The Ratio of X- and Y-Bearing Sperm in Ejaculates of Men with Three or More Children of the Same Sex

Purpose: The present study evaluated the proportions of X-bearing and Y-bearing sperm within the semen of donors who were the declared fathers of three or more sons or daughters. Methods: The proportions of sperm were determined using dual-color fluorescence in situ hybridization to identify the X and Y chromosomes. Results: The only difference observed was in semen volume. There was no increase in the proportion of Y-bearing sperm for men with only sons (49.7 ± 1.3%) or of X-bearing sperm for men with only daughters (44.8 ± 2.6%). Conclusions: A preponderance of either sons or daughters in a family cannot be explained simply by an altered ratio of X-bearing and Y-bearing sperm in the father's semen.     

多色引物原位标记技术快速检测精子染色体

目的:探索和评估应用于精子染色体数目异常检测的多色引物原位标记技术。方法:采用NaOH溶液对精子核进行去浓缩和变性,以非ddNTP阻断的单色及三色引物原位标记技术对正常男性精液标本的4条染色体进行检测。结果:成功地在2.5h内标记了同一精子核内的多条染色体,最佳的染色体同时标记通量为3条,单色以上标记达到99%。结论:该快速多色引物原位标记技术优化了之前的单色精子标记技术,在提高了检测通量的同时,检测也更为简便快速和经济可靠。     

Implications of Sperm Chromosome Abnormalities in Recurrent Miscarriage

Purpose: Our purpose was to assess the existence of sperm chromosome abnormalities in recurrent pregnancy loss in an assisted reproduction program. Methods: In this prospective study, 12 sperm samples from couples undergoing in vitro fertilization with two or more first-trimester spontaneous abortions were analyzed. Diploidy and disomy in decondensed sperm nuclei were assessed for chromosomes 13, 18, 21, X, and Y using two- and three-color fluorescence in situ hybridization. Results: Sex chromosome disomy in sperm samples from recurrent abortion couples was significantly increased compared to that from internal controls (0.84% vs 0.37%). In a subpopulation of seven couples who underwent oocyte donation, mean frequencies for sex chromosome disomy (1%) were even higher and diploidy (0.43%) was also significantly increased. Conclusions: These results suggest an implication of sperm chromosome abnormalities in some cases of recurrent pregnancy loss.     

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.     

引物原位标记技术快速诊断精子染色体非整倍性

目的:探索应用引物原位标记技术分析精子染色体非整倍性的可靠性。方法:采用3mol/LNaOH溶液对精子标本进行快速而有效的预处理,此步骤能够对精子细胞核同时进行去浓缩和变性,分析正常男性的8份精液标本的17条染色体和性染色体的非整倍性。结果:二体频率为0.28%至0.36%,染色体间在二体频率上无显著差异,但是21号染色体的不分离频率比其它染色体要高。结论:引物原位标记技术是一种简便、快速、经济的精子染色体非整倍性分析方法,具有较强的敏感性和特异性。     

Fluorescence In Situ Hybridization with Chromosome Paint Probes: A Novel Approach To Assess Aneuploidy in Human Sperm Nuclei

Purpose: Fluorescence in situ hybridization (FISH) using whole-chromosome paint probes was performed to evaluate disomy and diploidy frequency for chromosomes 1, 18, 19, and 22 in human sperm nuclei. Methods: Ten subjects of proven fertility and normal spermatic parameters were included in the study. A dual-color FISH method was carried out. Results: A total of 157,896 spermatozoa was scored. The mean frequencies of disomic sperm for chromosomes 1, 18, 19, and 22 were 0.22% (range, 0.19 to 0.28%), 0.24% (range, 0.14 to 0.37%), 0.22% (range, 0.17 to 0.30%), and 0.25% (range, 0.21 to 0.29%), respectively. The mean frequency of diploidy was 0.14% (range, 0.09 to 0.18%). No interindividual and interchromosomal variations in the aneuploidy frequency were observed between the different subjects. Conclusions: FISH with whole-chromosome paint probes provides a novel and efficient approach for disomy assessment in human sperm nuclei.     

