Sperm chromatin damage impairs human fertility‡

FERTILITY AND STERILITY威 VOL. 73, NO. 1, JANUARY 2000 Copyright ©1999 American Society for Reproductive Medicine Published by Elsevier Science Inc. Printed on acid-free paper in U.S.A.

Sperm chromatin damage impairs human fertility Marcello Spano`, Ph.D.,* Jens P. Bonde, Ph.D.,† Henrik I. Hjøllund, Ph.D.,† Henrik A. Kolstad, Ph.D.,† Eugenia Cordelli, B.Sc.,* Giorgio Leter,* and The Danish First Pregnancy Planner Study Team‡ ENEA CR Casaccia, Rome, Italy, and Aarhus University Hospital, Aarhus, Denmark Received November 23, 1998; revised and accepted August 26, 1999. Supported by grants from the Aarhus University Research Foundation and from the Italian Ministry of University and Scientific and Technological Research (MURST). Reprint requests: Marcello Spano`, Ph.D., Section of Toxicology and Biomedical Sciences, Department of the Environment, ENEA CR Casaccia, Via Anguillarese 301, 00060 Rome, Italy (FAX: ⫹39-0630486559; E-mail: spanomrc@casaccia .enea.it). * Section of Toxicology and Biomedical Sciences, ENEA CR Casaccia. † Department of Occupational Medicine, Aarhus University Hospital. ‡ The Danish First Pregnancy Planner Study is a collaborative follow-up study on environmental and biological determinants of fertility. The project is coordinated by the Steno Institute of Public Health, Aarhus University and is undertaken in collaboration with the Department of Growth and Reproduction, National University Hospital, Copenhagen. The team includes Jens Peter E. Bonde, Niels Henrik I. Hjøllund, Tina Kold Jensen, Tine Brink Henriksen, Henrik A. Kolstad, Erik Ernst, Aleksander Giwercman, Niels Erik Skakkebæk, and Jørn Olsen. 0015-0282/99/$20.00 PII S0015-0282(99)00462-8

Objective: To examine the relationship between sperm chromatin defects, evaluated by the flow cytometric (FCM) sperm chromatin structure assay (SCSA), and the probability of a pregnancy in a menstrual cycle (fecundability). Design: Follow-up study. Setting: The Section of Toxicology and Biomedical Sciences, ENEA Casaccia, Rome, Italy, and the Department of Occupational Medicine, Aarhus University Hospital, Aarhus, Denmark. Patient(s): Two hundred fifteen Danish first pregnancy planners with no previous knowledge of their fertility capability. Intervention(s): None. Main Outcome Measure(s): Semen samples were collected at enrollment to measure semen volume, sperm concentration, motility, and morphology (by microscopy), as well as chromatin susceptibility to in situ, acid-induced partial denaturation by the FCM SCSA. Time to pregnancy was evaluated during a 2-year follow-up period. Demographic, medical, reproductive, occupational, and lifestyle data were collected by questionnaire. Fecundability was correlated with SCSA-derived parameters. Result(s): Fecundability declines as a function of the percentage of sperm with abnormal chromatin and becomes small when aberrant cells are ⬎40%. Conclusion(s): Optimal sperm chromatin packaging seems necessary for full expression of the male fertility potential. The SCSA emerged as a predictor of the probability to conceive in this population-based study. (Fertil Steril威 2000;73:43–50. ©1999 by American Society for Reproductive Medicine.) Key Words: Human spermatozoa, chromatin structure, flow cytometry, fertility, time to pregnancy

The sperm nuclear condensation process involves a dramatic sequence of events including topological rearrangements, transition of DNAbinding proteins, alteration in transcription, loss of nucleosomal structure, and acquisition of a chromosomal organization consisting almost entirely of condensed chromatin (1, 2). Accumulating evidence indicates a negative correlation between disturbances in the organization of the genomic material into sperm nuclei and the fertility potential of spermatozoa. It is suspected that chromatin packaging anomalies in human spermatozoa can arise because of defective protamination (3, 4) and/or the presence of breaks in the DNA molecule. Links between increased sperm chromatin defects and decreased fertilization rates have been reported in the context of assisted fertili-

zation techniques (5–9). In addition, a number of studies have shown that spermatozoa with abnormal nuclear chromatin organization are more frequent in subfertile or in infertile than in fertile men (10 –18). The sperm chromatin condensation level can be evaluated by a variety of microscopybased approaches (16, 19 –22) and by computer interfaced flow cytometry (FCM) (10, 12, 13). Among the FCM techniques, interest has recently been focused on the sperm chromatin structure assay (SCSA), first described in 1980 (10). The SCSA exploits the metachromatic properties of acridine orange (AO) to monitor the susceptibility of sperm chromatin DNA to acid-induced denaturation in situ because the low pH treatment causes partial DNA denaturation only in sperm having altered chromatin 43

