FIGURE 1. Propidium Iodide Staining and Flow Cytometry. Acridine Orange Staining and Flow Cytometry. Statistical Analysis

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1 Using semen flow cytometry to evaluate association of ploidy status and chromatin condensation of spermatozoa with conventional semen parameters: Clinical application in intrauterine insemination Leandros Lazaros, Ph.D., a Apostolos Kaponis, M.D., a Georgios Vartholomatos, Ph.D., b Elissavet Hatzi, Ph.D., a Stefania Botsari, M.D., a Nikolaos Plachouras, M.D., a Georgios Makrydimas, M.D., a Konstantinos Zikopoulos, M.D., a Nikolaos Sofikitis, M.D., c and Ioannis Georgiou, Ph.D. a a Genetics and IVF Unit, Department of Obstetrics and Gynecology, b Laboratory of Hematology, Molecular Biology Unit, and c Department of Urology, Ioannina University School of Medicine, Ioannina, Greece Objective: To evaluate the association between the ploidy status and the nuclear chromatin condensation of spermatozoa with conventional semen parameters by using semen flow cytometry (SFC). The prognostic value of SFC on the successful outcome of intrauterine insemination (IUI) was examined. Design: Prospective study. Setting: Patients referred to the IVF Unit of Ioannina University School of Medicine. Patient(s): Ninety-two men with sperm count between and spermatozoa/ml and one hundred normozoospermic men were analyzed. Intervention(s): Conventional semen analysis and SFC analysis after acridine orange and propidium iodide staining. IUI performed in 92 couples. Main Outcome Measure(s): Evaluation of sperm maturity and ploidy. Correlation with conventional semen parameters. Result(s): An association of the spermatozoa ploidy status with sperm morphology and motility was revealed. The highest aneuploidy rates were observed when <15% of spermatozoa had normal morphology. An inverse relation was found between sperm morphology and maturity. The pregnancy rates were significantly lower when semen with <15% normal forms (9% vs. 25%), low percentage of mature spermatozoa, and increased total aneuploidy rate were used for IUI. Conclusion(s): Ploidy status and sperm maturity are critical parameters for evaluation of the fertilizing capacity of spermatozoa. SFC could be used to evaluate semen samples before IUI and potentially prognose the outcome. (Fertil Steril Ò 2011;95: Ó2011 by American Society for Reproductive Medicine.) Key Words: Chromatin condensation, flow cytometry, IUI, ploidy status Intrauterine insemination (IUI) is one of the most frequent treatments for couples with male subfertility. Although the treatment itself is less invasive and expensive than others, its efficacy has been challenged (1). Sperm concentration and motility are taken into account before an IUI procedure. However, increasing numbers of couples fail to achieve a pregnancy even though sperm parameters are normal, indicating that additional parameters should be evaluated for the determination of semen fertilizing capacity. Reproductive failures have been associated with cytogenetic abnormalities in germ cells of infertile individuals with normal karyotype. It is well known that a normal somatic karyotype evaluated Received December 17, 2009; revised April 30, 2010; accepted May 11, 2010; published online June 18, L.L. has nothing to disclose. A.K. has nothing to disclose. G.V. has nothing to disclose. E.H. has nothing to disclose. S.B. has nothing to disclose. N.P. has nothing to disclose. G.M. has nothing to disclose. K.Z. has nothing to disclose. N.S. has nothing to disclose. I.G. has nothing to disclose. Reprint requests: Ioannis Georgiou, Ph.D., Professor of Medical Genetics and Clinical Embryology, Laboratory of Medical Genetics and Human Reproduction, Department of Obstetrics and Gynecology, Medical School, University of Ioannina, Ioannina, Greece (FAX: þ ; igeorgio@uoi.gr). by standard cytogenetic analysis does not exclude the presence of aneuploid gametes (2). A significant correlation has been reported between the sperm aneuploidy rate and conventional semen parameters (3 6), suggesting that the ploidy status of spermatozoa might have a crucial role in their fertilizing capacity. Sperm maturity constitutes another major factor for the proper functioning of spermatozoa and an important cause of infertility (7). A significant correlation exists between sperm maturity and nuclear chromatin stability (8). The spermatozoa nuclear chromatin stability increases during spermiogenesis, owing to substitution of histones by protamines (9), a process called sperm chromatin condensation. Mature spermatozoa are those with totally condensed chromatin and complete epididymal maturation. Sperm maturity can be estimated by the degree of exclusion of the dye acridine orange, which produces green fluorescence when it binds doublestranded nucleic acids and red fluorescence when it binds singlestranded nucleic acids (10). Significant increases of fluorescence values were positively correlated with abnormal percentages of immature spermatozoa and a decreased fertilizing capacity of the semen samples (11). Semen flow cytometry (SFC) is able to detect the ploidy status and the maturity of spermatozoa. SFC has been used for size, cell compactness, and cytoplasmic structure analysis of spermatozoa 110 Fertility and Sterility â Vol. 95, No. 1, January /$36.00 Copyright ª2011 American Society for Reproductive Medicine, Published by Elsevier Inc. doi: /j.fertnstert

