Sperm Fluorescence In Situ Hybridization Research Articles

Assessment of sperm chromosomal abnormalities using fluorescence in situ hybridization (FISH): implications for reproductive potential.

Chromosomal abnormalities play an important role in male infertility, which is becoming a significant issue in human fertility. Aim of this study was to evaluate the incidence of spermatic aneuploidies and diploidies in human sperm, according to semen parameters. We performed semen analysis according to the 6th edition of WHO criteria in 50 male subjects; samples weredivided into normozoospermic (n = 23) or thosewith altered seminal parameters (n = 27). To assess chromosomal numerical alterations of sperm, fluorescence in situ hybridization (FISH) was used. A significant increase in aneuploidies and diploidies was observed in samples with altered seminal parameters. Furthermore, stratifying this group, we observed a significant increase in aneuploidies and total abnormalities in oligozoospermic, asthenoteratozoospermic (AT), and oligoteratoasthenozoospermic (OAT) samples compared to normozoospermic. Our results showed the correlation between altered seminal parameters and numerical chromosomal abnormalities, confirming that sperm FISH analysis could be an additional clinical tool to assess reproductive potential in infertile males. Moreover, our results point to the importance of updating the normality ranges for detecting chromosomal aneuploidies using FISH.

Sperm chromosomal abnormalities in infertile men with failed intracytoplasmic sperm injection (ICSI)

BackgroundMale infertility is a major health problem with multi-factorial etiology. Intracytoplasmic sperm injection (ICSI) has revolutionized the treatment of men with severely compromised semen parameters and improved their chances of achieving pregnancy. However, many concerns have been raised about ICSI safety; in part owing to utilizing aneuploid sperms from infertile men, which may be associated with an increased incidence of chromosomal aneuploidies in ICSI pregnancies. The aim of the study was to determine the incidence of sperm aneuploidies for chromosomes 13, 18, 21, X, and Y in semen of infertile males with failed ICSI. This will assist infertile couples and enable them to make informed decisions. For this purpose, sperm aneuploidy frequency for chromosomes 13, 18, 21, X, and Y was evaluated by multicolor fluorescence in-situ hybridization (FISH) in 35 patients with abnormal semen parameters, and 10 normozoospermic healthy men who served as controls.ResultsSperm aneuploidies for chromosomes 13, 18, 21, X, and Y in semen from infertile men with failed ICSI were detected at higher frequencies than controls. Patients had significantly higher disomy, nullisomy, diploidy, and polyploidy frequencies compared to controls.ConclusionsInfertile men with failed ICSI have a significantly increased frequency of sperm chromosome abnormalities compared to controls. Fluorescence in situ hybridization facilitated the study of numerical chromosomal abnormalities in human sperm nuclei (sperm FISH). Sperm FISH, prior to the application of assisted reproductive techniques, saves a considerable amount of time and resources.

P–257 An unknown cause lead to polyspermy in IVF cycles and 0PN zygotes in ICSI cycles in male patient

Abstract Study question The patient sperm has normal morphology and motility, which paternal factors cause the abnormal fertilization in IVF/ICSI and what is the underlying mechanism? Summary answer A genetic mutation of BEX1 and decreased PLC-zeta has been found in patient, which may provide novel insights of polyspermy and pronucleus formation during fertilization. What is known already In mammals, pronucleus formation, a landmark event for fertilization, is critical for embryonic development. Abnormal fertilization refers to the abnormal number of pronucleus and polar bodies in zygotes during in vitro fertilization, with an incidence of 5–15%, among which the incidence of polyspermy and 0PN is about 2–10% and 30%. However, the mechanisms underlying pronucleus formation still unclear. More research has focused on oocyte activation, while paternal relevant abnormal fertilizations have been rarely established. The mechanism of how sperm and/or substances carried by sperm influence the physiological process of fertilization is also unclear. Study design, size, duration In our study, we first work on the preliminary observation and analysis of sperm morphology, structure and sperm chromosome number, and then made further analysis at the genetic level to find out the cause of this particular phenotype in this patient. We use of zone-free golden hamster ova test the fertilizing capacity and rescue the pronucleus formation with SrCl2. Participants/materials, setting, methods The patient, golden hamster, Papanicolaou stain, scanning electron microscope (SEM), Transmission Electron Microscope (TEM), Fluorescence in situ hybridization (FISH), Whole Exome Sequencing (WES), IVF, ICSI, Assisted Oocyte Activation (AOA). Main results and the role of chance During 2016–2018, they did 4 cycle assistant reproduction technology. Cycle1, conventional IVF(C-IVF), 9 MII oocytes, 9 3PN zygotes; Cycle2, ICSI, 10 MII oocytes, 10 0PN zygotes; Cycle3, donor-oocytes C-IVF, 6 MII oocytes, 6 3PN zygotes, and the donor did C-IVF get normal zygotes and embryos; Cycle4, donor-sperm C-IVF, 7 MII oocytes, 4 2PN zygotes, 3 useable embryos. Remarkably, clinical examination about male shows normal sperm semen parameters. Papanicolaou stain and SEM shows that the sperm of the patient has normal morphology. The TEM data shows that the spermatozoa with normal head morphology and intact 9 + 2 sperm flagella structure. In the sperm FISH analysis, Chromosome ploidy is haploid. We performed WES on the male, after exclusion of frequent variants and application of technical and biological filters, two homozygous missense mutations were identified in BEX1 (c.191G>A [p. W64X]), which has been few reports of male infertility. The western blot result show that the PLC-zeta was decreased in patient. After 10mM SrCl2 assisted oocyte activation, the zygote has the pronucleus formation in ICSI. Limitations, reasons for caution At present, we only observe sperm related factors (morphology, structure, chromosome number, genetic mutation). Next step is to detect the substances sperm carried (e.g. RNA-seq, proteomics). In this case, what is of great concern to us is the inconsistencies of the abnormal fertilization during the conventional IVF and ICSI cycles. Wider implications of the findings: Many studies of fertilization mechanism, the main focus is on the maternal cytoplasmic factors, such as the Ca 2+ release initiate the fast block of oocytes. There are few reports about abnormal fertilization due to sperm factors. Our case may offer new insights for the study of fertilization. Trial registration number Not applicable

