Chromosome 15 aneuploidy in the sperm and conceptus of a sibling with variable familial expression of round-headed sperm syndrome

Objective: To characterize rates of chromosome aneuploidy in sperm from three siblings, one of whom had an IVF/ICSI conceptus with trisomy 15.

Design: Blind evaluation of the sperm chromosome aneuploidy rates, semen quality, and sperm ultrastructure.

Setting: IVF clinic and university-based andrology research laboratory.

Patient(s): Three brothers, two of whom underwent infertility evaluation and therapy.

Main Outcome Measure(s): Semen from three siblings was coded and blindly evaluated for standard World Health Organization semen quality variables and sperm ultrastructure. Sperm were decondensed and hybridized with fluorescent probes specific for chromosomes X, Y, 13, 15, 18, and 21, then evaluated microscopically to determine the aneuploidy rate for those chromosomes.

Result(s): Two siblings had increased round-headed morphology on standard morphology evaluation, which was confirmed using electron microscopy. The sperm aneuploidy rate was significantly increased for chromosome 15 in sibling 1, the father of a conceptus with trisomy 15. Aneuploidy rates were also slightly increased for chromosomes X, Y, and 18 in sibling 1.

Conclusion(s): This is the second report of increased sperm chromosome aneuploidy in infertile patients with round-headed sperm. Although ICSI is successful in treating this syndrome, the risk for aneuploidy of the conceptus may be increased. Other studies have reported an increased incidence of sperm chromosome aneuploidy in some infertile patients, but this is the first report of aneuploidy in both the sperm and conceptus of a patient undergoing IVF/ICSI.     

Increased chromosome X, Y, and 18 nondisjunction in sperm from infertile patients that were identified as normal by strict morphology: implication for intracytoplasmic sperm injection.

OBJECTIVE: To determine the incidence of nondisjunction for chromosomes X, Y, and 18 using fluorescence in situ hybridization (FISH) on morphologically normal sperm from infertile men who are candidates for ICSI. DESIGN: After standard hematoxylin staining, sperm with normal morphology were identified using Kruger's strict morphology criteria. The location of each normal-appearing sperm was recorded using an electronic microstage locator. Slides were subsequently subjected to FISH for detection of chromosomes X, Y, and 18 (control probe). Nuclei were relocated and analyzed under the fluorescent microscope. SETTING: University-affiliated IVF and intracytoplasmic sperm injection program. PATIENT(s): Men classified as infertile on the basis of abnormal strict morphology (<4% by Kruger's criteria). For controls, normal fertile men (n=6) were also analyzed. INTERVENTION(S): Semen smears were obtained retrospectively from infertile (n=8) and fertile (n=6) men. MAIN OUTCOME MEASURE(s): Ploidy of each cell was determined according to the number of signals detected for each probe. RESULT(S): Approximately 100-150 morphologically normal sperm were identified and located in each case. Subsequent FISH analysis of these normal sperm showed aneuploidy to range from 1.8% to 5.5% in the infertile group as compared with 0 to 2.6% among the control fertile group. Statistically significant differences in the incidence of aneuploidy for the sex chromosomes as well as for all three (X, Y, and 18) chromosomes was observed. CONCLUSION(S): Although 95% to 98% of the sperm were found to be normal for X, Y, and 18, our findings show that infertile couples undergoing ICSI are likely to be at an increased risk for having a genetically abnormal conceptus as compared with the fertile controls. These results demonstrate that normal morphology is not an absolute indicator for the selection of genetically normal sperm. Hence, observed pregnancy failures among ICSI patients may in part be due to the selection of aneuploid sperm.     

