Laura Gambera

Spermatogenesis in a man with complete deletion of USP9Y.

Deletions in the azoospermia factor region AZFa on the human Y chromosome and, more specifically, in the region that encompasses the ubiquitin-specific peptidase 9, Y-linked gene USP9Y have been implicated in infertility associated with oligospermia and azoospermia. We have characterized in detail a deletion in AZFa that results in an absence of USP9Y in a normospermic man and his brother and father. The association of this large deletion with normal fertility shows that USP9Y, hitherto considered a candidate gene for infertility and azoospermia, does not have a key role in male reproduction. These results suggest that it may not be necessary to consider USP9Y when screening the Y chromosome of infertile or subfertile men for microdeletions.

Infertile spermatozoa in a human carrier of robertsonian translocation 14;22.

OBJECTIVE To present the ultrastructural, functional, and chromosomal analyses of spermatozoa from an infertile man with normal phenotype and chromosomal translocation 14;22. DESIGN Case report. SETTING Regional Reference Center for Male Infertility in Siena, Italy. PATIENT(S) A 36-year-old man with primary infertility for 3 years and his parents. INTERVENTION(S) Family history and lymphocytic karyotypes, physical and hormonal assays, and semen analysis. MAIN OUTCOME MEASURE(S) Morphological sperm evaluation was performed by light, fluorescent, and electron microscopy; chromosomal constitution was examined by the fluorescence in situ hybridization (FISH) technique. The penetration ability of spermatozoa was checked by the hamster test. RESULT(S) The spermatozoa of the patient showed unusual ultrastructural defects. The nuclei were large, spheroidal, and generally uncondensed; the acrosomes were frequently absent or reduced; and the axonemes were often devoid of dynein arms or central singlet tubules. These characteristics are related to immaturity. The lymphocytic karyotype revealed a robertsonian translocation 14;22 in the sterile patient and his mother. FISH sperm analysis demonstrated a high frequency of diploidy for the chromosome 18,XY. The hamster penetration test gave negative results. CONCLUSION(S) The unusual structural sperm immaturity is associated with the translocation 14;22. This chromosomal anomaly may therefore negatively influence the spermatogenesis; an interchromosomal effect on meiosis segregation is also suggested.

Ultrastructural and DNA fragmentation analyses in swim-up selected human sperm.

Seventeen sperm samples were evaluated by transmission electron microscopy (TEM) before and after swim-up separation. DNA-fragmentation was tested by terminal d-UTP nick end labeling (TUNEL) in unselected and selected semen samples, and the results were analyzed in relation to sperm ultrastructural characteristics detected by TEM. A significant improvement in mean numbers and percentages of structurally normal sperm was observed after swim-up selection, corresponding to a significant decrease in the percentage of necrotic and apoptotic sperm, while the percentage of sperm with immature nuclei did not change significantly. TUNEL indicated a significant decrease in chromatin-fragmented sperm after swim-up. Swim up selection based on sperm motility excludes many sperm with ultrastructural evidence of necrosis (absent or reacted acrosome, disrupted chromatin, broken plasma membrane) and apoptosis (misshapen nuclei with marginated chromatin), as confirmed by TUNEL analysis. Nevertheless, immature sperm with elliptical or roundish nuclei, misshapen acrosomes and uncondensed chromatin remain part of fertilizing pool.

The impact of environmental exposure to perfluorinated compounds on oocyte fertilization capacity

In an attempt to assess the effect of perfluorinated compounds (PFC) on oocytes quality and fertilization rate, we studied follicular fluid (FF) PFC levels in 18 patients undergoing IVF-ET cycles. A significant correlation (R = 0.75; P < 0.001) was observed between FF PFC levels and fertilization rate. Moreover, patients with FF PFC contamination had significantly lower fertilization rate (p < 0.02) and number of embryos transferred (p < 0.02), compared to the PFC negative group.