Frequency of Sperm Aneuploidy in Oligoasthenoteratozoospermic (OAT) Patients by Comprehensive Chromosome Screening: A Proof of Concept.

Background:Embryonic aneuploidy usually results in implantation failure and miscarriage. Considering significantly high frequency of sperm aneuploidy reported in oligoasthenoteratozoospermia (OAT) using fluorescence in situ hybridization (FISH) in limited number of chromosomes and lack of comprehensive chromosome screening (CCS) in OAT, the aim of this study was applying CCS in OAT sperm and comparison of the results with FISH findings.Methods:Five OAT patients with normal blood karyotypes and history of implantation failure were included. The successfully amplified samples, each containing two sperm, were analyzed by array comparative genomic hybridization (aCGH). FISH was utilized mainly depending on the aneuploidies found by aCGH to assess their frequencies in total sperm population.Results:In aCGH for 30 sperm, aneuploidy was found in 66% of samples. Following the study of 4300 sperm by FISH, an average of 55.46% aneuploidy was observed. No pregnancy was resulted with normal partners.Conclusion:Using aCGH, some abnormalities were observed that are not typically considered in sperm FISH studies. Despite small sample size of the comprehensive study, like other similar studies, the frequency of aneuploidies was considerable and similar to FISH. Aneuploidies revealed by aCGH at single sperm resolution were different from sperm population detected by FISH. Considering high frequency of aneuploidy in OATs sperm, preimplantation genetic testing for aneuploidy (PGT-A) can be used for in transfer of chromosomally normal embryos.

Conventional ICSI improves the euploid embryo rate in male reciprocal translocation carriers.

To evaluate whether the morphologically normal spermatozoa selected for intracytoplasmic sperm injection (ICSI) under microscope had a higher rate of normal/balanced chromosome contents than that in the whole unselected sperm from reciprocal translocation carriers. Five hundred unselected spermatozoa from each of 40 male translocation carriers were performed with fluorescence in situ hybridization (FISH), to determine the rates of gametes with different meiotic contents of translocated chromosomes. Meanwhile, 3030 biopsied blastocysts from 239 male and 293 female reciprocal translocation carriers were detected with the microarray technique to analyze the rates of embryos with different translocated chromosome contents. The D3 embryo rate, blastocyst formation rate, and euploid rate of blastocysts were remarkably higher in male carriers than those in female (p = 0.001, p = 0.004, and p = 0.035, respectively). In addition, the percentages of alternate products, which contained normal/balanced chromosome contents, in embryos from male carriers were markedly higher than those in sperm FISH (p = 2.48 × 10-5 and p = 2.88 × 10-10), while the percentages of adjacent-2 and 3:1 products were lower than those in sperm FISH (p = 0.003 and p = 5.28 × 10-44). Moreover, consistent results were obtained when comparing the rates of products in embryos between male and female carriers. Specifically, the incidence of alternate products in male carriers was higher than those in female carriers (p = 0.022). However, no similar differences were seen between sperm and embryos of female carriers. ICSI facilitates the selection of spermatozoa with normal/balanced chromosome contents and improves the D3 embryo rate, blastocyst formation rate, and the euploid embryo rate in male carriers.

Simple FISH-based evaluation of spermatic nuclear architecture shows an abnormal chromosomal organization in balanced chromosomal rearrangement carriers.