Increased sperm nuclear DNA damage in normozoospermic infertile men: a prospective study

OBJECTIVE: To evaluate levels of sperm nuclear DNA damage in infertile men with normal and abnormal standard semen parameters. DESIGN: Prospective study. SETTING: Male infertility clinic. PATIENT(S): Ninety-two men seeking infertility treatment and 16 fertile volunteers. INTERVENTION(S): Standard semen analysis was performed according to the World Health Organization guidelines. MAIN OUTCOME MEASURE(S): Sperm DNA damage was assessed by sperm chromatin structure assay and the results expressed as %DFI. RESULT(S): Of the 92 patients, 21 (23%) had normal standard sperm parameters (concentration, motility, and normal sperm forms), while 71 (77%) had an abnormality in one or more of these parameters. The %DFI [median (25th and 75th percentiles)] in infertile men with normal sperm parameters [23 (15, 32)] was significantly higher than fertile donors [15 (11, 20)] (P=.02), but not significantly different from infertile men with abnormal sperm parameters [28 (18, 41)] (P=.27). CONCLUSION(S): The results of this study indicate that a significant increase in SCSA-defined DNA damage can be found in sperm from infertile men with normal standard sperm parameters. Therefore, sperm DNA damage analysis may reveal a hidden abnormality of sperm DNA in infertile men classified as idiopathic based on apparently normal standard sperm parameters.     

Incidence of sperm aneuploidy in relation to semen characteristics and assisted reproductive outcome.

OBJECTIVE: To evaluate the incidence of sperm aneuploidy in men screened for infertility and identify any eventual relation with assisted reproductive outcome. DESIGN: Controlled prospective study. SETTING: University hospital-based IVF program. PATIENT(S): Infertile couples who were screened for sperm aneuploidy and evaluated for IVF treatment. INTERVENTION(S): Fluorescence in situ hybridization was used to identify chromosomes 18, 21, X, and Y. The assisted reproductive techniques of IVF and intracytoplasmic sperm injection were used for infertility treatment. MAIN OUTCOME MEASURE(S): The incidence of sperm aneuploidy, semen parameters, fertilization rate, pregnancy characteristics, and rate of neonatal malformations were determined. RESULT(S): Oligozoospermic and teratozoospermic men had a significantly higher incidence of chromosomal abnormalities than men with normal semen parameters (2.7% vs. 1.8%). The increased frequency of sperm aneuploidy did not appear to affect pregnancy losses or the occurrence of neonatal malformations. CONCLUSION(S): Suboptimal semen samples had a higher incidence of aneuploidy. In this study, the increased frequency of chromosomal abnormalities did not have a direct effect on the fertilization rate, pregnancy characteristics, or neonatal outcome.     

Characterization of aneuploidy rates, protamine levels, ultrastructure, and functional ability of round-headed sperm from two siblings and implications for intracytoplasmic sperm injection

OBJECTIVE: To characterize and contrast protamine levels, chromatin decondensation, chromosome aneuploidy rates, and functional ability of round-headed sperm from two brothers with round-headed sperm syndrome. DESIGN: Analysis of semen samples from siblings with round-headed sperm syndrome and comparison with semen of fertile donors. SETTING: University school of medicine and laboratories. PATIENT(S): Two infertile siblings. MAIN OUTCOME MEASURE(S): Sperm aneuploidy rates of chromosomes X, Y, 13, 18, and 21, protamine 1 and 2 (P1-P2) ratios, Western blot evaluation of protamines, and chromatin decondensation rates. Additional measures include standard semen quality parameters, electron microscopy ultrastructure evaluation, analysis of acrosome morphology, and sperm penetration rates. RESULT(S): Aneuploidy rates were significantly increased in sibling no. 1 but not in sibling no. 2. The levels of P1 and P2 were decreased in sibling no. 1, and an unusual level of protamine precursors was present. Ultrastructural differences also were observed between the siblings. CONCLUSION(S): These data indicate profound differences in sperm from two siblings with complete round-headed sperm syndrome. Multigenic defects and/or variable expression of the syndrome may be responsible for the syndrome and necessitate individual screening of affected individuals because the pattern of expression appears highly variable.     