structure. On the other hand, sperm with normal chromatin packaging seem more resistant to the denaturation challenge. The SCSA rapidly identifies the cells with abnormal chromatin structure, and there are indications that their relative abundance could be indicative of impaired fertility (10, 11, 23–25). In addition, several studies have shown that the correlation between conventional andrological parameters and SCSA data is weak both in normal (23, 26) and pathological subjects (25). Therefore, SCSA parameters can be considered independent descriptors of semen quality and can be useful to complement the information derived from the classical andrological assessment. Because the withinperson variability of the SCSA parameters is much lower over time than the classical measures of human sperm (23, 26), chromatin damage assessment by SCSA has also been considered in recent reproductive toxicology studies (27, 28). At the present time, the preliminary conclusion is that poor-quality sperm chromatin structure, as evaluated by the SCSA that identifies sperm with loosely packaged chromatin and/or damaged DNA, may be indicative of male subfertility. This conclusion, however, needs to be corroborated by more stringent evidence. Previous data from animal experiments reported a strong correlation between SCSA data and fertility ranking in bulls (29) and boars (30). More recently, in a prospective study involving 165 American, presumably fertile, couples desiring to achieve pregnancy, SCSA data were the best predictors for whether a couple would not become pregnant (31). In this study to extend and reinforce this finding, we examined the relationship between sperm chromatin damage, assessed by using the FCM SCSA and the probability to conceive in a menstrual cycle, in a prospective population-based follow-up study involving a cohort of 215 Danish first pregnancy planners.

MATERIALS AND METHODS Population and Semen Sampling We invited members of four Danish trade unions to enroll in the study if they lived with a partner, had no reproductive history, and intended to stop contraception so that they could have a child. From 1992 to 1994, a total of 231 couples living in the western region of Denmark met these criteria. The couples were fully informed about the purpose of the study and provided written consent. The follow-up started when the couples discontinued contraception and was completed within 24 months. At enrollment both partners filled in a questionnaire on demographic, medical, reproductive, occupational, and lifestyle factors. A fresh semen sample was collected in a 50-mL polyethylene jar and examined within the first 2 hours, in accordance with World Health Organization guidelines (32) to determine semen volume, sperm concentration, motility, and morphology, as described earlier (33). Briefly, the 44

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Sperm chromatin and human fertility

smears are air-dried, fixed in ethanol-ether, and stained by a modified Papanicolau technique. Aliquots (0.2– 0.4 mL) of semen were frozen in cryotubes at ⫺80°C for later FCM SCSA analysis. Of the 231 couples initially enrolled, 3 were excluded because of azoospermia and 3 because of insufficient semen volume for the SCSA analysis; 10 were not analyzed because of technical reasons. The remaining 215 couples provided information on 1,326 menstrual cycles during followup, but only 1,301 cycles (838 cycles, months 1– 6; 463 cycles, months 7–24) were included in the analysis, because 25 cycles without reported sexual intercourse between cycle days 11 and 20 were excluded. During the first six menstrual cycles or until a pregnancy was recognized by the general practitioner, the women kept a diary on vaginal bleeding and sexual intercourse during the previous 24 hours, providing simple “yes/no” statements.

FCM SCSA The SCSA was applied following the procedure described elsewhere (23, 24, 26) with minor modifications. Briefly, at the end of the collection period, all samples were coded and shipped on dry ice to the ENEA FCM facility in Rome. On the day of analysis, the samples were quickly thawed in a 37°C water bath and used immediately. Cells (1–2 ⫻ 106) were treated with a low pH detergent solution containing 0.1% Triton X-100, 0.15 M NaCl, and 0.08 N HCl (pH 1.4) for 30 seconds and then stained with 6 mg/L of AO (chromatographically purified; Molecular Probes, Eugene, OR) in a phosphate-citrate buffer (pH 6.0). Cells were analyzed by a FACStar Plus flow cytometer (Becton Dickinson, San Jose, CA), equipped with an Ar ion laser (Innova 306; Coherent, Santa Clara, CA), tuned at 488 nm. Events (10,000) were accumulated for each measurement. Under these experimental conditions, when excited with a blue light source, AO intercalated with doublestranded DNA emits green fluorescence (530 ⫾ 30 nm), and AO associated with single-stranded DNA emits red fluorescence (⬎630 nm). Because it has been demonstrated that residual RNA molecules in the mature sperm do not interfere with the measurement (23), the ratio of red to green fluorescence reflects the level of single- vs. double-stranded DNA. Sperm chromatin damage was quantified by FCM measurements of the metachromatic shift from green (native, double-stranded DNA) to red (denatured, single-stranded DNA) fluorescence and displayed as red vs. green fluorescence intensity cytogram patterns. Scattergram analysis of raw data with each point representing the coordinate of red and green fluorescence intensity values for every individual sperm was performed with use of Becton Dickinson standard software. When cellular debris appears in the lower left hand corner, it is gated off and excluded from data analysis. The extent of DNA denaVol. 73, No. 1, January 2000

FIGURE 1 Representative green (FL1-H) vs. red (FL3-H) fluorescence bivariate cytogram (A) together with the corresponding ␣T histogram (B) of one sample measured by the FCM SCSA. The fraction of cells with abnormal chromatin (COMP ␣T) is boxed off in the cytogram and in the corresponding frequency histogram.