Interphasic DNA has a constant three-dimensional conformation, which is particularly striking for spermatic DNA, with distinct chromosomal territories and a constant chromosomal conformation. We hypothesized that this organization is fragile, and that an excess or a lack of chromosomal segments could hinder the genomic structure as a whole. Five human male chromosomal translocation carriers and five controls were included. Spermatic DNA spatial organization was studied, in both balanced and unbalanced spermatozoa, with two-dimensional fluorescent in situ hybridization (FISH) via analysis of chromosomes not implicated in the cases' translocations, compared to that of normal controls. Two parameters were studied: the distance between the two telomeric ends of chromosome 1, and the area of the chromosomal territories of chromosomes 1 and 17. Sperm FISH analysis of rearrangement carriers revealed changes in the nuclear architecture compared to that of controls. Inter-telomeric distance and chromosomal territories areas were both significantly increased. We show that an excess or lack of chromosomal segments can hinder the normal spatial nuclear architecture in sperm. These results show that nuclear architecture is a fragile assembly, and that local chromosomal abnormalities may impact the nucleus as a whole. This suggests a potential avenue for selection of spermatozoa prior to in vitro fertilization, not only in rearrangement carriers but also in the infertile population at large. Furthermore, we suggest that 2D-FISH could possibly be a useful tool in assessing spermatic nuclear organization in a way to evaluate male fertility.

The use of fluorescence in situ hybridization analysis on sperm: indications to perform and assisted reproduction technology outcomes.

To determine the consequences of an altered sperm fluorescence in situ hybridization (FISH) result for ART outcomes and the indications for a sperm FISH analysis. Data from 439 infertile men were collected. Bivariate analyses were performed to determine the association of men's age, seminal alterations, and sperm FISH indication, with the incidence of X, Y, 13, 18, and 21 sperm chromosomal abnormalities. A multivariate logistic regression analysis was performed to establish the most predictive variables for altered sperm FISH. Results from the IVF/ICSI cycles were collected for 248 out of 439 patients. Two distinct groups were established: 151 couples that used their own oocytes and 97 couples involved in egg donation programs. In both groups, ART outcomes were compared between normal and altered sperm FISH. Teratozoospermia and oligozoospermia were associated with sperm chromosome anomalies (p < 0.05). Indications for sperm FISH analysis with the highest predictability were teratozoospermia, male age, oligozoospermia, and implantation failure (AUC = 0.702). Embryo quality (p = 0.096), pregnancy rate (p = 0.054), and implantation rate (p = 0.089) were higher in own-oocytes couples with normal sperm FISH than in altered sperm FISH couples, although differences were not statistically significant. In donor-oocytes couples, in which high-quality embryos were transferred later than in own-oocytes couples (3.8 vs. 3.0 days), we did not identify differences in the ART outcome between normal and altered sperm FISH couples. In both groups, the possible interference of woman age was negligible. Sperm FISH is indicated in middle-aged oligoteratozoospermic patients with implantation failures in previous IVF/ICSI cycles. Sperm chromosome anomalies have a moderate detrimental impact on embryo quality, implantation, and pregnancy rates.

74. NORMAL LIVE BIRTH FROM TWO CARRIERS OF RARE INSERTIONAL TRANSLOCATIONS UNDERGOING PGT

Introduction Despite the use of more precise techniques that indicates greater incidence, meiotic segregation of interchromosomal insertion has rarely been studied. Theoretically, the risk to have a child at term with malformation or mental retardation is high but there are no tools to determine this risk. We have investigated the Preimplantation genetic diagnosis (PGT) management of patients with interchromosomal insertion (IT) by fluorescent in situ hybridization (FISH) to obtain a better understanding and improve the genetic counseling. Material & methods Two couples were enrolled in our PGT unit. The male of the first couple carries the IT 46,XY,ins(4;11)(q22;p12p13) and the female karyotype of the second couple is 46,XX,ins(2;10)(q21;q25.2q22). We have investigated, by FISH technique, the meiotic segregation analysis of the sperm of the IT male carrier and embryos providing from the two carriers. One FISH analysis round was performed for the male IT carrier. Five specific probes of chromosomes 4 and 11 were applied to nuclei from spermatozoa and blastomeres from embryo biopsy. An independent X2 test was used to compare abnormal spermatozoa from the patient and the control. Two FISH analysis rounds were performed for the female IT carrier. Six specific probes of chromosomes 2 and 10 were applied to blastomeres of PGT embryos. Result(s) The sperm-FISH segregation analysis showed a significantly increased percentage of unbalanced gametes (52.7%) compared with the control sperm (3.4%, p Conclusion(s) These results permit to reassure the physicians in the management of IT carriers and better guide the genetic counseling. PGT is a suitable diagnostic tool for IT carriers. Sperm FISH analysis contributes to a better IT behavior and segregation understanding. The reanalysis of not transferred whole embryos is essential to avoid misdiagnosis and therefore to contribute to a better IT behavior and segregation understanding. Several studies are needed to specify the risk to ITs carriers and to better assess the risk incurred by the couples.

Sperm chromosomal abnormalities and their contribution to human embryo aneuploidy.