Acrosin activity as a potential marker for sperm membrane characteristics in unexplained male infertility

OBJECTIVE: To evaluate sperm membrane system integrity in unexplained infertile male subjects with three consecutive conception failures on IUI even though semen clinical parameters were normal. DESIGN: Prospective study. SETTING: Medical biotechnology laboratory, School of Medical Science and Technology IIT Kharagpur, India. PATIENT(S): Twenty-nine patients with unexplained infertility, 17 normal proven-fertile healthy donors, and 21 infertile males with low motility but with other semen parameters remaining normal. INTERVENTION(S): Semen samples were collected from unexplained infertile patients as well as from healthy fertile donors after abstinence of 3-5 days and were analyzed according to World Health Organization guidelines. MAIN OUTCOME MEASURE(S): Release of 5'-nucleotidase (plasma membrane marker), lactate dehydrogenase (mitochondrial marker) and free acrosin, proacrosin, and total acrosin (acrosomal membrane marker). RESULT(S): Plasma membrane integrity and respiratory activity of sperm cells were comparable in all three groups. The proacrosin-acrosin system was adversely affected in unexplained infertile subjects despite high sperm motility. CONCLUSION(S): Total acrosin activity may be considered as a sensitive biochemical marker for clinical evaluation of unexplained infertility in males.     

Sperm aneuploidy and recurrent pregnancy loss

Experiments of double target in-situ hybridization were performed separately for chromosomes 1-17, 8-18 and sex chromosomes on sperm samples from 20 couples suffering from three or more recurrent first trimester abortions. For a subset of this study population, additional experiments of multicolour fluorescence in-situ hybridization for chromosomes 4, 7, 12, 13, 15, 18, 21, and 22, were performed on the bases of the available data from abortive tissue karyotyping. A markedly high rate of sperm disomy (14.5-15.5%) was scored in only two cases. For three other patients, the cumulative disomy rates for chromosomes 1, 17, 8, 18, X and Y also increased but at a lower level (7.8-9.5%). For the remaining 15 patients, the frequency of sperm aneuploidy was moderately increased or normal. Men with recurrent pregnancy loss (RPL) and poor semen quality had baseline sperm aneuploidy and diploidy rates higher than men with normal semen parameters (with or without RPL). Using probes for chromosomes 1, 17, 8, 18, X and Y, significantly elevated frequencies of sperm aneuploidy (not diploidy) were found in 10% of men with a history of RPL. Their rate of sperm aneuploidy was 30-34%. For the other men, changes in sperm aneuploidy were not thought to affect RPL. Poor semen quality per se impacted negatively on sperm aneuploidy and diploidy, thus making the interpretation of clinical data more difficult.     

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.     

Balanced chromosomal translocations in men: relationships among semen parameters, chromatin integrity, sperm meiotic segregation and aneuploidy

Purpose

To analyse relationships between semen parameters, sperm chromatin integrity and frequencies of chromosomally unbalanced, disomic and diploid sperm in 13 Robertsonian and 37 reciprocal translocation carriers and to compare the results with data from 10 control donors.

Methods

Conventional semen analysis, Sperm Chromatin Structure Assay and FISH with probes for chromosomes involved in the individual translocations and for chromosomes X, Y, 7, 8, 13, 18 and 21.

Results

Normal semen parameters were found in 30.8 % of Robertsonian and 59.5 % of reciprocal translocation carriers. The rates of unbalanced sperm were 12.0 % in Robertsonian and 55.1 % in reciprocal translocation carriers with no difference between normospermic patients and those showing altered semen parameters. Significantly increased frequencies of spermatozoa showing defects in chromatin integrity and condensation, aneuploidy for chromosomes not involved in a translocation and diploidy were detected in translocation carriers with abnormal semen parameters. Normospermic reciprocal translocation carriers showed an increase in chromosome 13 disomy compared to the control group. There was no relationship between gametic and somatic aneuploidy in 12 translocation carriers studied by FISH on sperm and lymphocytes. The frequency of motile sperm was negatively correlated with the frequency of sperm showing disomy, diploidy and defective chromatin condensation.

