High DNA Fragmentation Index Research Articles

Use of Testicular Sperm/Intracytoplasmic Sperm Injection Yields High Pregnancy Rates in Couples who Failed Multiple In Vitro Fertilization Cycles Owing to High Levels of Sperm DNA Fragmentation

Use of Testicular Sperm/Intracytoplasmic Sperm Injection Yields High Pregnancy Rates in Couples who Failed Multiple In Vitro Fertilization Cycles Owing to High Levels of Sperm DNA Fragmentation

Effects of different dilution levels on bull- and ejaculate related variability of plasmamembran integrity, acrosomal damage and DNA-integrity of cryopreserved bull spermatozoa

In this study the effects of three different dilution levels (60, 30 and 15 Mio/ml) on quality of cryopreserved bovine sperm were investigated. Particularly it was tested, if effects of dilution in regard to sperm quality were related to the factors bull or ejaculate. The following parameters were analysed in four ejaculates each of 42 bulls: percentage of plasma membrane intact sperm (PMI), percentage of sperm with acrosomal damage (AS) as well as percentage of sperm with a high DNA-fragmentation index (DFI%) following cryopreservation. PMI-values decreased (p < 0.05) with increasing degree of dilution. AS-values were not influenced (p > 0.05) by dilution level. There was a decline in DFI% (p <0.05) with increasing dilution of the ejaculate. Coefficient of variation for PMI, AS and DFI% were not (p > 0.05) influenced by dilution. The effects of the factor ejaculate on the variabilities of PMI and AS were higher than the effects of the factor bull. Variability of DFI% depended at the highest dilution level more on the factor bull and in the lowest dilution level more on the factor ejaculate. In conclusion, these results show that dilution of sperm has positive as well as negative effects on sperm quality. With increasing dilution the effect of the factor bull on plasma membrane- and acrosomal integrity.

Catalase supplementation on thawed bull spermatozoa abolishes the detrimental effect of oxidative stress on motility and DNA integrity

The potential protective effect of catalase supplementation during in vitro culture of frozen/thawed bull spermatozoa was investigated. Frozen/thawed semen collected from three fighting bulls was diluted in phosphate buffered saline (PBS) and incubated at 37 degrees C under different experimental conditions: Control, Catalase (CAT) (200 U/mL), Oxidant (OXI) (100 microm Fe(2+)/1 mm ascorbate), and Catalase + Oxidant (CAT/OXI). We assessed sperm motility, acrosomal integrity, viability and chromatin status (SCSA) at 0, 2 and 6 h of incubation. Our results showed that catalase abolished the effect of the oxidant, protecting spermatozoa against reactive oxygen species, and improving both sperm motility and chromatin status during incubation. The OXI treatment significantly reduced the percentage of motile sperm after 6 h of incubation. The statistical model also showed that there were differences in sperm motility between CAT/OXI (20.8 +/- 2.9%) and OXI (11.6 +/- 7.6%) (p < 0.001). There were no significant effects of OXI on sperm viability, acrosomal status or proportion of abnormal tails. %DFI (spermatozoa with moderate or high DNA Fragmentation Index) was significantly higher on OXI (p < 0.001). Catalase prevented DNA fragmentation even in the presence of the oxidant (%DFI: 30.3 +/- 0.8% OXI vs. 17.4 +/- 0.7% CAT/OXI). We conclude that catalase supplementation after thawing could protect bull spermatozoa against oxidative stress, and it could improve media used for processing thawed spermatozoa.

Sperm DNA fragmentation in subfertile men: the effect on the outcome of intracytoplasmic sperm injection and correlation with sperm variables