Interindividual Variations in the Disomy Frequencies of Human Spermatozoa and their Correlation with Nuclear Maturity as Evaluated by Aniline Blue Staining

Objective: To evaluate the importance of interindividual variations in the disomy frequencies of human sperm and their possible correlation with the principal semen parameters.

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

Setting: University-based laboratory for reproductive biology.

Patient(s): Fifty-seven human ejaculates selected at random from a population of men undergoing semen analysis.

Intervention(s): Semen specimens were analyzed, and sperm samples were prepared for fluorescence in situ hybridization.

Main Outcome Measure(s): Semen parameters, including necrozoospermia, global motility, sperm concentration, multiple abnormalities index, and teratozoospermia were evaluated, aniline blue staining was completed, and disomy frequencies for chromosomes 8, 15, 18, X, and Y were determined using fluorescence in situ hybridization.

Result(s): Noticeable differences in disomy frequencies between individuals were observed, and these frequencies were correlated with the degree of nuclear maturity.

Conclusion(s): We hypothesize that the positive correlation can be explained by an abnormality of chromosomal segregation at the time of meiosis that would cause disturbances during the transition of nucleoprotein or by one or several premeiotic abnormalities of chromatin that would perturb both the meiotic process and the construction of definitive proteins.     

Chromosome 21 Detection in Human Oocyte Fluorescence In Situ Hybridization: Possible Effect of Maternal Age

Purpose: The purpose of this study was to evaluate, among 100 uncleaved oocytes, the incidence of numerical and structural chromosome 21 and X abnormalities and to analyze the influence of various factors, such as in vitro (IVF) indications, follicle stimulation protocols, and women's age. Methods: We investigated 150 uncleaved oocytes from 128 patients after an IVF attempt. After cytogenetic analysis (Giemsa) 100 oocytes (66%) were selected for fluorescence in situ hybridization (FISH). Fluorescent probes for human chromosomes X and 21 were used simultaneously according to standard procedures for their hybridization and detection. Results and Conclusions: We analyzed by the FISH protocol 100 metaphase II oocytes with 22 to 25 chromosomes. Our results demonstrate a high rate of disomy for chromosome 21 in human oocytes. Among them, eight were disomic (8%) and three were nullosomic (3%) for chromosome 21. Only one disomy of chromosome X was noted. The various indications of IVF and the different folliculogenesis stimulating protocols did not seem to influence the results but suggested a correlation between the maternal age and the aneuploidy rate of chromosome 21.     

Evaluating sex chromosome content of sorted human sperm samples with use of dual-color fluorescence in situ hybridization

OBJECTIVE: Although most methods for selecting the sex of offspring by sorting spermatozoa are ineffective at shifting the ratio of Y- to X-containing cells, some commercial sources continue to offer such services. Our objective was to evaluate commercially “sorted” samples with use of dual-color fluorescence in situ hybridization and to identify variations in assessment by comparing motile and total sperm populations, donors, observers, and fluorescence in situ hybridization probes.STUDY DESIGN: Cryopreserved sperm from seven anonymous donors were processed as for insemination. Sperm cells from each total sample or motile subfraction were prepared for fluorescence in situ hybridization by incubation with disulfide-reducing agents to expand sperm nuclei. Two sets of X and Y chromosome–specific, fluorophore-labeled deoxyribonucleic acid probes were used. At least 400 nuclei from each preparation were classified independently by three blinded observers. Hybridization efficiency, aneuploidy, and sex chromosome content were evaluated in subsets of five unsorted, five female-oriented, and five male-oriented samples. Total and motile subfractions were compared with eight samples. Fluorescence in situ hybridization probes were compared in five paired unsorted samples.RESULTS: No differences were detected between washed samples and paired motile subfractions. No differences in hybridization and aneuploidy were detected between groups of sorted samples. The Y/X ratio was significantly different between the sorted groups. However, male-oriented samples had a lower Y/X ratio than female-oriented samples did. Observer and probe choice accounted for small but significant variations that did not alter conclusions about the Y/X ratio for sorted samples.CONCLUSION: In a series of 10 sorted samples from one commercial source, dual-color fluorescence in situ hybridization demonstrated a small but significant shift in the sex chromosome ratios among samples. However, this shift was opposite to that expected by the orientation of the sorted samples. (Am J Obstet Gynecol 1997;176:1172-80.)     