2 FIGURE 1 Association of mean ploidy rates in sperm cells with (A) sperm morphology and (B) sperm motility. Pearson correlation was used (A) between the hyperhaploidy, hypohaploidy, and diploidy rates and the percentage of normal forms of spermatozoa (P<.01) and (B) between the hyperhaploidy, hypohaploidy, haploidy, and diploidy rates and sperm motility (P<.01). MATERIALS AND METHODS Subjects The study population consisted of 92 men aged years, who were referred, prospectively, to the IVF Unit of the Department of Obstetrics and Gynecology, Medical School of Ioannina, Greece, for infertility treatment. A complete medical history was taken and physical examination performed. Men suffering from karyotypic abnormalities, hypogonadotropic hypogonadism, varicocele, or obstructive syndromes of the seminal tract and men under treatment with spermatogenesis/motility impairing medication were excluded. Cases in which female infertility factor was found were also excluded. IUI was performed as first-line treatment of their infertility. The Institutional Ethics Committee approved the study protocol in accordance with the Helsinki declaration, and each participant gave informed consent. Semen analysis was performed according to World Health Organization (WHO) guidelines (16). Men were asked to abstain from sexual activity for 2 5 days before IUI. Two independent investigators performed blind semen analysis. The average values from the two investigators were calculated. In the event of inconsistency (>10% difference) a third assessment was done. The sperm concentration was between and spermatozoa/ ml. The morphology was evaluated after Papanicolaou staining. All samples were processed with sperm gradient kit and sperm preparation medium (Medicult, Jyllinge, Denmark) for the IUI procedure. A standard GnRHantagonist protocol was used for ovarian stimulation as described by Zikopoulos et al. (17). One hundred normozoospermic men (sperm concentration spermatozoa/ml), age matched with the study population, participated in the study as a control group. Propidium Iodide Staining and Flow Cytometry The method of propidium iodide staining and SFC was used to study the ploidy status of human spermatozoa, according to a protocol described previously (18). Two semen samples from each patient were used for the analysis to ensure reliable results. For the analysis, Facscalibur Flow Cytometer (Becton Dickinson, San Jose, CA) was used. Red fluorescence (BP 650LP filter) emitted from individual cells was recorded from 10,000 cells per sample after excitation with a 488 nm argon laser using a logarithmic scale to allow cells of all ploidy status to appear as peaks in the resulting histograms. Acridine Orange Staining and Flow Cytometry The method of acridine orange staining and SFC was used to study abnormal chromatin condensation of human spermatozoa, according to a protocol described previously (11). Two semen samples from each patient were used for the analysis to ensure reliable results. The processed semen samples were screened using a Facscalibur Flow Cytometer (Becton Dickinson). Green fluorescence (BP 530/30 filter) and red fluorescence (BP 650LP filter) were measured for 30,000 counts/sample after excitation with a 488 nm argon laser. The Consort 40 (Becton Dickinson) was used to analyze the data. (12, 13). The intensity of the fluorescent signal has been correlated with chromatin condensation (14) and the identification of haploid cells in semen (15). In the present study, we performed SFC to evaluate the ploidy status and the nuclear chromatin condensation of spermatozoa. Statistical Analysis Statistical analysis was performed using the chi-squared test. Normal distribution of continuous parameters was tested by Kolmogorov-Smirnov test. Differences in continuous parameters between genotypes were assessed with the nonparametric Kruskal-Wallis test. A P value of <.05 was set as statistically significant. All analyses used the SPSS statistical package (version 14.0; SPSS, Chicago, IL). RESULTS Study Population Characteristics Men with sperm count between and spermatozoa/ ml and % motility were examined (n ¼ 92). All semen samples contained increased amounts of spermatozoa with abnormal morphology. Sperm concentration was inversely correlated with the percentage of abnormal forms (P<.01). When sperm concentration was spermatozoa/ml, 35% of the sperm cells had abnormal morphology. When sperm concentration was spermatozoa/ml, the prevalence of abnormal forms increased to 50%. One hundred normozoospermic men, with sperm concentration spermatozoa/ml and % motility, age-matched with the study population, were examined. Their semen samples contained significantly lower numbers of spermatozoa with abnormal morphology compared with the study population. Association of Sperm Cell Ploidies With Sperm Morphology and Motility The spermatozoa ploidy status analysis revealed peaks of hypohaploid, normal haploid, immature haploid (elongated spermatids), hyperhaploid, diploid, and tetraploid cells in the histograms of all samples. A significant correlation between these ploidies and sperm morphology was revealed (Fig. 1A). Hyper-, hypo-, and diploidies were observed more frequently in samples with high percentages of abnormal forms (P<.01). The highest rates of ploidies were observed when <15% of spermatozoa had normal morphology Fertility and Sterility â 111