In this work we reviewed 18years of experience using fluorescence in situ hybridization (FISH) for sperm aneuploidy testing. We evaluated parameters associated with increased numerical sperm chromosome abnormalities and determined the male contribution to embryo aneploidies in terms of reproductive outcome by increased sperm aneuploidy. This retrospective study analyzed data from 2008 sperm samples of infertile males undergoing FISH analysis because of clinical history of repetitive implantation failure, recurrent miscarriage, impaired sperm parameters, or mixed causes. Sperm concentration was the only sperm parameter associated with FISH results-we observed a gradual increase of abnormal sperm FISH results in males with decreasing sperm concentration. However, a great proportion of normozoospermic males also showed increased sperm aneuploidies, suggesting that sperm parameters alone do not enable identification of a substantial proportion of infertile males at risk of sperm aneuploidies. Regarding reproductive outcomes, couples with normal sperm FISH results for the male had similar outcomes regardless of conventional in vitro fertilization (IVF), intracytoplasmic sperm injection (ICSI), or preimplantation genetic testing for aneuploidies (PGT-A). However, couples with abnormal sperm FISH results for the male showed better clinical outcomes after PGT-A, suggesting a potential contribution of sperm to embryo aneuploidy. Moreover, PGT-A cycles showed better clinical outcomes when 24 chromosomes were analyzed by array comparative genome hybridization (aCGH) or next-generation sequencing (NGS) instead of only nine chromosomes analyzed by FISH. In conclusion, sperm FISH analysis offers clinical prognostic value to evaluate reproductive possibilities in infertile couples. Therefore, couples with abnormal sperm FISH results should be offered genetic counseling and presented with clinical options such as PGT-A.

Sperm aneuploidy and DNA fragmentation in unexplained recurrent pregnancy loss: a multicenter case-control study

BackgroundRecurrent pregnancy loss (RPL) is defined as the loss of at least three pregnancies in the first trimester. Although the most common cause is embryo aneuploidy, and despite female checkup and couple karyotyping, in about 50% of cases RPL remain unexplained. Male implication has little been investigated and results are discordant. In this context, we conducted a multi-center prospective case-control study to investigate male gamete implication in unexplained RPL.MethodsA total of 33 cases and 27 controls were included from three university hospitals. We investigated environmental and family factors with a detailed questionnaire and andrological examination, sperm characteristics, sperm DNA/chromatin status using the sperm chromatin structure assay (SCSA) and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) and sperm aneuploidy using fluorescence in situ hybridization (FISH). The Mann-Whitney test and the Wilcoxon or Fisher exact tests were used. A non-parametric Spearman correlation was performed in order to analyze the relationship between various sperm parameters and FISH and sperm DNA fragmentation results.ResultsWe found significant differences between cases and controls in time to conceive, body mass index (BMI), family history of infertility and living environment. In cases, total sperm motility and the percentage of morphologically normal spermatozoa were significantly decreased. No difference was found between cases and controls in sperm DNA fragmentation or chromatin integrity. In cases, spermatozoa with aneuploidy, hyperhaploidy and chromosome 18 disomy were significantly increased.ConclusionsThis prospective case-control study is one of the largest to examine environmental factors, sperm characteristics, sperm DNA fragmentation and chromatin, and chromosome anomalies in spermatozoa in relation to unexplained recurrent pregnancy loss. The originality of our study lies in the comprehensive andrological examination and search for risk factors and fertility history. Further studies are needed to confirm the links between unexplained RPL and a male family history of infertility or miscarriages. The increased sperm aneuploidy observed in unexplained RPL supports a male etiology. These data pave the way for further studies to demonstrate the value of preimplantation genetic screening in men with increased sperm aneuploidy whose partners experience unexplained RPL.

PD09-03 COST-EFFECTIVENESS OF SPERM FLUORESCENCE IN SITU HYBRIDIZATION TESTING FOR INFERTILE MEN WITH SUSPECTED SPERM ANEUPLOIDY

