Morphometric epididymal epithelium analysis in obstructive azoospermic men: Predictive values in fertilization rates

Purpose: Our purpose was to determine the obstructed epididymal epithelium morphometric state and to elucidate the influence of morphometric parameters on the fertilizing ability of aspirated spermatozoa. Methods: During MESA with IVF, epididymal tissue samples were carefully dissected, fixed, and prepared for morphometric analysis. The epithelium of the three regions was studied: head, body, and tail (when present). The total area/luminal area ratio, epithelium thickness, and stereocilium length were measured. Results: We have found significant differences in the total area/luminal area ratio, epithelium thickness, and stereocilium length in obstructed epididymal length >4 versus 0.5–2 cm (P0.01). Epithelium thickness and stereocilium length were significantly lower when no fertilization was observed in IVF. Conclusions: These results suggest that morphometric data of obstructed epididymis epithelium can be used as a means of explaining the failure of IVF with epididymal spermatozoa.     

Detection of aneuploidy rate for chromosomes X,Y and 8 by fluorescence in-situ hybridization in spermatozoa from patients with severe non-obstructive oligozoospermia

Purpose  

To evaluate the frequency of sperm nuclei disomy for chromosomes 8, X, and Y in patients with severe non-obstructive oligozoospermia and to assess possible correlations between sperm nuclei aneuploidy and semen parameters or a particular clinical phenotype.     

Percutaneous epididymal sperm aspiration and short time insemination in the treatment of men with obstructive azoospermia

Objective

To study the efficacy of percutaneous epididymal sperm aspiration (PESA) in combination with short time insemination to treat infertile men with obstructive azoospermia (OA).

Design

Paired randomized controlled trial in which each couple’s cohort of oocytes was divided into two equal groups.

Setting

Center for reproductive care.

Patients

Twenty men with OA.

Interventions

Motile spermatozoa were collected using PESA. Half of the oocytes were used for intracytoplasmic sperm injection (ICSI). The rest were inseminated briefly with PESA sperm in vitro fertilization (IVF). After 4–5 h, the remaining cumulus cells were removed mechanically for second polar body observation to decide whether to apply “rescue” ICSI (RE-ICSI).

Main outcome measures

Rates of oocyte maturation, fertilization, cleavage, and good quality embryos. Numbers of available embryos and good quality embryos were compared between PESA-IVF (using a short incubation protocol + rescue ICSI) group and PESA-ICSI group.

Results

In the short time insemination group, cumulus cells were dispersed by PESA spermatozoa. No second polar bodies were found, so RE-ICSI was done. PESA-IVF + RE-ICSI and PESA-ICSI outcomes were comparable in terms of fertilization rates, 2PN cleavage rate and good quality embryo rates with no statistically significant differences.

Conclusions

PESA sperm without centrifugation could disperse the cumulus cells but were infertile and therefore could substitute for synthetic hyaluronidase. The outcomes of PESA-IVF with rescue ICSI were equivalent to PESA-ICSI. Using spermatozoa obtained by PESA and IVF before RE-ICIS is a viable treatment for men with OA.     

Reproductive potential of fresh and cryopreserved epididymal and testicular spermatozoa in consecutive intracytoplasmic sperm injection cycles in the same patients

Objective: To determine if the cryopreservation of epididymal and testicular spermatozoa alters their reproductive potential by examination of patients who underwent consecutive cycles of ICSI using fresh and then cryopreserved spermatozoa.

Design: Retrospective review.

Setting: Tertiary care university hospital.

Patient(s): One hundred sixty-two consecutive cycles of ICSI were analyzed. Thirteen patients were identified as having undergone treatment with freshly retrieved epididymal spermatozoa; these patients subsequently underwent treatment with spermatozoa cryopreserved from that cycle. Eighteen patients underwent ICSI with freshly retrieved testicular spermatozoa; these patients subsequently underwent treatment with spermatozoa cryopreserved from that cycle.

Intervention(s): None.

Main Outcome Measure(s): Fertilization rates and pregnancy rates.

Result(s): The fertilizing capacity of epididymal spermatozoa remained unchanged after cryopreservation and subsequent thawing, with fertilization rates of 58% and 57% for fresh and cryopreserved spermatozoa, respectively. Testicular spermatozoa, however, showed a significant decrease in fertilizing capacity after cryopreservation when compared with freshly retrieved spermatozoa (52% and 71%, respectively). Pregnancy rates appeared unaffected by the cryopreservation of epididymal spermatozoa (fresh, 3/13; frozen, 2/13) or testicular spermatozoa (fresh, 2/18; frozen, 5/18).