To present the first UK data on sperm DNA fragmentation levels in subfertile men and fertile controls, the correlation with semen variables, and to assess the effect on the outcome of intracytoplasmic sperm injection (ICSI). In all, 56 subfertile men undergoing ICSI (28 with positive and 28 with a negative outcome for paternity) and 10 control fertile semen donors were recruited. The sperm DNA fragmentation index (DFI) was assessed on raw pre-preparation samples using the sperm chromatin structure assay. A mean of 5212 sperm were analysed per sample and DFI data are presented by fertility status, ICSI outcome and correlated with semen variables (assessed using World Health Organisation criteria). Total DFI was significantly higher in subfertile men than in fertile controls (mean and median of 22.8% and 17.0% vs 8.4% and 5.0%; P < 0.001), as was the proportion of both moderate DFI (16.4% and 13.0% vs 6.4% and 4.0%; P = 0.001) and high DFI (6.2% and 6.1 vs 2.0% and 1.0%; P = 0.01). This difference remained significant when the control men were compared only with the subfertile men with successful paternity. There was no significant difference in DFI in the subfertile men when analysed by ICSI outcome (mean and median of 24.5% and 17.0% vs 22.3% and 21.0% for successful and unsuccessful cycles, respectively; P = 0.94). There was a positive statistically significant correlation (r = 0.37; P = 0.02) between the DFI and sperm morphology. This study confirms a relationship between male subfertility and sperm DFI; we discuss the correct role for genetic testing of sperm in the evaluation of subfertile men. Although DNA fragmentation data might help to decide a suitable treatment, once it is decided to proceed with ICSI, DFI levels have no effect on the outcome.

Sperm chromatin structure assay parameters are not related to fertilization rates, embryo quality, and pregnancy rates in in vitro fertilization and intracytoplasmic sperm injection, but might be related to spontaneous abortion rates

To investigate the relationship between sperm chromatin structure assay (SCSA) parameters, DNA fragmentation index (DFI) and high DNA stainability (HDS), and outcomes of IVF and intracytoplasmic sperm injection (ICSI). Retrospective review and prospective study. Academic human reproduction laboratory. Two hundred twenty-three couples undergoing conventional IVF (n = 137) and ICSI (n = 86). Testing with SCSA on a semen aliquot taken from ejaculate used for assisted reproductive technology (ART). Conventional semen parameters, DFI, HDS, outcomes of IVF and ICSI. There were no significant differences in IVF and ICSI fertilization rate, good embryo rate, and pregnancy rate (PR) between high, moderate, and low DFI or HDS groups. Men with HDS >15% had significantly higher IVF abortion rates. There was a statistically insignificant trend toward an increased abortion rate in the high DFI (>27%) group. The DFI correlated negatively with sperm motility, and HDS correlated negatively with sperm morphology and concentration. Neither DFI nor HDS scores can provide independent information about embryo quality, fertilization, and PRs for infertility patients undergoing ART. Sperm DNA fragmentation probably affects sperm motility. The relationship between HDS and IVF abortion rates provides preliminary evidence that ICSI may be indicated for men with HDS >15%. The potential adverse effect of sperm DNA damage on the quality of postimplantation embryo and spontaneous abortion should be a concern.

Impact of cryopreservation and reactive oxygen species on DNA integrity, lipid peroxidation, and functional parameters in ram sperm

Assisted reproduction using frozen-thawed semen has practical advantages, although cryopreservation is detrimental to sperm fertility in most mammals. We examined the influence of cryopreservation and reactive oxygen species (ROS) on ram sperm DNA stability (using SCSA), lipid peroxidation (LPO), chlortetracycline fluorescence (CTC) patterns, motility and viability. In Experiment 1, DNA integrity, LPO, CTC, motility and viability tests were performed on fresh and cryopreserved sperm after 0, 6, and 24 hr in synthetic oviductal fluid (SOF). In Experiment 2, fresh sperm were incubated in serum-free SOF (SOF-S; 1, 4, and 24 hr) with 0, 50, 150, or 300 microM H2O2 then assayed. Cryopreservation increased the percentage of sperm with a high DNA fragmentation index (%DFI), decreased the percentages of motile and viable sperm at thawing (0 hr), but did not affect LPO. H2O2 (150 or 300 microM) increased %DFI after 24 hr. LPO or sperm viability were not affected by H2O2, although most motility parameters decreased. H2O2 decreased the percentage of chlortetracycline pattern F sperm at 4 hr and increased the percentage of acrosome-reacted sperm (pattern AR) after 1 hr. Pooled data of Experiment 2 showed LPO was positively correlated with SCSA (r = 0.29 to r = 0.59; P < 0.05 to P < 0.01), while most motility parameters and the percentage of viable sperm were negatively correlated with LPO (r = -0.30 to r = -0.38; P < 0.05 to P < 0.01). LPO was positively correlated with the percentage of pattern AR sperm (r = 0.33; P < 0.01). Cryopreservation and H2O2 promote DNA instability in ram sperm, though motility is a more sensitive indicator of oxidative stress than the other parameters investigated.