3 FIGURE 2 Scattergrams of acridine orange stained spermatozoa analyzed by flow cytometry, showing (A) a semen sample containing an increased percentage of spermatozoa in red population zones and (B) a normal reference semen pool. To define the nuclear abnormalities and to estimate the percentage of mature spermatozoa in every semen sample, the data were examined using two-dimensional scattergrams plotting red versus green fluorescence. The FL1-H axis represented the green fluorescence, the FL2-H axis the red fluorescence. (mean values 40.39%, 34.62%, and 19.23%, respectively). Samples with >30% normal morphology had an increased rate of hyperhaploidy equal to 7.69%. Hypo- and hyperhaploidy rates were inversely correlated with sperm motility (P<.01), whereas haploid rates were positively correlated with sperm motility (P<.01). Diploidy rate was not correlated with sperm motility (Fig. 1B). Control semen samples contained very low aneuploidy rates. When we compared the study population with the control group, significant differences in hyper- and hypohaploidy and diploidy rates were observed (data not shown). percentage of spermatozoa with abnormal morphology increased, the fluorescent intensities were increased and the percentage of mature spermatozoa was decreased (P<.01; Fig. 3). Control semen samples presented with less fluorescent intensities and higher percentage of mature spermatozoa ( %). The percentage of mature spermatozoa of each semen sample, which serves as a measure of the degree of chromatin condensation, was calculated as 100(window M)/(window T), where window M corresponded to spermatozoa with completed epididymal sperm maturation and window T to the major sperm band excluding cell debris and satellite populations (11). Association of Sperm Chromatin Condensation With Sperm Quality The SFC analysis after acridine orange staining revealed both green and red fluorescence emitted by all spermatozoa, but at different intensities. The flow cytometer stored the individual red and green fluorescence values for each of the 10,000 cells in every sample. Significant increases in green and red fluorescence values were positively correlated with a high percentage of spermatozoa with abnormal morphology (Fig. 2). Green or red fluorescence values were independent from sperm concentration and motility. Only sperm morphology was inversely correlated with the fluorescent intensities and subsequently with the percentage of sperm maturation. As the Association of Sperm Morphology, Ploidy Status, and Chromatin Condensation With IUI Outcome Semen samples with <15% normal morphology presented with increased aneuploidy rate and induced spermatozoa maturity. IUI failed to achieve pregnancy in 91% of them. Semen samples with >15% normal morphology achieved a significantly higher pregnancy rate (25% vs. 9%; P<.03), showing the correlation between sperm morphology and pregnancy rate after IUI. When semen aneuploidy rates and spermatozoa maturity were studied individually, strong associations were found with pregnancy rates (P<.01; Fig. 4). Pregnancy rates were positively correlated with the percentage of mature spermatozoa and inversely correlated with the total aneuploidy rate. 112 Lazaros et al. Evaluating semen quality with flow cytometry Vol. 95, No. 1, January 2011

4 FIGURE 3 Association of the mean percentage of mature spermatozoa with sperm morphology. Pearson correlation was used between the percentage of mature spermatozoa and the percentage of abnormal forms of spermatozoa (P<.01). FIGURE 4 Association of IUI pregnancy rates with (A) the total aneuploidy rate and (B) the percentage of mature spermatozoa. Chi-square test was used to compare the positive and negative pregnancy outcomes (A) in every category of total aneuploidy rate (P<.01) and (B) in every category of percentage of mature spermatozoa (P<.01). DISCUSSION Basic semen analysis is the most important test for the evaluation of conventional semen parameters. In the present study, semen samples with concentrations between and spermatozoa/ ml were analyzed before IUI, and elevated amounts of spermatozoa with abnormal morphology were found. The abnormal morphology has been associated with flaws in sperm cell genetic constitution (5), pointing to the ploidy status analysis of spermatozoa as necessary. The SFC analysis after propidium iodide staining revealed an increased aneuploidy rate in the present study population. Hyper- and hypohaploidies and diploidies were the most frequent abnormal ploidies, with a higher frequency in samples with increased percentages of spermatozoa with abnormal morphology, proving the strong association between abnormal sperm cell morphology and ploidy. These findings are in accordance with a study by Weissenberg et al. (19), who demonstrated that semen samples with enlarged head and one-head two-tail spermatozoa are correlated with very high rates of aneuploidy (15% 99%) and diploidy (19% 40%). The total aneuploidy rate of morphologically abnormal spermatozoa was 4.4-fold higher than that of spermatozoa with normal morphology in normozoospermic men (20). The hypo- and hyperhaploidy and diploidy rates have been found to be significantly increased in semen samples with poor quality due to defects in