Conclusion(s): This study offers further evidence that motile epididymal spermatozoa retain their fertilizing capacity after cryopreservation. The data presented on testicular spermatozoa suggest that although cryopreservation may reduce the fertilizing capacity of testicular spermatozoa, there is no decrease in pregnancy rates.     

A comparison of the morphology of testicular, epididymal, and ejaculated sperm from fertile men and men with obstructive azoospermia

Objective: To assess the morphology of testicular, epididymal, and ejaculated sperm.

Design: Morphology of the three types of sperm was assessed by using Tygerberg strict criteria.

Setting: The Regional Fertility Center, Royal Maternity Hospital, Belfast, Northern Ireland, United Kingdom.

Patient(s): Thirty-two men with obstructive azoospermia and 10 fertile men.

Intervention(s): Trucut needle testicular biopsy and percutaneous epididymal sperm aspiration under local anesthetic.

Main Outcome Measure(s): Percentages of normal sperm and sperm with head, midpiece, and tail defects for testicular, epididymal, and ejaculated sperm. Testicular sperm morphology in men with obstructive azoospermia was compared with that of fertile men.

Result(s): The percentage of normal testicular sperm (4.3%) differed significantly from the percentages of normal epididymal (10.8%) and ejaculated sperm (9.6%). Testicular sperm morphology in men with obstructive azoospermia did not differ from that in fertile men.

Conclusion(s): Tygerberg strict criteria are not suitable for the assessment of testicular sperm morphology.     

Aneuploidy rates for chromosomes X/Y and 18 among preselected spermatozoa in men with severe teratospermia

Eight infertile men with various degrees of oligoasthenoteratozoospermia and repeated implantation failure were selected for this study due to exceptionally high rates of sperm aneupoidy in their ejaculates. All subjects had normal physical examination, karyotype and serum FSH concentration. Prior to IVF treatment, spermatozoa was collected, processed, micromanipulated and tested for chromosomes X, Y and 18 using fluorescence in-situ hybridization. Aneupoidy rates for chromosomes X, Y and 18 were determined among sperm population selected for normal morphology using high-order magnification light microscopy. A second group of fast motile spermatozoa were collected using an intracytoplasmic sperm injection pipette from the medium–oil interface from microdroplets. The average aneuploidy rates for the three chromosomes were 7.6% (395/5182) in the sperm specimen before selection, 8.7% (116/1326) in the normal morphology selected group and 4.3% (59/1388; P < 0.001) in the fast motile selected group. In conclusion, high-magnification light microscopy aimed at selection of spermatozoa with normal morphology did not affect the aneuploidy rate. On the other hand, fast motile spermatozoa harboured significantly less chromosomal abnormalities (P < 0.001). Preselection of the most rapid sperm subpopulation for intracytoplasmic sperm injection may improve the qualities of the fertilizing spermatozoon.Eight infertile men with various degrees of oligoasthenoteratozoospermia and repeated implantation failure were selected for the study due to exceptionally high rates of sperm aneupoidy in their ejaculates. All subjects had normal physical examination, karyotype and serum FSH concentration. Prior to IVF treatment, spermatozoa was collected, processed, micromanipulated and tested for chromosomes X, Y and 18 using fluorescence in-situ hybridization. Aneupoidy rates for chromosomes X, Y and 18 were determined among sperm population selected for normal morphology using high-order magnification light microscopy. A second group of fast motile spermatozoa were collected using an ICSI pipette from the medium–oil interface from microdroplets. The average aneuploidy rates for the three chromosomes were 7.6% (395/5182) in the sperm specimen before selection, 8.7% (116/1326) in the normal morphology selected group and 4.3% (59/1388, P < 0.001) in the fast motile selected group. In conclusion, high-magnification light microscopy aimed at selection of spermatozoa with normal morphology did not affect the aneuploidy rate. On the other hand, the fast motile spermatozoa harboured significantly less chromosomal abnormalities. Pre-selection of the most rapid sperm subpopulation for ICSI may improve the qualities of the fertilizing spermatozoon.     