Chromosomal Aberrations and Aneuploidies of Spermatozoa

Chromosomal abnormalities are relevant causes of human infertility, affecting 2 -14 % of infertile males. Patients with seminal anomalies could be affected by improper meiotic recombination and increased sperm chromosome aneuploidy. Since the transmission of a haploid chromosomal asset is fundamental for embryo vitality and development, the study of sperm chromosomes has become fundamental because intracytoplasmic sperm injection allows fertilization in cases of severe male infertility.In this chapter we summarize the data on the incidence of sperm aneuploidy, detected by fluorescence in situ hybridization (FISH), in infertile men with normal or abnormal karyotype. The possibility of reducing sperm chromosomal imbalance is also reported.Among control males, the lowest aneuploidy rate was detected (range: 0.09 -0.14 % for autosomes; 0.04 -0.10 % for gonosomes). In infertile patients with normal karyotype, the severity of semen alteration is correlated with the frequency of aneuploidy, particularly for X and Y chromosomes. Among patients with abnormal karyotype, 47,XXY and 47,XYY carriers showed a high variability of sperm aneuploidy both for gonosomes and autosomes. In Robertsonian translocation carriers, the increase in aneuploidy rate was particularly evident for total sex disomy, and resulted mainly from interchromosomal effect (ICE). In reciprocal translocation carriers, a high percentage of unbalanced sperm (approximately 50 %) was detected, perhaps mostly related to ICE.Sperm chromosomal constitution could be analyzed to obtain more accurate information about the causes of male infertility. It would be worthwhile to evaluate the benefits of a therapy with recombinant Follicle Stimulating Hormone (rFSH) on sperm chromosome segregation in selected infertile males.

Correlation between oocyte preincubation time and pregnancy rate after intracytoplasmic sperm injection.

Background and aim. Both nuclear and cytoplasmic maturation of oocytes have to be completed in a coordinated manner to ensure optimal conditions for fertilization. This is well known for in vitro fertilization, but is debated for intracytoplasmic sperm injection (ICSI). It has been reported that preincubation of oocytes prior to ICSI is associated with improved maturation of oocytes, fertilization and embryo quality. Therefore, in the present study, we evaluated the fertilization rate, embryo quality and pregnancy rate in relation to incubation times of metaphase-II oocytes before ICSI. Method. We analyzed 135 selected ICSI cycles. Subjects were assigned to six groups according to oocyte incubation time before ICSI: 2–4 h, 5 h, 6 h, 7 h, 8 h and 9–12 h. Results. We observed that the fertilization rate increased slightly at short (2 to 6 h) and then decreased at longer preincubation times (7 to 12 h). Concomitantly, cleavage rate increased up to 6 h of preincubation and decreased significantly in the groups in which ICSI was carried out after 7 to 12 h of incubation. With regard to clinical pregnancy rate, we observed a significant increase from 2 to 5 h of preincubation, when this parameter reached its maximum value (35%), tapering to 33% after 6 h and then dropping sharply to 12 h. Conclusions. These data confirm that the most appropriate incubation time for mature oocytes before ICSI is 5–6 h. This time improves embryo quality and pregnancy rate in ICSI cycles.

Successful multiple pregnancy achieved after transfer of frozen embryos obtained via intracytoplasmic sperm injection with testicular sperm from an AZFc-deleted man

OBJECTIVE To describe a case of successful triplet pregnancy after testicular sperm extraction (TESE) from a man with AZFc deletion and intracytoplasmic sperm injection (ICSI). DESIGN Case report. SETTING University hospital. PATIENT(S) A 38-year-old man affected by complete AZFc deletion and azoospermia. INTERVENTION(S) Spermiogram, Y-chromosome microdeletion screening, TESE for sperm recovery from testicular tissue on the same day as ICSI, transfer of frozen-thawed embryos, vaginal ultrasound examination. MAIN OUTCOME MEASURE(S) The Y chromosome genetic status of an azoospermic patient who underwent TESE and ICSI, the fertilization and pregnancy outcome. RESULT(S) The patient was found to be azoospermic, and the deletion screening showed complete AZFc deletion. After TESE, the recovered testicular sperm were selected for ICSI. Three good quality embryos were obtained and were frozen due to ovarian hyperstimulation syndrome in the female partner. After transfer of the thawed embryos, a triplet pregnancy was diagnosed by vaginal ultrasonography at the seventh week of gestation. Two male and one female healthy babies were born. CONCLUSION(S) This is the first report of a successful triplet pregnancy after the transfer of frozen-thawed embryos in a couple in whom the male partner was azoospermic and a carrier of complete AZFc deletion. This deletion should not adversely affect a mans TESE retrieval prognosis or the fertilization, cleavage, and implantation of embryos. The offspring were healthy, although the two sons inherited the AZFc deletion.