Spano`. Sperm chromatin damage. Fertil Steril 2000.

turation can conveniently be expressed by the function ␣T which is the ratio of red to total (red plus green) fluorescence intensity (34), thus representing the amount of denatured, single-stranded DNA over the total cellular DNA.

␣T values can range between 0 and 1 but, for practical reasons, they are generally and conveniently reported spanning between 0 and 1,024 channels of fluorescence. ␣T was calculated with use of the ListView software (Phoenix Flow Systems, San Diego, CA), for each sperm cell in a sample, and the results are expressed as the mean (X ␣T), the standard deviation (SD ␣T) of the ␣T distribution, and as the proportion of cells with high ␣T values, usually called cells outside the main population (COMP ␣T). For a more detailed description of this data analysis procedure, the reader is referred elsewhere (35). Practically, DNA in sperm with abnormal chromatin structure exhibits increased red fluorescence intensity, which eventually results in a broadening of the ␣T function (higher SD ␣T), a shift toward higher mean (⫻ ␣T) values, and a larger percentage of COMP ␣T. Standard deviation ␣T represents the variability of chromatin structure abnormalities within the sperm population. In addition, other parameters considered in the SCSA analysis were the mean channel (⫻ green) and standard deviation (SD green) of the green fluorescence intensity distribution. The green fluorescence intensity distribution reflects double-stranded DNA content and/or degree of sperm chromatin condensation, which excludes DNA stainability. For the flow cytometer set up and calibration aliquots were used from a normal human ejaculate sample, not from the Danish samples, retrieved from our laboratory reposiFERTILITY & STERILITY威

tory. Calibration aliquots were thawed and measured at each start-up of the flow cytometer and after every 10 samples, to ensure standardization and stability of the instrument from sample to sample and from day to day. The instrument high voltage of the green and red fluorescence intensities were set to have a mean ␣T value of 220 ⫾ 10 channels relative to the calibration control sample.

Statistical Analysis Time to pregnancy was analyzed by logistic regression analysis of the total number of observed cycles with the outcome pregnant or not pregnant. Time of fertilization is a discrete biological variable, because ovulation takes place only once during a menstrual cycle. Thus, logistic regression is equivalent to discrete survival analysis. The method is based on the analysis of the likelihood and therefore is valid for discrete survival data that have the same probability distribution as a series of Bernoulli trials (36). Therefore, measures of association for fecundability odds ratios were estimated by discrete survival analysis of the outcome pregnant/not pregnant of the total number of observed cycles (n ⫽ 1,301). The fecundability odds ratios describing the relation between cycle outcome (pregnancy, yes/no) and the separate SCSA values were adjusted by menstrual cycle number (categorized variables) and several potential confounding factors (listed in Table 3, see Results). Models were fitted by including SCSA values and one or more conventional semen characteristics to evaluate the significance of one characteristic adjusted for the effects of others. The measures of SCSA were entered as continuous variables (one-unit increment) and as grouped variables based on the quintiles of the 45

TABLE 1

TABLE 2

Characteristics of the 215 Danish first pregnancy planner couples. Mean (⫾SD) age (y) Male partner Female partner Mean (⫾SD) body mass index (kg/m2) Male partner Female partner Last contraceptive method (%) Oral contraception Condom Intrauterine device Other methods Percentage of patients with menstrual cycle length (d) ⬍25 25–33 ⬎34 Unknown Urogenital disorders (%) Male partners* Female partners† Tobacco smoking (%) Male nonsmokers Female nonsmokers Alcoholic beverages (⬎10 drinks/wk) (%) Male partners Female partners Percentage of menstrual cycles in which couples had sexual intercourse at least once between days 14 and 16 Mean (⫾SD) days of sexual abstinence before semen sample Percentage of fever in the previous month

24.3 ⫾ 2.9 22.6 ⫾ 3.4 35.3 48.4 2.8 13.5

1.9 75.3 20.5 2.3

Semen volume (mL)* Sperm concentration (⫻106/mL) Normal sperm (%)† Motile sperm (%) SCSA parameter ⫻ ␣T SD ␣T COMP ␣T (%) ⫻ green SD green

174 215 210 215 215 215 215 215 215

3.49 77.6 40.8 37.9 227.4 78.0 14.0 475.3 119.4

3.30 57.0 42.5 33.0 221.9 73.2 10.7 468.1 116.7

Range 0.5–9.0 0.3–408 13.0–63.0 10.0–97.0 195.5–370.0 36.2–176.4 3.2–88.1 401.7–607.0 78.7–187.5

Note: The SCSA parameters ⫻ ␣T, SD ␣T, ⫻ green, and SD green are expressed in fluorescence channel units. * Forty-one fresh samples (19.1%) with recorded spillage were excluded. † Five smears were unsuitable for morphology scoring. Spano`. Sperm chromatin damage. Fertil Steril 2000.