Assessing the Chromosome Copy Number in Metaphase II Oocytes by Sequential Fluorescence in Situ Hybridization

Purpose: Aneuploidy in oocytes is the main cause of failed embryo implantation and of miscarriage. At present, only limited data on the prevalence of aneuploidy in freshly collected human oocytes are available and all studies have been performed with conventional methods for karyotyping. In this feasibility study, multiple-hybridization fluorescence in situ hybridization (FISH) was evaluated as an alternative method to determine the number of chromosomes in oocytes. Methods: Fifty-two spare oocytes were collected from 23 patients treated with gonadotropins for intrauterine insemination or intracytoplasmic sperm injection. A conventional dual color FISH approach using mixtures of chromosome-specific standard alpha-satellite probes was applied consecutively to the chromosomes of the same metaphase II oocyte. Mixtures of three to six probes were designed in order to allow chromosome identification based on signal color and centromeric index. Results: One hybridization cycle was possible in 52 uninseminated metaphase II oocytes, two hybridizations in 43 oocytes (82.7%), three hybridizations in 30 oocytes (57.6%), four hybridizations in 27 oocytes (51.9%), and five hybridizations in 15 oocytes (28.8%). Altogether, 591 chromosomes could be marked (47.4% of the entire chromosome complement, 11.4 chromosomes per oocyte). The most important single factor contributing to technical failure was loss of the oocyte from the slide. Conclusions: This feasibility study demonstrates that multiple-hybridization FISH can be used for the assessment of a larger proportion of the chromosome complement in oocyte as compared to previous studies based on FISH.     

Meiotic behavior of the sex chromosomes in a 45,X/46,X,r(Y)/46,X,dic r(Y) patient whose semen was assessed by fluorescence in situ hybridization

OBJECTIVE: To determine the meiotic behavior of a ring Y chromosome in a semen sample from a 45,X/46,X,r(Y)/46,X,dic r(Y) patient and the possible interchromosomal effects of the ring on other chromosome pairs. DESIGN: Retrospective analysis. SETTING: Universitat Autònoma de Barcelona. PATIENT: An oligoasthenoteratozoospermic patient who presented for infertility consultation. MAIN OUTCOME MEASURE(S): The sex chromosome content of spermatogenic cells, meiotic figures, and spermatozoa in the ejaculate and the possible interchromosomal effects on chromosomes 13, 18, and 21 were analyzed by using multicolor fluorescence in situ hybridization. Germ-cell aneuploidies were scored. RESULT(S): X0 cells are meiotically incompetent. All meiotic figures were exclusively XY, and 80% showed unpaired sex chromosomes. A high proportion of postreductional cells were XY (45.5%) or nullisomic for sex chromosomes (13.92%). This percentage decreased in spermatozoa to 14.89% and 27.66%, respectively. A statistically significant increase in X-bearing versus Y-bearing cells both in postreductional cells (23.9% vs. 14.3%) and spermatozoa (41.9% vs. 19.3%) was also observed. Evidence for an interchromosomal effect on chromosome 21 was detected. CONCLUSION(S): Data suggest that this patient had a generalized increase incidence of chromosome anomalies, underscoring the importance of incorporating screening for sperm aneuploidies in genetic analysis of affected patients.     

Effect of maternal age on spontaneous abortion during the first trimester in Northeast China

Objective: We aimed to determine the relationship between fetal chromosomal abnormalities and maternal age among spontaneous first trimester abortions in women in Northeast China.