Conclusions

Abnormal semen parameters can serve as indicators of an additional risk of forming spermatozoa with defective chromatin and aneuploidy in translocation carriers.     

Elevated sperm chromosome aneuploidy and apoptosis in patients with unexplained recurrent pregnancy loss

OBJECTIVE: To evaluate sperm chromosome aneuploidy and semen quality in 24 partners of women with unexplained recurrent pregnancy loss and to analyze the data in relation to sperm apoptosis data. METHODS: Semen quality parameters and sperm chromosome aneuploidy for chromosomes X, Y, 13, 18, and 21 were evaluated in the recurrent pregnancy loss patients, fertile controls, and a control group of men from the general population. RESULTS: The mean aneuploidy rate in the recurrent pregnancy loss group was 2.77 +/- 0.22, significantly higher (P <.005) than in either the general population (1.48 +/- 0.12) or in fertile (1.19 +/- 0.11) control groups. In the recurrent pregnancy loss patients, the percentage of aneuploid sperm was correlated to the percentage of apoptotic sperm (r =.62, P <.001). Normal morphology was diminished in the patient group, compared with the general population group (P <.01) and the donor group (P <.001). CONCLUSIONS: These data indicate that some recurrent pregnancy loss patients have a significant increase of sperm chromosome aneuploidy, apoptosis, and abnormal sperm morphology. This study demonstrates a new possible cause of recurrent pregnancy loss.     

Defective function of a nongenomic progesterone receptor as a sole sperm anomaly in infertile patients.

OBJECTIVE: To compare the function of a novel nongenomic progesterone (P) receptor on the human sperm surface (mediating the P-induced acrosome reaction) in spermatozoa from fertile donors and from infertile patients. To examine the possible implication of defective P receptor function as an etiologic factor in unexplained male infertility. DESIGN: Progesterone binding and P effects were assessed in sperm from infertile patients and compared with corresponding parameters for sperm from healthy donors. SETTING: Private hospital, medical research center, and a university-based andrological laboratory. PATIENTS, PARTICIPANTS: Sperm samples were from infertile patients (no pathology detected in their wives) attending our infertility clinic and from healthy sperm donors. INTERVENTIONS: None. MAIN OUTCOME MEASURES: Progesterone binding sites were visualized with a fluorescein-labeled protein-P conjugate. Indo 1-AM (a fluorescent indicator of intracellular free Ca2+) was used to measure P-induced Ca2+ influx. Progesterone-induced acrosome reaction was monitored after acrosomal staining with Pisum sativum agglutinin. RESULTS: Among 8 patient sperm samples with normal spermiogram values (of 53 examined), 5 showed a reduced percentage of P-binding spermatozoa and an abnormal response to the hormone in terms of Ca2+ influx and the acrosome reaction. CONCLUSIONS: Defective function of a sperm surface P receptor is described in some cases of male infertility. The observed fluorescence microscopic patterns of hormone binding may be used to further investigate receptor activity in unexplained male infertility.     

Aneuploidy frequencies in semen fractions from ten oligoasthenoteratozoospermic patients donating sperm for intracytoplasmic sperm injection.