You have accessJournal of UrologyInfertility: Epidemiology & Evaluation I1 Apr 2018PD09-03 COST-EFFECTIVENESS OF SPERM FLUORESCENCE IN SITU HYBRIDIZATION TESTING FOR INFERTILE MEN WITH SUSPECTED SPERM ANEUPLOIDY Taylor P. Kohn, Alexander W. Pastuszak, Jaden R. Kohn, Katherine M. Rodriguez, Dolores J. Lamb, and Larry I. Lipshultz Taylor P. KohnTaylor P. Kohn More articles by this author , Alexander W. PastuszakAlexander W. Pastuszak More articles by this author , Jaden R. KohnJaden R. Kohn More articles by this author , Katherine M. RodriguezKatherine M. Rodriguez More articles by this author , Dolores J. LambDolores J. Lamb More articles by this author , and Larry I. LipshultzLarry I. Lipshultz More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2018.02.600AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Men with recurrent spontaneous abortions (SAB) or implantation failures (IF) may have abnormal sperm aneuploidy as diagnosed by fluorescent in situ hybridization (FISH). In vitro fertilization (IVF) with pre-implantation genetic screening (PGS) is the most effective assisted reproductive technique (ART) for men with sperm aneuploidy, yet the cost-effectiveness of FISH testing in the context of available genetic testing of the embryo remains unclear. Here we examine the cost effectiveness of sperm FISH testing in the context of ART. METHODS Couples with a history of either 2 IVF failures or 2 SABs without known female factor were surveyed via phone call to document subsequent pregnancy attempts. Of 98 couples surveyed, 80 men underwent FISH testing while 18 did not. Men were grouped by normal FISH, abnormal FISH, and not tested for FISH, and outcomes of pregnancies conceived by natural intercourse, IVF, intracytoplasmic sperm injection (ICSI), and IVF/PGS were assessed. The cost-effectiveness for each category and reproductive method, as well as the overall cost of performing or not performing FISH prior to assisted reproduction, were determined. RESULTS Of the 80 men who underwent FISH, 78 had abnormal results, while 2 were normal. Success and failure rates, costs, and cost-effectiveness of each reproductive method in our sample are shown in Table 1a. The costs per pregnancy for men without FISH testing using PGS with IVF were $30,268 and $49,600, respectively. For men with normal and abnormal FISH, PGS after IVF was more cost-effective than either IVF alone or ICSI with IVF (Table 1a). The total costs, associated with FISH testing in men with suspected sperm aneuploidy was $17,351 for FISH testing prior to attempted conception and $16,318 for bypassing FISH and proceeding directly to the reproductive method of choice - more than cost of the FISH testing (Table 1b). CONCLUSIONS PGS after IVF is more cost-effective than IVF alone, or ICSI with IVF for men with possible sperm aneuploidy. Sperm FISH testing adds additional cost especially when the rate of rate of ″abnormal″ is so high. Thus, couples utilizing assisted reproductive techniques may consider utilizing sperm FISH only when a sperm donor would be considered, or proceeding directly to PGS without undergoing sperm FISH testing. © 2018FiguresReferencesRelatedDetails Volume 199Issue 4SApril 2018Page: e222-e223 Advertisement Copyright & Permissions© 2018MetricsAuthor Information Taylor P. Kohn More articles by this author Alexander W. Pastuszak More articles by this author Jaden R. Kohn More articles by this author Katherine M. Rodriguez More articles by this author Dolores J. Lamb More articles by this author Larry I. Lipshultz More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Semi-automated scoring of triple-probe FISH in human sperm using confocal microscopy.

Structural and numerical sperm chromosomal aberrations result from abnormal meiosis and are directly linked to infertility. Any live births that arise from aneuploid conceptuses can result in syndromes such as Kleinfelter, Turners, XYY and Edwards. Multi-probe fluorescence in situ hybridization (FISH) is commonly used to study sperm aneuploidy, however manual FISH scoring in sperm samples is labor-intensive and introduces errors. Automated scoring methods are continuously evolving. One challenging aspect for optimizing automated sperm FISH scoring has been the overlap in excitation and emission of the fluorescent probes used to enumerate the chromosomes of interest. Our objective was to demonstrate the feasibility of combining confocal microscopy and spectral imaging with high-throughput methods for accurately measuring sperm aneuploidy. Our approach used confocal microscopy to analyze numerical chromosomal abnormalities in human sperm using enhanced slide preparation and rigorous semi-automated scoring methods. FISH for chromosomes X, Y, and 18 was conducted to determine sex chromosome disomy in sperm nuclei. Application of online spectral linear unmixing was used for effective separation of four fluorochromes while decreasing data acquisition time. Semi-automated image processing, segmentation, classification, and scoring were performed on 10 slides using custom image processing and analysis software and results were compared with manual methods. No significant differences in disomy frequencies were seen between the semi automated and manual methods. Samples treated with pepsin were observed to have reduced background autofluorescence and more uniform distribution of cells. These results demonstrate that semi-automated methods using spectral imaging on a confocal platform are a feasible approach for analyzing numerical chromosomal aberrations in sperm, and are comparable to manual methods. © 2017 International Society for Advancement of Cytometry.