Comparison of sex chromosome aneuploidy in spermatozoa of fertile men and those requiring ICSI treatment detected by fluorescence in situ hybridization

OBJECTIVE: To compare the frequencies of aneuploidy for chromosomes X, Y and 18 in spermatozoa of infertile and fertile males, using 3-color fluorescence in situ hybridization. METHODS: Twelve infertile patients who underwent intracytoplasmic sperm injection treatment at Queen's Medical Centre, Nottingham were studied. Three fertile men served as controls. Aneuploidy frequencies in both groups were compared using 2-sample t-tests. RESULTS: A total of 26,615 ad 93,649 cells were scored in the control and infertile groups respectively. The frequencies of diploidy, sex chromosome disomy and chromosome 18 disomy in the fertile (0.11, 0.28 and 0.11%) compared to the infertile males (0.05, 0.18 and 0.06%) were not statistically significantly different. CONCLUSION: Our preliminary data do not indicate an increased risk from paternal origin sex chromosome aneuploidies in ICSI. However, we recommend further investigations of the cytogenetic constitution of spermatozoa from severe male factor patients.     

A study of aneuploidy and DNA fragmentation in spermatozoa of three men with sex chromosome mosaicism including a 45,X cell line

Meiotic segregation of mosaic males with a 45,X cell line has been little examined. In this study, we evaluated the risk of aneuploid gametes using fluorescence in situ hybridization (FISH) and DNA fragmentation in ejaculated spermatozoa of three men with sex chromosome mosaicism including a 45,X cell line. Triple- and dual-color FISH were performed. Sperm DNA fragmentation was detected using the TUNEL assay. A significantly increased frequency of XY disomic spermatozoa was observed for patients (P)1 and P2. A significant increase in diploidy and autosomal aneuploidy was found in P2 and P3, respectively. The rate of DNA fragmentation was not different from that observed in a control group. Data from the literature are scarce (only 3 cases reported), making comparison of the present data difficult, especially as the frequencies of the cell lines comprising the mosaicism differed between patients. Furthermore, the proportion of the different cell lines can differ from one tissue to another in the same patient. Whether the relative levels of the several cell lines present in the mosaicism can influence the rate of aneuploid spermatozoa remains unknown.     

Sperm meiotic segregation, aneuploidy and high risk of delivering an affected offspring in carriers of non-Robertsonian translocation t(13;15)

Purpose

To determine the percentage of unbalanced spermatozoa and an interchromosomal effect in two carriers of balanced translocations t(13;15)(q32;q26) and t(13;15)(q32;p11.2).

Methods

Sperm nuclei analysis by fluorescent in situ hybridization for detection of percentage of unbalanced spermatozoa and sperm with disomy of chromosomes X, Y, 8, 18, 21 and diploidy.

Results

The incidence of unbalanced spermatozoa was 50.5 % and 44.6 % in patient 1 (P1) and patient 2 (P2), respectively. Partial disomy of chromosome 13 was detected in 13.4 % and 21.3 % of sperm in P1 and P2, respectively. The unbalanced karyotype der(15)t(13;15) was found previously in a son of P1 and in two adult relatives, and prenatally in the family of P2. This demonstrates a high risk of delivering an affected offspring. Significantly increased frequencies of chromosomes 8, 18, X and XY disomy and diploidy were observed in P2, which might either indicate an interchromosomal effect or be related to his asthenoteratozoospermia.

Conclusions

Since the proportions of unbalanced spermatozoa and the risk of delivering an affected offspring are high, prenatal or preimplantation genetic diagnosis is recommended for such patients.     

Molecular and cytogenetic characterization of a structural rearrangement of the Y chromosome in an azoospermic man

OBJECTIVE: To better define an abnormal karyotype found in a male with primary infertility. DESIGN: Case report. SETTING: Molecular and cytogenetics unit in a university-affiliated hospital. PATIENT(S): A 41-year-old, azoospermic, but otherwise healthy male. INTERVENTION(S): Lymphocytic karyotype and genetic counseling. MAIN OUTCOME MEASURE(S): Metaphases were studied by standard G- and Q-banding, followed by fluorescence in situ hybridization (FISH) and polymerase chain reaction to analyze specific Y chromosome regions. RESULT(S): Chromosomal analysis and FISH allowed us to define the propositus's karyotype as 45,X/46,X,idic(Yp)/46,XY (71%, 26%, and 3% of analyzed metaphases, respectively). Molecular analysis of azoospermic factor (AZF) regions showed deletion of AZFb and AZFc. CONCLUSION(S): A 45,X/46,X,idic(Yp) mosaicism is associated with a very broad spectrum of phenotypes, including patients with Ullrich-Turner syndrome, patients with various degrees of genital ambiguity, or normal males. In the presence of a normal masculinization in otherwise healthy males azoospermia is a distinct feature that can be explained by partial deletion of AZF regions.