Methods: We evaluated 497 chorionic villi samples from patients with a history of spontaneous abortion during the first trimester. We divided the samples into five groups according to the maternal age: <25, 25–29, 30–34, 35–39, and ≥40 years. We identified chromosomal abnormalities by fluorescence in situ hybridization.

Results: Among the 497 chorionic samples of spontaneous abortion, 180 (36.22%) had fetal chromosomal abnormalities. Patients aged ≥40 years had a significantly higher percentage (60.61%) of fetal chromosomal abnormalities compared with the other groups. More women in the ≥40 and 35- to 39-year groups had a history of three consecutive miscarriages and 10 kinds of abnormalities. The most frequent aneuploidy was trisomy 22, followed by trisomy 16.

Conclusions: These results revealed that the kinds of fetal abnormalities, numbers of abortions, and chromosomal abnormality rates increased with increasing maternal age. The most common trisomy types in spontaneous abortions were closely related to maternal age. We hypothesize whether the larger probability of chromosomal abnormalities is due to increased mutation rate with maternal age, or due to a worse in-utero conditions.     

Preimplantation genetic diagnosis for aneuploidy screening in patients with unexplained recurrent miscarriages

OBJECTIVE: To determine the aneuploidy rate in embryos of women with idiopathic recurrent miscarriages and to evaluate whether preimplantation genetic diagnosis for aneuploidy screening could be a feasible approach to improve the possibility of successful pregnancy in these couples. DESIGN: Prospective cohort study. SETTING: Tertiary university referral center. PATIENT(S): Women (n = 49) with recurrent idiopathic miscarriages. INTERVENTION(S): In vitro fertilization with preimplantation genetic diagnosis for aneuploidy screening. MAIN OUTCOME MEASURE(S): Ongoing pregnancy rate (PR) and aneuploidy rate. RESULT(S): The aneuploidy rate was, respectively, 43.85% and 66.95% in the younger and older group. The ongoing PR per cycle was 25.71% in the younger and 2.94% in the older patients. CONCLUSION(S): There is no therapeutic evidence to prescribe IVF with or without preimplantation genetic diagnosis for aneuploidy screening for this heterogeneous group of patients.     

Is cytogenetic diagnosis of 46,XX karyotype spontaneous abortion specimens erroneous? Fluorescence in situ hybridization as a confirmatory technique

AIM: Performing the standard cytogenetic technique on spontaneous abortion material is still a valuable tool, but finding a normal 46,XX karyotype can confuse investigators and lead to a problem in diagnosis. This is mainly because it is possible for the female or male conceptus to retain contaminating maternal cells. To address this possibility, we used fluorescence in situ hybridization technique (FISH). X (DXZ1: p11.1-q11.1 region) and Y (DYZ3: p11.1-q11.1 region) chromosome alpha-satellite probes were employed to confirm the karyotypes previously diagnosed as 46,XX by our cytogenetic laboratory, or to verify the occurrence of 'Y chromosome component'. METHODS: Besides conventional long-term tissue cultures and G-bands by trypsin using Giemsa (GTG) bandings, FISH analyses were also performed. RESULTS: A total of 134 spontaneous abortion specimens (singleton gestations) were referred for cytogenetic evaluation, of which 125 specimens were successfully karyotyped. Of these, 20.8% (26/125) had chromosome aberrations; 88.5% (23/26) of these aberrations were numerical and 11.5% (3/26) were structural. The most prevalent numerical anomalies were trisomies 15, 16 and 21, tetraploidies, triploidies and monosomy X. FISH results were obtained for 45 out of 92 cases with 46,XX, of which 2 (4.4%) showed XY signals. CONCLUSIONS: For accurate cytogenetic evaluation of spontaneous abortion materials, an additional technique such as FISH is required in order to confirm the cytogenetic results or to provide an estimate of the error rate in the analysis of miscarriages.     