DNA fragmentation assessment by flow cytometry and Sperm–Bos–Halomax (bright‐field microscopy and fluorescence microscopy) in bull sperm

The aim of this study was to find the relationship between fertility (as 90-day non-return rates) and DNA fragmentation assessed by two techniques [sperm chromatin structure assay (SCSA) and Sperm-Bos-Halomax (SBH)]. Furthermore, other quality parameters were achieved (motility, morphological abnormalities, cytoplasmic droplets, viability, capacitation and acrosomal and mitochondrial status) and their correlations with fertility were analysed. Bulls were divided into three fertility groups: high [non-return rate (NRR) >or= 80], medium (80 < NRR >or= 70) and low (70 < NRR > 40). The results of this study indicate that there is a good correlation between fertility and different parameters of sperm quality (SBH and SCSA parameters, % of spermatozoa with head, neck and total abnormalities, and % of spermatozoa with proximal cytoplasmic droplets) and differences between fertility groups were observed in some of them (SBH and SCSA parameters and % of spermatozoa with head, neck and total abnormalities). In this sense, SBH parameters rendered good correlations with fertility (r = -0.42 using bright light microscope and r = -0.47 with fluorescence). Also, standard deviation of DNA fragmentation index (SD-DFI) and DFIh (cells with High DNA fragmentation index) showed good correlations with fertility (r = -0.41 and r = -0.29). No correlations were observed between SCSA and SBH parameters. A multiple regression shows that four parameters (% of proximal cytoplasmic droplets, % of intact acrosomes in total population, SD-DFI and percentage of fragmented DNA detected by bright light microscope) present a good predictive value of the fertility of sperm samples (r(2) = 0.34, p < 0.001).

P-619: Characterization of four sperm cell populations resolved and flow cytometry sorted by the Sperm Chromatin Structure Assay (SCSA®)

1) characterize by light microscope image analysis and neutral pH Comet analysis the four SCSA derived sperm populations consisting of: Main (no DNA damage detected), Moderate (moderately damaged sperm) and High (highly damaged sperm) DNA Fragmentation Index (% DFI) and High DNA Stainability (HDS is a measure of immature chromatin). 2) Estimate the selection frequency of the moderate DFI populations for ICSI patients in comparison to the more natural selection of sperm by the eggs for the routine IVF patients. Retrospective. ART patients undergoing routine IVF or ICSI 1) SCSA- sort the four sperm populations for: a) computer image analysis of Feulgen stained sperm for morphology and morphometry, and b) neutral pH Comet- defined DNA strand breaks. 2) Statistical frequency selection estimation of the Moderate DFI sperm population by ICSI technicians vs more natural selection of sperm by eggs in routine IVF resulting in pregnancy/miscarriage. The morphology/morphometry features of the Moderate DFI sperm population were equally normal as the Main population sperm but one-half were Comet positive. The High DFI sperm population showed cell morphology/morphometry features with considerable signs of necrosis/apoptosis and were 100% Comet positive. Semen samples derived from the routine IVF pregnancies had significantly lower % moderate DFI values in comparison to the % moderate DFI population pregnancies from the ICSI pregnancies (P<0.001). Sperm with normal morphology/morphometry may have single and double strand DNA breaks. The SCSA detects both single-strand DNA breaks (moderate DFI and neutral Comet negative) and 100% sperm with single and double strand DNA breaks (neutral Comet positive). Since one- half of the Moderate DFI sperm have DNA double strand breaks identified by the neutral pH Comet assay but with normal morphology/morphometry, the Moderate DFI population is likely to be picked out as frequently for ICSI injection as the main population without SCSA-identifiable DNA damage. The high DFI population is likely detected as abnormal and not used for ICSI. To date, six ICSI studies have shown a relationship between elevated sperm DNA fragmentation and increased miscarriage rates. These Moderate DFI sperm may be responsible for decreased pregnancy and increased spontaneous abortion rates, as well as the possibility for increased birth defects seen at birth or latent genetic defects seen in later life.