OBJECTIVE: To determine aneuploidy frequencies in pellet and swim-up semen fractions from 10 infertile men with severe oligoasthenoteratozoospermia (OAT) who were donating sperm for intracytoplasmic sperm injection and to determine whether the swim-up isolation method would successfully separate aneuploid from haploid sperm. DESIGN: Prospective study. SETTING: Infertility clinic and molecular genetics laboratory. PATIENT(S): Ten patients with severe OAT. INTERVENTION(S): Cytogenetic analyses by fluorescence in situ hybridization to determine aneuploidy frequencies for chromosomes 1, 13, 18, 21, X, and Y in sperm from swim-up and pellet fractions. MAIN OUTCOME MEASURE(S): Gametic aneuploidy was scored in sperm fractions separated by the swim-up technique and clinical results after intracytoplasmic sperm injection were tabulated. RESULT(S): In all cases, chromosome aneuploidy levels in patients were significantly greater than in controls. The type and percentage of aneuploid sperm for all patients with OAT found in both swim-up and pellet fractions were not different, with the exception of diploid sperm, which remained in the pellet fraction. After ET, 2 (20%) of 10 couples achieved successful pregnancies. CONCLUSION(S): The data show significantly higher rates of diploidy, autosomal disomy and nullisomy, sex chromosome disomy and nullisomy, and total aneuploidy in sperm from all separated fractions obtained from all patients with OAT versus controls. This patient population with OAT may be at increased risk of producing aneuploid offspring.     

不育症患者精子X,Y及18染色体的荧光原位杂交分析

目的:分析不育症患者精子X,Y,18染色体的荧光原位杂交情况。方法:在男性不育症组中,2例无精子症患者从附睾抽吸获取精子、3例从睾丸获取精子、2例严重少精症患者从射出精液中找到精子。选择5例正常射出精液者作为对照组。应用荧光原位杂交法(FISH)检查精子X,Y以及18号染色体,比较两组精子染色体非整倍体的发生率。结果:不育症组睾丸精子、附睾精子的非整倍体率无差别,但不育症组精子与正常男性组精子比较,非整倍体总发生率、性染色体二体性率及缺对染色体率明显增高(2.8％vs0.58％,0.81％vs 0.19％,2.1％vs0.37％),P<0.001。结论:无精子症与严重少精子症患者的精子比正常精子具有更高的染色体非整倍体率,需要进行大样本的研究,为不育症患者的治疗和遗传咨询提供有效的证据。     

Single cell analysis of DNA in more than 10,000 individual sperm from men with abnormal reproductive outcomes

Purpose

This pilot study sought to (1) validate the use of a novel technology for single-sperm-cell genome sequencing (Sperm-seq) in infertile men who may not have optimal quantity or quality of sperm for genomic analysis and (2) compare these results to fertile donors.

Methods

Infertile men undergoing IVF with female partners with a previous history of failed fertilization with ICSI (FF) or poor blastulation of embryos (PB) were recruited from a large IVF center. Sperm-seq was used to analyze thousands of individual sperm and was carried out at an affiliated university research institute. Global aneuploidy rate, crossover locations, and crossover frequencies were assessed in the infertile population, and compared with a control group of 20 fertile donors, which were analyzed previously at the same laboratory.

Results

Eight patients were initially included, but 3 samples did not yield high-quality genomic data for analysis. A total of 10,042 sperm were analyzed from 5 patients, 2 in the FF group, and 3 in the PB group. The global aneuploidy rate among the samples was 2–4%, similar to the control group. Likewise, crossover locations and frequencies were similar.

Conclusion

Sperm-seq provides a robust analysis but may not be applicable to all male infertility cases due to technical limitations. This group of male infertility patients did not have higher rates of aneuploidy or abnormal crossover patterns compared to a fertile donor population. Our data may suggest that FF and PB phenotypes may not be related to sperm aneuploidy or meiotic errors but rather to other intrinsic nuclear anomalies.

