tissue to restore ovarian function has been established, reports of pregnancy are still very scarce. Here, we describe the second published full-term spontaneous pregnancy after an orthotopic and heterotopic transplantation of cryopreserved ovarian tissue in a 31-year-old woman previously treated by conditioning therapy for bone marrow transplantation for Hodgkin's disease. This birth gives compelling evidence for the graft origin of the gamete and confirms the efficacy of ovarian tissue transplantation in restoring human natural fertility after oncological treatment. This case report stresses the importance of proposing the ovarian tissue cryopreservation procedure to all young patients who require potentially sterilizing treatment, with all alternative options to preserve fertility being duly taken into consideration., SCOPUS: ar.j, info:eu-repo/semantics/published

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.

For couples where one or both partners are infected with human immunodeficiency virus or hepatitis C, the doors to receiving fertility care are opening as a result of better antiviral medication, better long-term prognosis and consequent changes in attitude. In line with this, fertility centres electing to treat couples with blood-borne viral (BBV) infection need to re-examine their policies and procedures to ensure the safety of their staff and both non-infected and infected patients during assisted reproduction treatments. At a time when the European Tissue Directive aims to introduce quality standards for assisted reproduction throughout Europe, we highlight the risks involved when treating patients with known BBV infections and argue that safety cannot be met with any certainty unless samples from such patients are handled within a separate high security laboratory or laboratory area, technically adapted to ensure minimal cross-contamination risk to uninfected gametes and embryos.

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.

BACKGROUND: Little is known about the extent and timing of selection against the embryos that are carriers of unbalanced translocations. METHODS: Fluorescence in-situ hybridization (FISH) with probes for chromosomes 13, 14 and 18 was performed, mostly on day 3, on 69 human embryos which were then allowed to develop further in culture to day 5, from five carriers of Robertsonian translocation (RT) t(13;14). RESULTS: Twelve normal/ balanced blastocysts were replaced in seven consecutive cycles (day 5). Three cycles resulted in clinical pregnancies. The proportion of blastocysts displaying a normal/balanced karyotype was 56%, while only the 20% of blocked embryos were normal/balanced (χ 2 : P < 0.05). All the embryos analysed on day 5, except one, displayed mosaicism. The percentages of diploid cells for chromosomes 13 and 14 were significantly lower than for chromosome 18 (chromosome 13: 49.0 28.0; chromosome 14: 53.0 31.8; chromosome 18: 75.7 20.4; Mann–Whitney test: P < 0.01). The embryos displaying ≥62% of diploid cells for at least two of the three chromosomes analysed, more frequently reached the blastocyst stage (blocked embryos: blastocysts chromosome 13: 43.1 30.3, 64.9 29.0; chromosome 18: 64.9 29.0, 83.0 12.9; Mann–Whitney test: P < 0.01). CONCLUSIONS: Normal/balanced embryos developed better but the proportion of abnormal blastocysts was still high. Preimplantation genetic diagnosis is recommended to select normal/balanced embryos from RT t(13;14) carriers.

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.

A first elective transfer policy of two embryos based solely on embryo morphology was compared to a more restrictive policy transferring two embryos to all patients aged < 35 years with less than three previous cycles to reduce the incidence of multiple pregnancies. With a significant reduction in the number of triple transfers from 72.4 to 44.3%, the delivery rates were similar for both policies, 31 and 32.1%. However, the multiple pregnancy rates per transfer significantly decreased from 12.5 to 7.8% (P < 0.05). Of 99 pregnancies, only 24.2% were multiple including 1% of triplets compared to 40.7% multiple pregnancies including 6.7% of triplets for the first policy. Forty-eight transfers of two average embryos with the new policy were compared to 264 transfers of three average embryos with the old policy. Multiple pregnancy rates per transfer were significantly reduced by a third from 23 to 8% (P < 0.05) without a reduction of the pregnancy rates (42 and 48%). This study demonstrated that elective transfer of two embryos reduced the number of multiple pregnancies without impairing the pregnancy rates even with the transfer of average embryos.

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.

The purpose of our study was to determine the involvement of the L-arginine-NO system in preeclampsia. We studied 26 patients with preeclampsia and 27 normotensive pregnancies. Maternal and cord plasma, urine and amniotic fluid were assayed for nitric oxide metabolites (nitrite and nitrate) using the Griess reaction. Sections of placenta and fetal membranes were immunostained with polyclonal anti-endothelial and anti-neuronal nitric oxide synthase antibodies. The concentration of nitrate in the amniotic fluid of preeclamptic patients (median 10.3 mumol/mg creatinine) was significantly higher (P0.001) than in the normotensive group (5.6 mumol/mg creatinine). Nitrate concentrations in maternal and cord plasma and in urine were similar in the two groups. Endothelial cells of the villi of preeclamptic placentas showed a higher positivity in endothelial nitric oxide synthase immunostaining with respect to normotensive controls. Our results indicate that feto-placental NO production is not reduced in preeclampsia. In contrast, the increased concentrations of NO metabolites in amniotic fluid and the positive immunostaining of endothelial nitric oxide synthase in the placental villi suggest that the placental L-arginine-NO system is up-regulated in preeclampsia.

Flow cytometric (FCM) DNA content measurements were carried out on testicular monocellular suspensions obtained from mice exposed per os to a single dose of 50, 100, 300, 600, and 900 mg/kg body weight (b.w.) of 2-methoxyacetic acid (MAA) in order to investigate its cytotoxic action on germ cells. The effects of MAA were evaluated 2, 7, 14, 28, and 45 d after treatment in terms of altered cell type ratios in FCM fluorescence distribution histograms. Testis weight and histological tissue sections were also analyzed. MAA induced marked changes in the relative percentages of tetraploid and haploid cells, indicating the occurrence of cytotoxic damage on primary spermatocytes. Multiparameter FCM analysis showed that, besides its action on nucleic acid synthesis, MAA can also affect the cellular energy metabolism reflected in an altered mitochondrial mass distribution on round spermatids surviving the MAA treatment. This study demonstrates that rapid and unique FCM procedures can be usefully applied in reproductive toxicology.