12.6 6.5 62.3 69.3 34.9 11.2

75.2 4.1 ⫾ 3.9 9.8

Spano`. Sperm chromatin damage. Fertil Steril 2000.

distributions. Model fits were examined by the HosmerLemeshow goodness-of-fit test (37). The unadjusted relation between SCSA parameters at start of follow-up and likelihood of pregnancy in a menstrual cycle was outlined graphically by means of smoothing with local linear regression of binomially distributed data. The 95% confidence limits (CI) were calculated by generalized additive models (SAS/PROC Insight; SAS Institute Inc., Cary, NC, 1998). Spearman nonparametric correlation was used to determine correlations between semen characteristics and SCSA parameters. The analyses were conducted with use of the SAS statistical package (SAS/STAT User’s Guide, Release 6.03; SAS Institute Inc.).

RESULTS Figure 1 shows a representative example of the outcome from FCM SCSA analysis of human sperm samples. Figure Spano` et al.

No. of samples Mean Median

28.4 ⫾ 2.9 25.8 ⫾ 2.7

* Epididymitis, gonorrhea, orchitis, and a few other rare disorders. † Salpingitis, ovarian cysts, gonorrhea, perforated appendix, and a few other rare disorders.

46

Descriptive statistics of semen characteristics in the 215 Danish first-pregnancy planners.

Sperm chromatin and human fertility

1A refers to the bivariate cytogram (green vs. red fluorescence intensity) of AO-stained sperm after acid-induced denaturation, and each dot represents one single individual cell. The position of dots depends on their relative green and red fluorescence intensities. The corresponding ␣T distribution is shown in Figure 1B. The relevant SCSA parameters have been calculated on this histogram, namely, ⫻ ␣T, the mean channel value of the main peak, and SD ␣T, the peak standard deviation. Furthermore, the fraction of cells with abnormal chromatin (COMP ␣T) is represented by sperm that have higher red fluorescence intensities and accumulate in the histogram region located to the right of the main peak. Couple characteristics are displayed in Table 1, and the descriptive statistics of the semen parameters evaluated by the microscopic analysis and by the FCM SCSA are listed in Table 2. The proportion of women who became pregnant within the follow-up period of 6 and 12 menstrual cycles was 57.2% and 70.2%, respectively. The fecundability was 15% in the first cycle. The unadjusted probability of pregnancy in a menstrual cycle across the entire range of SCSA values obtained from the initial semen samples is outlined in Figure 2A–C, and the corresponding confounder adjusted measures of association are given in Table 3. We found statistically significant inverse relationships between fecundability and the SCSA parameters ␣T and COMP ␣T. Fecundability continuously decreased as a function of increasing values of ⫻ ␣T and increasing frequency of COMP ␣T sperm. When ⫻ ␣T was ⬎240 channels and COMP ␣T was ⬎20%, fecundability started dropping and became very small for ⫻ ␣T values of ⱖ280 or when the COMP ␣T fraction was ⱖ40%. The association between COMP ␣T and fecundability in Vol. 73, No. 1, January 2000

the first cycle of follow-up was even stronger. As shown in Figure 2D, the probability of fathering a child sharply declined for COMP ␣T of ⬎20% and was negligible when this fraction added up to 40%. Therefore, if a certain individual has a high fraction of sperm with abnormal chromatin, this makes this individual a good candidate not to conceive. On the other hand, the SCSA parameter ⫻ Green was not related to fecundability. The adjustment for period of abstinence did not change the fecundability odds ratios (data not shown). It is worth noting that ⫻ ␣T and COMP ␣T were highly significantly related to fecundability, even after adjustment for the possible female partner-related confounding factors listed in Table 3. In addition, ⫻ ␣T and COMP ␣T contributed to prediction of fecundability independently of sperm concentration. The fecundability odds ratios in models also including sperm concentration was for ⫻ ␣T 0.989 (95% CI 0.978 – 0.999, P⫽.0311) and for COMP ␣T 0.958 (95% CI 0.920 – 0.998, P⫽.0398). However, in models including the proportion of normal sperm cells in addition to sperm concentration, the contributions were of borderline significance (P⫽.061 and .052, respectively).

DISCUSSION SCSA measurements reflect the heterogeneity of the nuclear chromatin conformational organization in the different sperm cells of the ejaculate. Human semen samples exhibit a wide variability in chromatin packaging levels (23, 26, 31), presence of endogenous nicks in the DNA (16, 21, 38, 39), and length of the histone-associated regions (40). Together with densely packed nuclei of most spermatozoa, there are, also in normal samples, a variable number of sperm with a variety of chromatin defects. The SCSA can provide an objective assessment of sperm chromatin integrity within a semen sample, rapidly identifying and evaluating the fraction of cells showing increased susceptibility to DNA denaturation. The samples classified in this way can be considered abnormal if the number of cells with abnormal chromatin structure, the COMP ␣T fraction, would exceed a certain value. In previous studies involving SCSA analysis on healthy volunteers, the reported mean COMP ␣T percentages were centered around 16.8 ⫾ 7.2% (23), 9.4% with an upper limit at 16% (25), and 15.0 ⫾ 10.6% (26). However, in these studies, no associations have been attempted with the fertility capabilities of the individuals. The degree of heterogeneity compatible with the normal fertility potential has been addressed in the present study and in another independent study (31). The results of both studies are consistent with the finding that sperm chromatin structure is reflective of fertility potential, which deteriorates when the percent of COMP ␣T sperm is ⬎30%. Our data demonstrate that a high proportion of sperm with chromatin abnormalities, measured by the SCSA for enFERTILITY & STERILITY威