Viability and DNA fragmentation in differently sorted boar spermatozoa

Sperm cell defense against DNA damage relies on two factors: the tight packaging of chromatin, based on condensation and substitution of histones with protamines, and the antioxidant agents present in seminal plasma. These defenses are extremely important as mature sperm is unable to repair DNA damage and even if a successful fertilization occurs, embryo undergoes apoptosis at the time of genomic activation. Sex-sorting exposes spermatozoa to stress sources such as high pressure, laser beam and electrical charge. The aim of this work was to determine how sorting procedures affect viability and DNA integrity in boar spermatozoa, by using the newly developed Sperm- Sus-Halomax ®. Four sperm populations were considered: CONTROL (no treatment), REAL (sex-sorted semen), BULK (semen sorted without sex separation) and NO LASER (semen only exposed to the high pressure, but including also cells normally discarded from sex-sorting). A significantly ( P = 0.019) lower viability in NO LASER (64.71%) than in CONTROL (78.6%) and REAL (80.5%) groups was found; this was accompanied by a significantly ( P = 0.001) higher DNA fragmentation index (DFI) in NO LASER group (6.86%) respect to CONTROL (3.30%) and REAL (3.42%) groups. BULK group did not show any difference in viability or DFI as compared to the other groups. In conclusion, we may believe that sex-sorting procedure as a whole does not affect either viability or DFI and that shear mechanical forces are a relevant source of DNA damage for sorted semen.

EVALUATION OF SPERM NUCLEAR MORPHOLOGY IN SPECIMENS WITH ABNORMAL SPERM CHROMATIN STRUCTURAL ASSAYS

Two recent tests have claimed to identify the subfertile male even when other semen parameters were normal: the sperm chromatin structure assay (SCSA) and abnormal sperm nuclear morphology using much higher magnification. The present study attempted to determine if having a high (> 30%) DNA fragmentation index (DFI), thus resulting in an abnormal SCSA test, is associated with a greater likelihood of sperm with abnormal nuclei. Four males with high DFI scores (57.6%, 65.4%, 31.0%, and 35.3%) had their nuclei evaluated by a complex microscope set-up that magnifies the sperm at least 6000x. The corresponding % of normal nuclei was 0%, 20.0%, 23.7% and 40.0%. The mean and median % of normal nuclei was 20.9+/-16.43 and 21.8, respectively. More studies of similarly matched refractory in vitro fertilization cases, where males have normal DFI scores, are needed to determine if having a high DFI index is associated with a lower percentage of normal nuclei.

Clinical aspects of sperm DNA fragmentation detection and male infertility

Over the past 25 years, various methods have been developed to measure sperm DNA strand breaks in situ. Currently, there are four major tests of sperm DNA fragmentation, including the Comet, Tunel, sperm chromatin structure assay (SCSA) and the acridine orange test (AOT). The Comet assay is a light microscope technique where the sperm cells are mixed with melted agarose and then placed on a glass slide. The cells are lysed and then subjected to horizontal electrophoresis. The Tunel assay, another light microscope technique, transfers labeled nucleotide to the 3′OH group of a broken DNA strand with the use of terminal deoxynucleotidyl transferase. The fluorescence intensity of each scored sperm is determined as a “yes” or “no” for sperm on a light microscope slide or by channels of fluorescent intensity in a flow cytometer. The light microscope-based AOT, uses the metachromatic properties of acridine orange to stain sperm cells. The SCSA treats sperm with low pH to denature DNA at the sites of DNA strand breaks, followed by acridine orange (AO) staining of green for native DNA and red for denatured DNA as measured by flow cytometry (FCM) as well as % sperm with high DNA stainability (HDS: immature sperm with intact DNA related to decreased fertilization rates). The SCSA method has defined a 27–30% DNA fragmentation index (DFI) as the point in which a man is placed into a statistical category of taking a longer time to in vivo pregnancy, intra uterine insemination (IUI) and more routine in vitro fertilization (IVF) cycles or no pregnancy. Current data suggest that intracytoplasmic sperm injection (ICSI) may help overcome the diminished pregnancy prognosis with high DFI over the other ART or natural methods.

EFFECT OF AN ABNORMAL SPERM CHROMATIN STRUCTURAL ASSAY (SCSA) ON PREGNANCY OUTCOME FOLLOWING (IVF) WITH ICSI IN PREVIOUS IVF FAILURES

A high DNA fragmentation index (DFI) when performing the sperm chromatin structural (SCSA) assay was claimed to be so specific for male subfertility that even IVF and ICSI did not result in live pregnancies. The present study was designed to corroborate or refute these findings. The SCSA test was performed on the male partner from couples failing to have a successful pregnancy despite at least 2 previous IVF attempts. In contrast to the aforementioned studies, ongoing pregnancies were found despite working with a group of recalcitrant patients. Nevertheless, a high DFI score was associated with a trend for lower ongoing pregnancy rates especially related to a high miscarriage rate. Other more recent studies seem to support our conclusions. A high DFI score should influence a patient to choose IVF as a therapeutic modality sooner, especially with ICSI.