spermatogenesis (21). These abnormalities in sperm morphology differentiation and chromosome segregation are associated phenomena (22). The final sperm morphology differentiation begins after the second meiotic division of the spermatozoa when the nucleus is haploid. Alterations in the mechanism of meiosis lead to aneuploid spermatozoa with morphologic abnormalities, explaining why semen samples with increased rates of abnormal forms present increased aneuploidy rates (3, 6). We could suggest that abnormal sperm morphology is a marker of abnormal spermatogenesis, which in turn is associated with elevated sperm aneuploidy. Sperm ploidies were inversely correlated with sperm motility. The hypo- and hyperhaploidy rates were reduced as sperm motility increased. Vegetti et al. (4) reached the same conclusion by using fluorescence in situ hybridization (FISH) techniques. We suggest that the increased progressive motility is probably associated with a high concentration of normal haploid sperm cells, as determined from SFC analysis after propidium iodide staining. An intriguing finding of the present study was that semen samples that met the WHO criteria regarding morphology (>30% normal morphology) appeared with a high hyperhaploidy rate (7.69%; Fig. 1A). The high incidence of aneuploidies detected in the present study with SFC compared with those obtained by FISH techniques can be explained by the increased sensitivity of this method. SFC offers a screening of aneuploidies observed in all chromosomes. In contrast, the aneuploidy rates obtained from FISH are based on the analysis of specific chromosomes, excluding a significant amount of aneuploidies (23, 24). In addition, SFC is a very fast method with very low cost (one-tenth of the cost for FISH). On the other hand, FISH techniques are suitable for determination of specific aneuploidies of specific chromosomes that SFC cannot offer. In the present study, sperm cell maturity was evaluated in all of the semen samples. The maturation of spermatozoa with entirely condensed chromatin in their nucleus involves a group of structure rearrangements, transcription alterations, and replacement of histones by protamines. All of these processes have been found to be incomplete in many ejaculated spermatozoa from infertile men (25), where increased DNA stainability has been reported. Acridine orange staining followed by SFC (11) was performed to estimate the Fertility and Sterility â 113

5 percentage of sperm cells with complete epididymal maturation and totally condensed nuclear chromatin. We found that significant increases in green and red fluorescence values were positively correlated with high percentages of spermatozoa with abnormal morphology. Spermatozoa with abnormal morphology have uncondensed chromatin in their nucleus and the increased fluorescence values were markers for spermatozoa immaturity. The increased intensity of green fluorescence is perhaps the result of an abnormal exchange of histone for transition proteins and protamines (26). The intensities of green or red fluorescence were independent from sperm concentration and motility in samples with grayzone sperm concentrations ( spermatozoa/ml). In such semen samples, sperm concentration and motility did not constitute markers for sperm cell maturity. Only morphology and chromatin condensation could offer a reliable evaluation in those borderline samples. The same conclusion was reached by Sakkas et al. (27), who examined whether morphology and chromatin anomalies in human spermatozoa influence fertilization after intracytoplasmic sperm injection (ICSI). They demonstrated that poor chromatin packaging and damaged DNA might contribute to failure of sperm decondensation, resulting in failure of fertilization. These findings emphasized the importance of evaluating the chromatin of sperm samples used for assisted reproductive technologies. Semen samples with increased aneuploidies and immaturity of spermatozoa yielded poor pregnancy rates after IUI. A majority of semen samples that did not lead to a pregnancy had increased percentages of immature spermatozoa with <15% normal morphology and high aneuploidy rates (Fig. 4). Sperm chromosomal abnormalities (28), high sperm aneuploidy frequency (21), and impaired spermatozoa maturation (27) were associated with decreased fertilization and pregnancy rates after ICSI. It seems logical to suggest that sperm morphology, spermatozoa ploidy status, and maturity are crucial for a successful outcome of IUI. The performance of SFC to predict a poor IUI outcome when sperm concentration and motility are within lower normal limits would be useful. According to Good clinical treatment in assisted reproduction, composed by the European Society for Human Reproduction and Embryology in June 2008, the number of IUI attempts should not exceed nine cycles, and the majority of pregnancies usually occur during the first six attempts. Sperm concentration and motility are the major criteria taken into account before an IUI procedure. However, the present results show that critical parameters for the evaluation of the fertilizing capacity of spermatozoa, in semen samples with concentration between 15 and spermatozoa/ml, should be the sperm morphology, the nuclear chromatin condensation, and the ploidy of spermatozoa. 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