hanced DNA susceptibility to acid-induced denaturation in situ, is strongly associated with reduced fecundability. Fecundability started declining rapidly as the COMP ␣T fraction increased ⬎20%. Because it is reasonably expected that the fecundability in the first cycles is predicted more precisely by samples collected immediately before follow-up, the relationship between sperm chromatin integrity and the probability of fathering a child resulted much stronger when we considered this time window. In the first cycle, no pregnancy occurred when COMP ␣T was ⬎40%. Therefore, our data strongly support and corroborate the results of a recently published SCSA prospective study involving 165 American presumably fertile couples who were discontinuing contraception to achieve pregnancy (31). In that study, COMP ␣T emerged as the best indicator to predict whether a couple would not become pregnant. In fact, all couples with COMP ␣T values ⬎30% experienced either delayed pregnancy or no pregnancy at all. In general, SCSA values from couples conceiving during the first months were significantly different compared with the SCSA values obtained from individuals conceiving at later times or not conceiving, thus providing strong evidence that sperm chromatin structure was reflective of fertility potential. Furthermore, in our experimental conditions, another ␣T parameter resulted associated with fertility potential. We have observed that the higher the ⫻ ␣T values the lower the probability to conceive. Again, this trend has also been observed in the study quoted above (31) where statistically significant higher values of ⫻ ␣T were reported in cases of delayed pregnancy or no pregnancy at all. On the other hand, the SCSA parameter ⫻ green, the mean green fluorescence intensity that reflects DNA stainability, was not related to fecundability. It has been shown that the progressive chromatin packaging, as the human sperm transverse the epididymus, can be followed by a reduction of DNA stainability relative to round spermatids because of the marked decrease of DNA-binding sites available for the fluorochrome binding (41) and that lack of appropriate sperm maturation resulted in an increased DNA stainability (24). The highly motile subpopulation of spermatozoa, selected by swim-up or Percoll gradient centrifugation methods, was also characterized by a marked improvement of chromatin structure features and reduced DNA stainability, compared with the whole sperm population in the ejaculate (17, 42, 43). Altogether, even though the underlying mechanisms are not fully elucidated, these observations indicate that a correct chromatin packaging around the protamine core seems a necessary condition for the optimal expression of the male gamete fertility potential. However, this condition does not seem mandatory for a successful fertilization as demonstrated by intracytoplasmic sperm injection where normal fertilization and pregnancy rates can also be achieved with 47

FIGURE 2 The unadjusted relation between fecundability and SCSA parameters. (A), ⫻ ␣T (1,301 cycles). (B), COMP ␣T (1,301 cycles). (C), ⫻ green (1,301 cycles). (D), COMP ␣T (first 215 cycles). The relationships are graphically represented by smoothing linear regression for binomially distributed data. The circles indicate the observed frequency of pregnancy in decentils of the SCSA parameters. The dotted lines are the 95% CI.

Spano`. Sperm chromatin damage. Fertil Steril 2000.

cells that have not completed spermiogenesis, such as epididymal and testicular spermatozoa (44).

reproduction techniques and, therefore, evaluated mainly on subjects with serious infertility problems.

The ␣T parameters seem more associated with the presence of DNA breaks in the mature sperm (39). A correlation between sperm chromatin abnormalities and the presence of DNA strand breaks has been shown to exist (21, 39, 40 45– 48) and, in particular, in human sperm samples, it has recently been demonstrated that the percentage of sperm with DNA strand breaks identified by the COMET assay are strongly correlated with the proportion of COMP ␣T cells (38). Thus, the ␣T parameters, most probably, appear to reflect chromatin alterations associated with DNA nicks. It is of interest that associations between increased DNA fragmentation and decreased fertilization rates and/or embryo cleavage have been reported after IVF (5, 8), subzonal insemination (6), and intracytoplasmic sperm injection (7, 9, 49). It should be pointed out, however, that these studies, performed to evaluate the influence of chromatin structure defects on the sperm-fertilizing capabilities and/or embryo development, have been tested in the context of assisted

We have also found that SCSA data contributed to prediction of fecundability independently of sperm concentration. The correlation analysis confirmed that the SCSA data are weakly associated with the parameters of the conventional semen quality assessment. For example, Spearman correlation coefficients were ⫺0.23 (P⬍.0006) and ⫺0.25 (P⬍.0003) for COMP ␣T vs. sperm concentration and vs. percent morphologically normal forms, respectively. These results are in close agreement with those obtained in other studies (23, 26, 31).