Separating X-Bearing Human Spermatozoa Through a Discontinuous Percoll Density Gradient Proved to Be Inefficient by Double-Label Fluorescent In Situ Hybridization

Purpose: Double-label fluorescence in situ hybridization (FISH) was used to evaluate the efficiency of separating X-and Y-chromosome-bearing spermatozoa through 12-step discontinuous Percoll gradients. Methods: Liquefied normal semen samples from 10 healthy donors were overlaid onto 25% Percoll and centrifuged. Parts of the sperm pellet were saved as control, while the remaining portion was separated by 12-step Percoll gradient. After centrifugation, the spermatozoa in the 80% Percoll layer were collected. The X:Y ratio of the control and separated spermatozoa was verified by double-label FISH (CEP SOX/SGYprobes) and scored blindly by one observer. Differences in the X: Y ratios between matched groups were analyzed by paired t testa. Results: The overall average labeling efficiency was 99.2%. A significant enrichment (P = 0.02) of X-bearing spermatozoa was obtained in Percoll separated fractions (mean X:Y ratio = 52.2:46.4) compared with the control group (X:Y ratio = 49.5:48.4). Discontinuous Percoll gradients also decreased the proportion of aneuploid spermatozoa (from 1.0 to 0.8%), but the differences were nonsignificant. Conclusions: Discontinuous Percoll separation did increase the X:Y ratio significantly, but the enrichment of X-bearing spermatozoa is insufficient for clinical use in preconceptional sex selection.     

Chromatin Packaging as an Indicator of Human Sperm Dysfunction

Purpose: Understanding the causes of fertilization failure is an important research field in assisted reproductive programs. The present study aimed to evaluated the possible relationship between chromatin packaging quality (CMA₃ staining) and (i) normal morphology and (ii) its ability to predict the functional integrity of spermatozoa in both in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI) treatment programs. Methods: Semen of 140 men from IVF and ICSI couples were analyzed for sperm concentration, motility, morphology, and chromatin packaging (CMA₃). For CMA₃ classification, two cutoff values were used, namely, 44.5%±13 and 1 SD above the mean, i.e., 57.5% (rounded off to 60%). IVF and ICSI data were stratified using three basic cutoff values for CMA₃ staining, namely, <44%, >44–60%, and >60%. Results: Based on CMA₃ results patients were divided into four groups, namely, group A, <44% CMA₃ (n = 15, IVF); group B, 44% and <60% CMA₃ (n = 39, IVF); group C, 60% CMA₃ (n = 45 IVF); and group D, 60% CMA₃(n = 41 ICSI). During receiver operator characteristic analyses the estimated cutoff value for CMA₃ staining, to distinguish between <4% and 4% morphology groups, was 60%. The area under the curve was 0.89, sensitivity of 75%, and specificity of 100%. When IVF rates of >60% and <60% were used, the optimal CMA₃ value for prediction of fertilization success again was recorded at 60%. The area under the curve was 0.76, sensitivity of 81.5%, and specificity of 63.6%. Conclusions: Chromatin packaging assessments should be included as a complementary assay to the sequential diagnostic approach of the male-factor patients.     

人精子2种扫描电镜样品制备方法的比较

目的:比较人精子2种扫描电镜样品制备方法的效果。方法:10例精液标本,同一例标本分别采用扫描电镜一般样品制备程序(方法A,A组)和本实验室方法(方法B,B组)处理。方法:B组减少了固定后洗涤精子、脱水和醋酸异戊脂置换的时间,增加了多级乙醇浓度梯度。观察2组开裂精子率的差别。结果:应用2种标本制备程序处理的各例标本,均可观察到开裂或断裂的精子。裂开部位可发生在精子头部、颈部或尾部,以发生在头部为多见,其次为颈部。A组的开裂精子率为28.9±16.2%,B组的为14.1±7.7%,B组的显著少于A组(P<0.01)。结论:方法B的程序更适合于人精子标本的制备,可减少精子表面的开裂或结构断裂,有利于人精子超微形态学观察。     