Sperm aneuploidies after human recombinant follicle stimulating hormone therapy in infertile males.

Errors in sperm chromosome segregation are frequently observed in infertile males. It would therefore be useful to develop methods for reducing the rate of aneuploidy in spermatozoa. Thirty-one males were selected with an elevated frequency of total sperm aneuploidy of sperm chromosomes 18, X and Y by fluorescence in-situ hybridization (FISH): 22 were treated with 150 IU of recombinant FSH on alternate days for 3 months and the other nine (controls) did not receive any hormonal treatment. Before therapy, FISH analysis demonstrated an increased frequency of diploidy (0.663 +/- 0.09%), disomy (0.412 +/- 0.03%) and total aneuploidy (1.30 +/- 0.12%) in the 22 males. Sperm analyses revealed reduced progressive motility (26.73 +/- 2.3%) and a reduced percentage of spermatozoa with normal morphology (23.86 +/- 5.3%). After 90 days of therapy, a significant reduction in aneuploidies (mean total aneuploidy: 0.86% +/- 0.11; P = 0.005) was obtained, as well as an improvement in functional and structural sperm characteristics. In untreated patients, no significant change in semen parameters and frequency of total aneuploidy was observed between baseline (1.054 +/- 0.06%) and 90 days later (1.080 +/- 0.05%). It is therefore suggested that deranged meiotic segregation in spermatozoa could be reduced by FSH treatment.

Protein modification as oxidative stress marker in normal and pathological human seminal plasma

Abstract Objective Our study aims to assess the oxidative stress status of seminal plasma from normozoospermic, azoospermic, and leukocytospermic males, since abnormal sperm and leukocytes in human ejaculates are the main source of reactive oxygen species (ROS) which lead to oxidative damages. For this purpose we applied a biochemical approach to the assessment of the oxidative stress status by using two-dimensional (2D) electrophoresis to check the level of protein oxidation after specific labeling of free thiol (–SH) groups. Methods Seminal plasma samples from normal and pathological males were analyzed by a luminol-based chemiluminescent assay. The same samples after specific labeling of free –SH groups with 3-N-maleimidopropionyl biocytin, were analyzed by 2D electrophoresis and computer-assisted semiquantitative determination of the amount of free –SH groups. Results Using a standard chemiluminescence assay, we demonstrated a high, low and normal level of ROS, respectively, in seminal plasma from leukocytospermic, azoospermic, and normozoospermic subjects. By 2D electrophoresis and streptavidin blotting of specifically labeled free –SH groups of proteins, we detected in the same samples a higher level of oxidated –SH groups comparable between azoospermic and leukocytospermic samples, whereas a significantly higher level of free –SH groups was detected in normozoospermic subjects. Discussion Our results demonstrated that a pathological oxidative stress status in seminal plasma may be revealed by the levels of the protein free –SH groups, both in the presence or absence of cells.

Characterisation of three systematic sperm tail defects and their influence on ICSI outcome

This study characterized three cases of systematic sperm tail defects using electron microscopy and immunolocalisation of centrin 1 and tubulin and explored their impact on ICSI outcome. Structural sperm tail defects of possible genetic origin were suspected as the eosin test revealed a sperm viability of >70% despite severe asthenozoospermia or the absence of motility. In Patient 1, 80%–85% of axoneme cross sections was incomplete. The fluorescent signal of tubulin was weak along the entire tail; the signal of centrin 1 was normal. After ICSI, a female healthy baby was born. Patient 2 showed spermatozoa with tails reduced in length at different levels, axonemal and periaxonemal alterations and fragility of head‐tail junction. Centrin 1 was altered in 80% of sperm. After ICSI, no embryos were obtained. Patient 3 showed tails reduced in length at light and fluorescence microscopy; ultrastructural study revealed a condition of dysplasia of fibrous sheath with heterogeneity of tails’ length. The signal for centrin 1 was altered in 50% of spermatozoa; two embryos were transferred without pregnancy. The correct diagnosis of sperm pathology is important in case of systematic sperm defects as it enables the clinician to improve patients management and to provide an adequate genetic counselling.