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Sperm chromatin and human fertility

It is worth noting that, in a recent study of 430 firstpregnancy planners (215 of whom have been analyzed by the FCM SCSA in the present work), fecundability increased with increasing sperm concentration up to 40 ⫻ 106/mL, whereas higher sperm densities were not associated with a further increase of fecundability (50). We have also found that fertility of men, with sperm densities of ⬍40 ⫻ 106/mL, Vol. 73, No. 1, January 2000

TABLE 3 Adjusted fecundability odd ratios for SCSA parameters among 215 Danish first-pregnancy planners. SCSA parameter*

No. of cycles

⫻ ␣T ⱕ216 217–223 224–240 241–279 ⱖ280 COMP ␣T ⱕ8 8–11 12–19 20–39 ⱖ40 ⫻ green ⱕ444 445–468 469–506 507–551 ⱖ552

1,301 336 317 330 262 56 1,301 332 319 326 266 58 1,301 328 334 321 233 62

Fecundability (%)

13.7 12.6 13.6 8.0 1.8 15.1 11.3 13.5 7.5 1.7 11.0 10.5 14.0 10.7 16.1

Odds ratio 0.985 1.000 0.918 1.060 0.550 0.170 0.967 1.000 0.671 0.939 0.427 0.129 1.000 1.000 0.880 1.400 0.960 1.470

95% C.I.

P value

0.976–0.995

.004

0.58–1.46 0.66–1.70 0.31–0.96 0.02–1.28 0.950–0.990

.718 .809 .035 .085 .002

0.42–1.08 0.60–1.48 0.24–0.76 0.02–0.97 0.996–1.004

.100 .785 .004 .047 .930

0.53–1.47 0.86–2.28 0.55–1.69 0.67–3.23

.635 .182 .894 .337

Note: Adjustment for cycle number (dichotomized variables) and female characteristics (age [ⱖ30 years: yes/no], urogenital disorders [yes/no], length of menstrual cycle [⬎33 days: yes/no], oral contraception as last method of birth control [cycle 1: yes/no; cycle 2: yes/no; cycle 3: yes/no], current smoking [yes/no]). * SCSA parameters were entered as continuous variables (one unit increment) and as grouped variables based on the quintiles of the distributions. ⫻ ␣T and ⫻ green are expressed in fluorescence channel units, whereas COMP ␣T is in %. Spano`. Sperm chromatin damage. Fertil Steril 2000.

was greatly impaired by even moderate levels of sperm chromatin damage (data not shown). This article represents one of the first efforts aimed at verifying whether sperm chromatin integrity could be predictive of the human fertility potential in the normal population, here represented by a cohort of 215 young, Danish first-pregnancy planners. Our epidemiological approach presents several advantages. The follow-up of couples without previous reproductive experience provided an unbiased estimate of the fecundability related to sperm quality characteristics. The couples were scrutinized to ascertain attempts to get pregnant and at least one unprotected intercourse had to take place during the midmenstrual cycle. Female causes of subfertility can confound the above associations if related to semen characteristics, but all results remained unchanged after adjustment for female urogenital disorders and other potential confounding factors. The fecundability in the first cycle of this population was 15%, somewhat lower than that observed in a few other follow-up studies, but the median sperm concentration was in agreement with findings from recent Danish occupational sperm studies (33). The use of oral contraception by most of the women in our sample is a likely explanation of the apparently low fecundability rate. In conclusion, we have demonstrated that poor-quality FERTILITY & STERILITY威

sperm chromatin structure, as assessed by the FCM SCSA, is highly indicative of male subfertility, regardless of the number, the motility, and the morphology of spermatozoa. These results corroborate the use of the SCSA in the andrology laboratory because this technique can help to identify additional defective samples, thus complementing the results from conventional semen quality assessment. References 1. Ward WS. The structure of the sleeping genome: implications of sperm DNA organization for somatic cells. J Cell Biochem 1994;55:77– 82. 2. Kramer JA, Krawetz SA. RNA in spermatozoa: implications for the alternative haploid genome. Mol Hum Reprod 1997;3:473– 8. 3. Balhorn R, Reed S, Tanphaichitr N. Aberrant protamine 1/protamine 2 ratios in sperm of infertile human males. Experientia 1988;44:52–5. 4. De Yebra L, Ballesca JL, Vanrell JA, Bassas L, Oliva R. Complete selective absence of protamine P2 in humans. J Biol Chem 1993;268: 10553–7. 5. Hoshi K, Katayose H, Yanagida K, Kimura Y, Sato A. The relationship between acridine orange fluorescence of sperm nuclei and the fertilizing ability of human sperm. Fertil Steril 1996;66:634 –9. 6. Bianchi PG, Manicardi GC, Urner F, Campana A, Sakkas D. Chromatin packaging and morphology in ejaculated human spermatozoa: evidence of hidden anomalies in normal spermatozoa. Mol Hum Reprod 1996; 2:139 – 44. 7. Sakkas D, Urner F, Bianchi PG, Bizzaro D, Wagner I, Jaquenoud N, et al. Sperm chromatin anomalies can influence decondensation after intracytoplasmic sperm injection. Hum Reprod 1996;11:837– 43. 8. Sun JG, Jurisicova A, Casper RF. Detection of deoxyribonucleic acid fragmentation in human sperm: correlation with fertilization in vitro. Biol Reprod 1997;56:602–7. 9. Lopes S, Sun JG, Jurisicova A, Meriano J, Casper RF. Sperm deoxyribonucleic acid fragmentation is increased in poor-quality semen samples and correlates with failed fertilization in intracytoplasmic sperm injection. Fertil Steril 1998;69:528 –32.