MP35-09 SPERM ANEUPLOIDY IS ASSOCIATED WITH WORSE GENERAL HEALTH IN INFERTILE MEN

You have accessJournal of UrologyInfertility: Epidemiology & Evaluation II1 Apr 2017MP35-09 SPERM ANEUPLOIDY IS ASSOCIATED WITH WORSE GENERAL HEALTH IN INFERTILE MEN Taylor P. Kohn, Alexander W. Pastuszak, Katherine M. Rodriguez, Zachary J. Solomon, Matthew F. Cherches, Dolores J. Lamb, and Larry I. Lipshultz Taylor P. KohnTaylor P. Kohn More articles by this author , Alexander W. PastuszakAlexander W. Pastuszak More articles by this author , Katherine M. RodriguezKatherine M. Rodriguez More articles by this author , Zachary J. SolomonZachary J. Solomon More articles by this author , Matthew F. CherchesMatthew F. Cherches More articles by this author , Dolores J. LambDolores J. Lamb More articles by this author , and Larry I. LipshultzLarry I. Lipshultz More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2017.02.1099AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Numerous studies have examined the relationship between male infertility and men′s health but most have not investigated specific causes of infertility and their health impact. Here we examine the relationship between general male health and sperm aneuploidy. METHODS We used the Charlson Co-Morbidity Index (CCI) to assess and compare the health status of men with abnormal sperm aneuploidy to five groups of men who received care at an academic infertility clinic: men with normal sperm ploidy (normal FISH), men with genetic abnormalities that may cause infertility (genetic infertile), men presenting for infertility with varicocele (varicocele), men with idiopathic infertility (idiopathic) and proven-fertile men. Sperm ploidy was assessed using fluorescence in situ hybridization (FISH). Genetic infertile men had proven Y chromosome microdeletions or abnormal karyotype. CCI for men with sperm FISH results was completed via telephone survey and CCI for genetic infertile men was extracted from intake forms. Men who were diagnosed with a varicocele or idiopathic infertility had not conceived after 12 months. Men with varicoceles, men with idiopathic infertility, and fertile controls completed the CCI in clinic. Fertile controls included men who presented for vasectomy and had fathered a child in the past 5 years. RESULTS 402 men were included in this analysis: 92 with an abnormal sperm FISH results, 7 with a normal sperm FISH test, 49 genetic infertile men, 85 men with idiopathic infertility, 86 infertile men with varicocele, and 83 fertile controls. Mean age, semen density, and CCI of all groups are described in Table 1. Of note, malignancy was common in our cohort, exceeding rates in the SEER database for comparable age (Table 1). Controlling for age, we explored infertility groups as a predictor of elevated CCI score, and found that only men with abnormal sperm FISH (p=0.002) had increased risk of a higher CCI score. Age also increased the risk for a higher CCI score, with each additional year raising the CCI by 0.0117 points (p=0.009). CONCLUSIONS Men with elevated sperm aneuploidy have nearly double the prevalence of general health issues, particularly malignancy, when compared with men with infertility of other etiologies. These findings further support the relationship between genetic causes of male infertility and general health. © 2017FiguresReferencesRelatedDetails Volume 197Issue 4SApril 2017Page: e460-e461 Advertisement Copyright & Permissions© 2017MetricsAuthor Information Taylor P. Kohn More articles by this author Alexander W. Pastuszak More articles by this author Katherine M. Rodriguez More articles by this author Zachary J. Solomon More articles by this author Matthew F. Cherches More articles by this author Dolores J. Lamb More articles by this author Larry I. Lipshultz More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Meiotic segregation analyses of reciprocal translocations in spermatozoa and embryos: no support for predictive value regarding PGD outcome

Translocation heterozygotes have an increased risk of producing gametes with unbalanced chromosome content. This often leads to reproductive problems such as infertility, repeated miscarriages or birth of an affected child. To increase the chances of having a healthy live-born child, translocation heterozygotes often opt for preimplantation genetic diagnosis (PGD). The aim of this study was to investigate whether there is a correlation between chromosome segregation in spermatozoa from translocation heterozygotes and the number of balanced embryos produced during PGD that may be used to predict the PGD outcome. Ten male reciprocal translocation heterozygotes that went through PGD at a Stockholm PGD centre were included. We analysed 1000 spermatozoa from each patient and between 3 and 29 embryos from the total of PGD cycles that the couples went through. Fluorescence in-situ hybridization (FISH) analysis of spermatozoa and embryos was performed with the same DNA probes. We found that the proportion of balanced spermatozoa was much higher than the proportion of balanced embryos during PGD. Our results indicate that a sperm FISH analysis prior to PGD is not a reliable predictor of the PGD outcome. PGD is a valuable reproductive alternative for translocation heterozygotes with reproductive problems and should be offered to these couples.

Genetic counseling for men with recurrent pregnancy loss or recurrent implantation failure due to abnormal sperm chromosomal aneuploidy.

The purpose of this study is to review recurrent pregnancy loss (RPL) due to sperm chromosomal abnormalities and discuss the genetic counseling that is required for men with sperm chromosomal abnormalities. The literature was reviewed, and a genetic counselor lends her expertise as to how couples with RPL and sperm chromosomal abnormalities ought to be counseled. The review of the literature was performed using MEDLINE. Sperm fluorescence in situ hybridization (FISH) can be used to determine if disomy or unbalanced chromosomal translocations are present. In men with aneuploidy in sperm or who carry a chromosomal translocation, pre-implantation genetic screening (PGS) combined with in vitro fertilization (IVF) and intra-cytoplasmic sperm injection (ICSI) can increase chances of live birth. In men with abnormal sperm FISH results, the degree of increased risk of abnormal pregnancy remains unclear. Genetic counselors can provide information to couples about the risk for potential trisomies and sex chromosome aneuploidies and discuss their reproductive and testing options such as PGS, use of donor sperm, and adoption. The provision of genetic counseling also allows a couple to be educated about recommended prenatal testing since pregnancies conceived with a partner who has had abnormal sperm FISH are considered to be at increased risk for aneuploidy. We review the literature and discuss genetic counseling for couples with RPL or recurrent implantation failure due to increased sperm aneuploidy.