     

Correlations between two markers of sperm DNA integrity, DNA denaturation and DNA fragmentation, in fertile and infertile men

OBJECTIVE: To evaluate two different assays of human sperm DNA integrity, DNA denaturation (DD) and DNA fragmentation (DF), and to correlate these with standard semen parameters. DESIGN: Prospective, observational study. SETTING: University infertility clinic.Patient(s): Forty consecutive semen samples from 33 nonazoospermic men presenting for infertility evaluation and 7 fertile men presenting for vasectomy. Intervention(s): Assessment of sperm concentration, motility, morphology, DD and DF. MAIN OUTCOME MEASURE(S): Sperm DD and DF in fertile and infertile men. RESULT(S): The mean (+/-SE) rates of DD and DF were significantly higher in infertile subjects compared to fertile controls, respectively: 25.4 +/- 3.0 vs. 10.2 +/- 2.3 (P=.028) and 27.6 +/- 2.5 vs. 13.3 +/- 2.5% (P=.016). DF and DD correlated strongly (r = 0.71, P<.0001). Also, DD and DF correlated negatively with standard semen parameters (concentration, motility, and morphology), the strongest correlation being with sperm motility. CONCLUSION(S): The strong correlation between sperm DD and DF, and the higher levels of sperm DNA damage in infertile compared with fertile men, indicate that male infertility is associated with poor sperm DNA integrity. Although infertile men may father children with assisted conception, fertilization with DNA-damaged spermatozoa may increase the risk of genetic disease in the offspring.     

Low acrosin activity in a subgroup of men with idiopathic infertility dose not correlate with sperm density, percent motility, curvilinear velocity, or linearity

The authors compared sperm acrosin activity with sperm density and cell motion characteristics in 21 normal fertile men and 25 patients with unexplained infertility. Under standardized and optimized conditions of abstinence and semen sample processing, we measured sperm acrosin activity in washed sperm from direct aliquots of semen and in aliquots of semen filtered through glasswool to remove dead cells and debris. Using washed sperm from semen, sperm acrosin levels in infertile men (median, 44 microIU/10(6) sperm) were significantly lower than values measured in fertile men (median, 67 microIU/10(6) sperm, P less than 0.01). After glasswool filtration, sperm acrosin activity was higher for both fertile and infertile men. Using washed sperm, 7 of 25 patients had acrosin activity consistently below values measured for fertile men; after glasswool filtration, values for 8 of 14 patients were below the normal range. For either fertile or infertile men, sperm acrosin activity showed no correlation with sperm density, percent motility, or either motion characteristic of curvilinear velocity (Vc40 microns/sec) or linearity (L3); and further, the low sperm acrosin activity of some infertile patients did not correlate with the motion co-characteristics measured at Vc40/L3, and the majority of patients with slower and/or less directional sperm had normal acrosin activity. From our data, we therefore conclude that sperm acrosin activity is independent of sperm motion characteristics.     

Estimation of aneuploidy levels for 8, 15, 18, X and Y chromosomes in 97 human sperm samples using fluorescence in situ hybridization

Objective: To estimate the mean frequency of aneuploidy levels of chromosomes 8, 15, 18, X, and Y in human sperm, while minimizing the effect of individual factors by analyzing sperm samples from a large set of patients.

Design: Prospective randomized analysis of sperm nuclei by fluorescence in situ hybridization.

Setting: University-based laboratory for reproductive biology.

Patient(s): One hundred two patients with a large distribution of sperm parameters, randomly selected from volunteers who had presented seeking a semen analysis.

Intervention(s): The sperm samples were prepared for fluorescence in situ hybridization.

Main Outcome Measure(s): The disomy frequencies for chromosomes 8, 15, 18, and sex chromosomes were determined using fluorescence in situ hybridization.

Result(s): The mean frequencies of disomy for autosomes were 0.18% for chromosome 8, 0.06% for chromosome 15, 0.2% for chromosome 18, and 0.24% for gonosomes (XX, 0.04%; YY, 0.05%; XY, 0.15%).

Conclusion(s): This study confirms other previous evaluations on restricted numbers of patients. Our results seem to confirm a relative equiprobability of disomy frequencies concerning the different chromosomal pairs during male meiosis.     