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10. Evenson DP, Darzyinckiewicz Z, Melamed MR. Relation of mammalian sperm chromatin heterogeneity to fertility. Science 1980;240: 1131–3. 11. Evenson DP, Melamed MR. Rapid analysis of normal and abnormal cell types in human semen and testis biopsies by flow cytometry. J Histochem Cytochem 1983;31:248 –53. 12. Spano` M, Calugi A, Capuano V, De Vita R, Go¨hde W, Hacker U, et al. Andrological sperm analysis by flow cytometry and sizing. Andrologia 1984;16:367–75. 13. Engh E, Clausen OP, Scholberg A, Tollefsrud A, Purvis K. Relationship between sperm quality and chromatin condensation measured by sperm DNA fluorescence using flow cytometry. Int J Androl 1992;15: 407–15. 14. Foresta C, Zorzi M, Rossato M, Varotto A. Sperm nuclear instability and staining with aniline blue: abnormal persistence of histones in spermatozoa in infertile men. Int J Androl 1992;15:330 –7. 15. Engh E, Scholberg A, Clausen OPF, Purvis K. DNA flow cytometry of sperm from normospermic men in barren couples and men of proven fertility. Int J Fertil Menopausal Stud 1993;38:305–10. 16. Hughes CM, Lewis SEM, McKelvey-Martin VJ, Thompson W. A comparison of baseline and induced DNA damage in human spermatozoa from fertile and infertile men, using a modified comet assay. Mol Hum Reprod 1996;2:613–9. 17. Golan R, Shochat L, Weissenberg R, Soffer Y, Marcus Z, Oschry Y, et al. Evaluation of chromatin condensation in human spermatozoa: a flow cytometric assay using acridine orange staining. Mol Hum Reprod 1997;1:47–54. 18. Troiano L, Granata AR, Cossarizza A, Kalashnikova G, Bianchi R, Pini G, et al. Mitochondrial membrane potential and DNA stainability in human sperm cells: a flow cytometry analysis with implications for male infertility. Exp Cell Res 1998;241:384 –93. 19. Krzanowska H. Toluidine blue staining reveals changes in chromatin stabilization of mouse spermatozoa during epididymal maturation and penetration of ova. J Reprod Fertil 1982;64:97–101. 20. Tejada RI, Mitchell JC, Norman A, Marik JJ, Friedman S. A test for the practical evaluation of male fertility by acridine orange fluorescence. Fertil Steril 1984;42:87–91. 21. Bianchi PG, Manicardi GC, Bizzaro D, Bianchi U, Sakkas D. Effect of deoxyribonucleic acid protamination on fluorochrome staining and in situ nick-translation of murine and human spermatozoa. Biol Reprod 1993;49:1083– 8. 22. Francavilla S, Cordeschi G, Gabriele A, Gianaroli L, Properzi G. Chromatin defects in normal and malformed human ejaculated epididymal spermatozoa: a cytochemical ultrastructural study. J Reprod Fertil 1996;106:259 – 68. 23. Evenson DP, Jost LK, Baer RK, Turner TW, Schrader SM. Individuality of DNA denaturation patterns in human sperm as measured by the sperm chromatin structure assay. Reprod Toxicol 1991;5:115–25. 24. Evenson DP, Jost LK. Sperm chromatin structure assay: DNA denaturability. In: Darzynkiewicz Z, Robinson JP, Crissman HA, eds. Flow cytometry, Part B. 2nd ed. Orlando: Academic Press, 1994:159 –75. 25. Fosså SD, De Angelis P, Kraggerud SM, Evenson D, Theodorsen L, Clausen OP. Prediction of posttreatment spermatogenesis in patients with testicular cancer by flow cytometric sperm chromatin structure assay. Cytometry (Communications in Clinical Cytometry) 1997;30: 192– 6. 26. Spano` M, Kolstad H, Larsen SB, Cordelli E, Leter G, Giwercman A, et al. The applicability of the flow cytometric sperm structure chromatin assay to epidemiological studies. Hum Reprod 1998;13:2495–505. 27. Larsen SB, Giwercman A, Spano` M, Bonde JP, ASCLEPIOS Study Group. A longitudinal study of semen quality in pesticide spraying Danish farmers. Reprod Toxicol 1998;12:581–9. 28. Potts RJ, Newbury CJ, Smith G, Notarianni LJ, Jefferies TM. Sperm chromatin damage associated with male smoking. Mutat Res 1999;423: 103–11. 29. Ballachey BE, Saacke RG, Evenson DP. The sperm chromatin structure assay: relationship with alternate tests of sperm quality and heterospermic performance of bulls. J Androl 1988;9:109 –15. 30. Evenson DP, Thompson L, Jost L. Flow cytometric evaluation of boar semen by the sperm chromatin structure assay as related to cryopreservation and fertility. Therio 1994;41:637–51. 31. Evenson DP, Jost LK, Marshall D, Zinaman MJ, Clegg E, Purvis K, et al. Utility of the sperm chromatin structure assay (SCSA) as a diag-

50

Spano` et al.