Meiotic and pedigree segregation analyses in carriers of t(4;8)(p16;p23.1) differing in localization of breakpoint positions at 4p subband 4p16.3 and 4p16.1.

PurposeThe purpose of this study was to compare meiotic segregation in sperm cells from two carriers with t(4;8)(p16;p23.1) reciprocal chromosome translocations (RCTs), differing in localization of the breakpoint positions at the 4p subband—namely, 4p16.3 (carrier 1) and 4p16.1 (carrier 2)—and to compare data of the pedigree analyses performed by direct method.MethodsThree-color fluorescent in situ hybridization (FISH) on sperm cells and FISH mapping for the evaluation of the breakpoint positions, data from pedigrees, and direct segregation analysis of the pedigrees were performed.ResultsSimilar proportions of normal/balanced and unbalanced sperm cells were found in both carriers. The most common was an alternate type of segregation (about 52 % and about 48 %, respectively). Unbalanced adjacent I and adjacent II karyotypes were found in similar proportions about 15 %. The direct segregation analysis (following Stengel-Rutkowski) of the pedigree of carriers of t(4;8)(p16.1;p23.1) was performed and results were compared with the data of the pedigree segregation analysis obtained earlier through the indirect method. The probability of live-born progeny with unbalanced karyotype for carriers of t(4;8)(p16.1;p23.1) was moderately high at 18.8 %—comparable to the value obtained using the indirect method for the same carriership, which was 12 %. This was, however, markedly lower than the value of 41.2 % obtained through the pedigree segregation indirect analysis estimated for carriers of t(4;8)(p16.3;p23.1), perhaps due to the unique composition of genes present within the 4p16.1–4p 16.3 region.ConclusionsRevealed differences in pedigree segregation analysis did not correspond to the very similar profile of meiotic segregation patterns presented by carrier 1 and carrier 2. Most probably, such discordances may be due to differences in embryo survival rates arising from different genetic backgrounds.

Increased chromosome 16 disomy rates in human spermatozoa and recurrent spontaneous abortions

To investigate if unexplained recurrent spontaneous abortions (RSA) are associated with increased rates of aneuploidy in spermatozoa of RSA partners ("RSA-men"). Case-control study. Academic research center. Patients enrolled at the Hormone and Fertility Center and controls at the Department of Urology (LMU-Munich). Sperm samples of 11 partners of unexplained RSA cases evaluated for elevated diploidy and disomy levels of chromosomes 1-22, X, and Y by multicolor sperm fluorescence in situ hybridization (FISH). Aneuploidy rates obtained in RSA-men compared with controls from the literature and internally; an increase of the aneuploidy rate was considered statistically significant, when it differed ≥ 2 standard deviations from the mean baseline level in controls. Our sperm FISH data on RSA men showed increased disomy rates for at least three chromosomes in more than 60% of patients but no statistically significant increase of the overall mean sperm disomy or diploidy rate. In particular, meiotic errors involving chromosome 16 contributed to increased sperm disomy in more than 60% of our patients. These data suggest that among paternal meiotic errors nondisjunction of chromosome 16 might have similar relative influence on fetal aneuploidy compared with maternal chromosome 16 disomy.

Comprehensive investigation in patients affected by sperm macrocephaly and globozoospermia.

The aim of this study was to provide a comprehensive genetic/phenotypic characterization of subjects suffering infertility owing to sperm macrocephaly (n = 3) or globozoospermia (n = 9) and to investigate whether the patients' genetic status was correlated with the alteration of various sperm parameters. AURKC was sequenced in case of sperm macrocephaly while the DPY19L2 status has been analyzed by multiple approaches including a novel qPCR-based copy number assay in case of globozoospermia. Globozoospermic patients were also analyzed for SPACA1, a novel candidate gene herein tested for the first time in humans. The effect of the patients' genetic status was interrogated by implementing the molecular screening with the characterization of several sperm parameters: (i) routine sperm analysis, integrated with transmission electron microscopy; (ii) sperm fluorescent in situ hybridization (FISH) analysis; (iii) sperm DNA fragmentation (DF) analysis. Moreover, for the first time, we performed microsatellite instability analysis as a marker of genome instability in men with sperm macrocephaly and globozoospermia. Finally, artificial reproductive technology (ART) history has been reported for those patients who underwent the treatment. Macrocephalic patients had an AURKC mutation and >89% tetraploid, highly fragmented spermatozoa. DPY19L2 was mutated in all patients with >80% globozoospermia: the two homozygous deleted men and the compound heterozygous showed the severest phenotype (90-100%). The newly developed qPCR method was fully validated and has the potential of detecting also yet undiscovered deletions. DPY19L2 status is unlikely related to FISH anomalies and DF, although globozoospermic men showed a higher disomy rate and DF compared with internal reference values. No patient was mutated for SPACA1. Our data support the general agreement on the negative correlation between macro/globozoospermia and conventional intracytoplasmic sperm injection outcomes. Microsatellites were stable in all patients analyzed. The comprehensive picture provided on these severe phenotypes causing infertility is of relevance in the management of patients undergoing ART.