Novel association between sperm reactive oxygen species production, sperm morphological defects, and the sperm deformity index

OBJECTIVE: To examine the relationship between sperm reactive oxygen species (ROS) production and sperm morphology in a group of infertile men and healthy fertile donors. DESIGN: A prospective clinical study. SETTING: Male infertility clinic, Glickman Urological Institute, The Cleveland Clinic Foundation, Cleveland, Ohio, and the Reproductive Medicine Unit, Liverpool Women's Hospital, United Kingdom PATIENT(S): Thirty-nine infertile men and 13 healthy fertile donors (control). INTERVENTION(S): Standard semen analysis, seminal leukocyte concentration, assessment of sperm morphology, and measurement of sperm ROS production. MAIN OUTCOME MEASURE(S): Levels of sperm ROS production, percentages of different sperm morphological abnormalities, and the sperm deformity index (SDI) scores. RESULT(S): A significant negative correlation was observed between sperm ROS production and the proportion of sperm with normal morphology and borderline morphology. Reactive oxygen species production was positively correlated with the proportion of sperm with amorphous heads, damaged acrosomes, midpiece defects, cytoplasmic droplets, tail defects, and SDI scores. Logistic regression analysis identified a two-variable model including SDI and percentage sperm motility, which correctly identified 84% of individuals with high seminal ROS and 85% of individuals with low seminal ROS. The model had an overall accuracy of 85%. CONCLUSION(S): The standard semen analysis to assess sperm motility, sperm morphology, and the SDI scores is a useful tool in identifying infertile men with high seminal ROS in infertility clinics where facilities for measuring levels of seminal ROS are not available.     

Efficient combined FISH and PRINS technique for detection of DAZ microdeletion in human sperm

Intracytoplasmic sperm injection (ICSI) now offers an effective therapeutic option for men with male infertility and is believed to allow transmission of genetically determined infertility to the male offspring. Transmission of DAZ (Deleted in Azoospermia) microdeletion is one of the major concerns for oligo and severe oligozoospermia patients. Screening of the Y chromosome microdeletion in the diagnostic work-up of infertile men is mainly done using polymerase chain reaction (PCR) on blood leukocytes. However, there are evidences showing that presence of DAZ in somatic cells might not be indicative of its presence in germ cell lineage. In this report we are going to describe a combined Primed in situ labeling (PRINS) and fluorescence in situ hybridization (FISH) technique to show the localization of DAZ gene as well as Y chromosome centromere on sperm nuclei. PRINS is a combination of FISH and in situ polymerization provides another approach for in situ chromosomal detection. In the present study the PRINS primers specific for DAZ genes and traditional direct labeled centromere FISH probes for Y and X chromosomes were used in order to simultaneously detect DAZ genes and sex chromosome aneuploidy in sperm samples.     

Novel associations between specific sperm morphological defects and leukocytospermia

OBJECTIVE: To examine the relationship between leukocyte concentrations in semen and sperm morphology in a group of infertile men and healthy fertile donors. DESIGN: A prospective clinical study. SETTING: Male infertility clinic at a tertiary care teaching hospital and a reproductive medicine unit at a Women's Hospital in the United Kingdom. PATIENT(S): Fifty-six infertile men and 13 healthy fertile sperm donors (control). INTERVENTION(S): Standard semen analysis, seminal leukocyte concentration, and the assessment of sperm morphology and sperm deformity index (SDI), applying Tygerberg's strict criteria. MAIN OUTCOME MEASURE(S): Granulocyte concentrations in semen, percentages of different sperm morphological abnormalities, and SDI scores. RESULT(S): Leukocyte concentrations were statistically significantly and negatively correlated with the proportion of sperm with damaged acrosomes, cytoplasmic droplet, tail defects, and SDI scores with normal and borderline morphology. The percentage sperm motility was significantly and negatively correlated with leukocytic concentration in semen. However, the leukocytic concentration was not significantly correlated with sperm concentration. CONCLUSION(S): This is the first study to report a significant positive correlation between leukocytospermia and sperm tail defects, acrosomal damage, and high SDI scores. These observations suggest that leukocytospermia is associated with compromised sperm structural integrity.