Sperm chromatin and human fertility

32. 33.

34. 35.

36. 37. 38.

39.

40.

41.

42.

43.

44.

45.

46.

47.

48.

49.

50.

nostic and prognostic tool in the human fertility clinic. Hum Reprod 1999;14:1039 – 49. World Health Organization. Laboratory manual for the examination of human semen and sperm-cervical mucus interaction. 3rd ed. New York: Cambridge University Press, 1993. Bonde JP, Hjøllund NH, Jensen TK, Ernst E, Kolstad H, Henriksen TB, et al. A follow-up study of environmental determinants of fertility among 430 Danish first-pregnancy planners: design and methods. Reprod Toxicol 1998;12:19 –27. Darzynckiewicz Z, Traganos F, Sharpless T, Melamed MR. Thermal denaturation of DNA in situ as studied by acridine orange staining and automated cytofluorometry. Exp Cell Res 1975;90:411–28. Evenson D, Jost L, Gandour D, Rhodes L, Stanton B, Clausen OP, et al. Comparative sperm chromatin structure assay measurements on epiillumination and orthogonal axes flow cytometers. Cytometry 1995;19: 295–303. Sheike T, Jensen TK. A discrete survival model with random effects: an application to time to pregnancy. Biometrics 1997;53:318 –29. Hosmer D, Lemeshow S. Applied logistic regression. New York: John Wiley & Sons, 1989. Aravindan GR, Bjordahl J, Jost LK, Evenson DP. Susceptibility of human sperm to in situ DNA denatured is strongly correlated with DNA strand breaks identified by single-cell electrophoresis. Exp Cell Res 1997;236:231–7. Sakkas D, Manicardi G, Bianchi PG, Bizzaro D, Bianchi U. Relationship between the presence of endogenous nicks and sperm chromatin packaging in maturing and fertilizing mouse spermatozoa. Biol Reprod 1995;52:1149 –55. Gardiner-Garden M, Ballesteros M, Gordon M, Tam PP. Histone- and protamine-DNA association: conservation of different patterns within the beta-globin domain in human sperm. Mol Cell Biol 1998;18: 3350 – 6. Golan R, Cooper TG, Oschry Y, Oberpenning F, Schulze H, Shochat L, et al. Changes in chromatin condensation of human spermatozoa during epididymal transit as determined by flow cytometry. Hum Reprod 1996;11:1457– 62. Molina J, Castilla JA, Gil T, Hortas ML, Vergara F, Herruzo A. Influence of incubation on the chromatin condensation and nuclear stability in human spermatozoa by flow cytometry. Hum Reprod 1995; 10:1280 – 6. Spano` M, Cordelli E, Leter G, Lombardo F, Lenzi A, Gandini L. Nuclear chromatin variations in human spermatozoa undergoing swimming up and cryoconservation evaluated by the flow cytometric sperm chromatin structure assay. Mol Hum Reprod 1999;5:29 –37. Silber SJ, Van Steirteghem AC, Liu J, Nagy Z, Tournaye H, Devroey P. High fertilization and pregnancy rate after intracytoplasmic sperm injection with spermatozoa obtained from testicle biopsy. Hum Reprod 1995;10:148 –52. Gorczyka W, Traganos F, Jesionowska H, Darzynkiewicz Z. Presence of DNA strand breaks and increased sensitivity of DNA in situ to denaturation in abnormal human sperm cells: analogy to apoptosis of somatic cells. Exp Cell Res 1993;207:202–5. Manicardi GC, Bianchi PG, Pantano S, Azzoni P, Bizzaro D, Bianchi U, et al. Presence of endogenous nicks in DNA of ejaculated human spermatozoa and its relationship to chromomycin A3 accessibility. Biol Reprod 1995;52:864 –7. Sailer BL, Jost LK, Evenson DP. Mammalian sperm DNA susceptibility to in situ denaturation associated with the presence of DNA strand breaks as measured by the terminal deoxynucleotidyl transferase assay. J Androl 1995;16:80 –7. Manicardi GC, Tombacco A, Bizzaro D, Bianchi U, Bianchi PG, Sakkas D. DNA strand breaks in ejaculated human spermatozoa: comparison of susceptibility to the nick translation and terminal transferase assays. Histochem J 1998;30:33–9. Sakkas D, Urner F, Bizzarro D, Manicardi G, Bianchi PG, Shoukir Y, et al. Sperm nuclear DNA damage and altered chromatin structure: effect on fertilization and embryo development. Hum Reprod 1998; 13(4 Suppl.):11–9. Bonde JP, Ernst E, Jensen TK, Hjøllund NH, Kolstad H, Henriksen TB, et al. Relation between semen quality and fertility: a populationbased study of 430 first-pregnancy planners. Lancet 1998;352: 1172–7.

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