畸形精子症患者精子染色体非整倍体的研究

目的:研究畸形精子症患者精子染色体的非整倍体率。方法:应用18号、X和Y染色体着丝粒探针,采用荧光原位杂交(FISH)技术比较畸形精子症患者(畸精组,n=18)和生育力正常且精子正常形态率、浓度、活力等均正常男性(对照组,n=5)精子中18号、X和Y染色体的非整倍体率。结果:畸精组共计数精子58 178条,对照组共计数精子16 369条。畸精组和对照组杂交效率分别为97.5%和98.3%;染色体非整倍体类型主要有二体(XX18、YY18、XY18、Y1818和X1818)和二倍体(1818XX、1818YY、1818XY)。畸精组和对照组的18号染色体二体率分别为0.29±0.16%和0.03±0.02%,性染色体二体率分别为0.65±0.24%和0.05±0.02%,二倍体率分别为0.14±0.12%和0.04±0.03%。18号、X和Y染色体非整倍体率组间均有统计学差异(P<0.05)。结论:与生育力和精液各参数均正常男性相比,畸形精子症患者18号、X和Y染色体非整倍体率明显升高。     

Incidence of Chromosomal Abnormalities from a Morphologically Normal Cohort of Embryos in Poor-Prognosis Patients

Purpose: Preimplantation genetic diagnosis of aneuploidy was performed on the embryos yielded by 70 poor-prognosis patients, with the aim of transferring those with a normal chromosomal complement, thus possibly increasing the chances of pregnancy. Methods: Multicolor fluorescence in situ hybridization (FISH) was applied for the simultaneous detection of chromosomes X, Y, 13, 16, 18, and 21. Inclusion criteria were (1) a maternal age of 36 years or older (n = 33), (2) three or more previous in vitro fertilization cycles (n = 20), and (3) an altered karyotype (n = 17). Results: A total of 412 embryos underwent FISH, resulting in 234 (57%) that were chromosomally abnormal. Euploid embryos were available for transfer in 59 patients, generating 19 pregnancies (32%), with an implantation rate of 19.9%. Conclusions: High rates of chromosomally abnormal embryos in poor-prognosis patients can determine repeated in vitro fertilization failures when embryo selection is performed on the basis of morphological criteria alone. Hence, the FISH analysis could represent the prevailing approach for the identification of embryos possessing full potential for developing to term.     

Relationship of Total Motile Sperm Count and Percentage Motile Sperm to Successful Pregnancy Rates Following Intrauterine Insemination

Purpose:This study sought (i) to investigate the relationship between postwash total motile sperm count and postwash percentage motile sperm in predicting successful intrauterine insemination and (ii) to determine the minimal postwash total motile sperm count required to achieve pregnancy with intrauterine insemination.Methods:Five hundred four women, who underwent 1636 intrauterine insemination cycles with their partner's sperm for infertility treatment from 1993 through 1995, were included in this retrospective study. All patient charts were reviewed for age, infertility etiology, ovarian stimulation regimens, semen characteristics, and treatment outcome. To determine the relationship between total motile sperm count and intrauterine insemination outcome, patients were grouped as (1) less than 0.5 million, (2) 0.5 to 1 million, (3) 1 to 5 million, (4) greater than 5 million, and (5) greater than 20 million.Results:Similar live birth rates (per cycle) were seen among the postwash total motile sperm count groups: group 1, 3.5%; group 2, 2.4%; group 3, 7.0%; group 4, 6.9%; and group 5, 7.0% (P = 0.37). However, regardless of the postwash total motile sperm count, the postwash motility predicted intrauterine insemination success at a cutoff value of 40%.Conclusions:The percentage of postwash sperm motility, and not the postwash total motile sperm count, can predict successful intrauterine insemination outcome. Such information can be useful in counseling patients regarding their chance of success with intrauterine insemination and in determining when alternate methods of assisted reproduction may be a better approach.