Contraindication of ART following a sperm FISH analysis, even though only 12% of the spermatozoa had enlarged heads

We report on a couple with a five-year history of idiopathic primary infertility. Two early miscarriages had followed intrauterine insemination (IUI). The man's fertility was then re-evaluated, in order to establish whether or not IUI was the best treatment option. Although the semen parameters were normal (sperm concentration: 89 million/ml; progressive motility: 40%; percentage of typical forms: 20%), a computer-assisted sperm morphology analysis with strict criteria found that 12% of the spermatozoa had enlarged heads. All of the latter had a normal form and none had multiple flagella. Using fluorescence in situ hybridization (FISH) analysis, we found that the proportion of aneuploid and diploid spermatozoa was 78% for the sample as a whole and 68% for normally-shaped spermatozoa with a normal-sized head. Although treatment options are well documented for men with macrocephalic sperm head syndrom, there is no consensus on individuals with a low but non-negligible proportion of spermatozoa with enlarged heads. Here, our FISH results contraindicated the use of assisted reproductive technology with the man's sperm. The couple decided to resort to donor sperm.

Risk evaluation and preimplantation genetic diagnosis in an infertile man with an unbalanced translocation t(10;15) resulting in a healthy baby

Balanced chromosome structural rearrangement, including reciprocal translocation, Robertsonian translocation and inversion, is an important cause of male infertility that results in azoospermia, oligozoospermia or asthenozoospermia [1]. Unbalanced translocation between two autosomes, which usually results in mental retardation or multiple congenital anomalies, is rare in infertile patients. To our knowledge, only the t(15;18) unbalanced translocation has been reported, in two healthy infertile brothers. Although fluorescence in situ hybridization (FISH) analysis showed that chromosomal normal sperms existed in their semen, these two brothers and their partners chose donor insemination rather than preimplantation genetic diagnosis (PGD) [2]. Whether patients with this kind of rare karyotype could deliver a healthy baby is unclear. Here, we report a man with severe oligoasthenoteratozoospermia carrying a derivative chromosome 10 resulting from the rare unbalanced translocation t(10;15). Based on risk evaluation by sperm FISH, we performed PGD for the couple and successfully established a normal pregnancy. Our results show that patients with such an abnormal karyotype can produce their own offspring, and emphasize the importance of risk evaluation using sperm FISH for infertile male patients. Patient data A couple presented to our reproduction center due to primary infertility that had persisted for 5 years. The woman showed no obvious cause of infertility on examinations, which included: (1) clinical findings: physical examination and medical history evaluation,such as menstrual history, obstetrical history, sexual history, family history, personal and lifestyle history; (2) biochemical tests: liver and renal function; blood cholesterol, blood triglycerides and blood glucose; and immunologic function; and (3) hormonal assay: estradiol, testosterone, progesterone, follicle stimulating hormone (FSH), luteinizing hormone, prolactin, 17-hydroxypregnenolone and thyroid stimulating hormone (TSH); (4) other diagnosis test such as hysterosalpingography. Her karyotype was also normal. However, the man was found to have severe oligoasthenoteratozoospermia after three routine sperm analyses and one sperm morphology examination following the WHO guidelines (Table 1). Conventional G-banding (G-bands after trypsin and Giemsa, GTG) analysis showed that he had only 45 chromosomes, with one derivative chromosome comprising chromosomes 10 and 15 (Fig. 1). Table 1 Semen analysis Fig. 1 a Partial karyotype of the patient showing normal chromosome 10,15 and der(10). b ideogram of the normal chromosome 10,15 and rearranged chromosome. Red, green and aqua bands show the position and color of the FISH probes used for preimplantation genetic ... The couple then accepted a genetic counseling and asked for further fertility evaluation. Oligoasthenoteratozoospermia might have been the cause of the primary infertility in this couple. The husband carried a rare unbalanced translocation comprising chromosome 10 and chromosome 15 that had not been reported previously. The genetic risk was unclear for this kind of abnormal karyotype. His chromosomal structure was similar to that of a Robertsonian translocation; therefore, the meiosis pattern might also be similar to a Robertsonian translocation. Sperm chromosome constitution can be obtained by sperm FISH analysis and might predict the probability of producing an embryo with normal karyotype. If chromosomally normal sperms are present in semen at a high prevalence, PGD can be performed to select embryos with balanced chromosomes for transfer and deliver a healthy baby. This study was approved by the Institutional Review Board of Central South University and Reproductive and Genetics Hospital of Citic-Xiangya, and the patients’ written informed consent was obtained.