265 SPERM DNA FRAGMENTATION AND PREGNANCY OUTCOME

Sperm DNA integrity is obviously important for normal embryo development and pregnancy outcome. Over the past 25 years, various methods have been developed to measure sperm DNA strand breaks in situ. The Sperm Chromatin Structure Assay (SCSA) treats sperm with low pH to denature DNA at the sites of DNA strand breaks, followed by acridine orange (AO) staining of green for native DNA and red for denatured DNA, as measured by flow cytometry (FCM), as well as % sperm with high DNA stainability (HDS: immature sperm with intact DNA related to decreased fertilization rates). FCM-sorted sperm from each SCSA-defined population (normal, moderate, and high DNA fragmentation and HDS sperm) show that the moderate DNA fragmentation index (DFI) population has the same image analysis characteristics as normal sperm without significant comets. Thus, an ICSI technician is not likely to differentiate between a normal and a moderate DFI sperm. The TUNEL assay uses an enzyme to add a fluorochrome-labeled base to a 3′-OH broken DNA strand. Both light microscopy and flow cytometry are used for measuring the % and extent of DNA fragmentation but cannot measure the level of HDS. For the COMET assay, sperm are suspended in an electrophoretic gel, placed on a glass microscope slide, digested with proteases and RNAse, subjected to an electric field, and then stained with a DNA dye. The % of comet positive sperm is scored, but the extent of fragmentation is difficult to define and the % HDS cannot be determined. Small pieces of fragmented DNA migrate in the gel forming a “comet.” All three methods have been used for both research and clinical diagnosis and as prognosis for livestock (bulls, boars, rams, stallions) and humans. Light microscope techniques suffer from a lack of statistical soundness needed for clinical decisions as well as present a potential bias in selection of sperm for measurements. Due to the thousands of sperm randomly selected for flow cytometry measurements, the data are statistically robust. Data from all three kinds of measurements in over a hundred manuscripts clearly show that sperm DNA fragmentation has a negative impact on embryo growth and pregnancy. Infertile animals may have nearly all of the sperm with fragmented DNA. Fertility ratings in bulls and boars are clearly related to the percent and extent of DNA fragmentation. Threshold levels for fertile/sub fertile/infertile differ for different species. Likewise different methods/laboratories have suggested various threshold levels to characterize a man with a highly fertile to low/very poor potential. The range of sperm with fragmented DNA is from ∼2% to 100%. The SCSA method has defined a 27–30% DFI as the point in which a man is placed into a statistical category of taking a longer time to achieve in vivo pregnancy, more intrauterine insemination and routine IVF cycles, or no pregnancy. Current data suggest that ICSI may help overcome the diminished pregnancy prognosis with high DFI over the other ART or natural methods.

Live births after IVF in men with a DNA fragmentation index of 30% or greater as determined by the sperm chromatin structure assay (SCSA™)

Evaluate the implantation and birth frequency in couples where the male partner had a DFI of ≥ 30 %. Retrospective review of IVF cycles with a DNA fragmentation index (DFI) ≥ 30%. Semen samples provided by men undergoing IVF were subjected to SCSA™ testing within 0–180 days of IVF. Samples were produced by masturbation into specimen cups (Mckesson Medi-pak #37-2469P) and processed within 15–300 minutes. Samples were evaluated for count and motility and frozen neat and stored for one to fourteen days in liquid nitrogen Dewars. Frozen samples were sent to SCSA™ Diagnostics, Inc, Brookings, SD for testing. Results were available within 7 days. Men with a high DFI (≥ 30%) were advised to use Fertility Blend™ supplement for men and couples were counseled to add an additional embryo to the transfer if available. In vitro fertilization (IVF), embryo culture and embryo transfer were performed by processes standard to the industry. Sperm donation was not suggested as an option for IVF treatment. Data presented includes men with DFI’s ≥ 30%, pregnancy and birth data where derived from only the first cycle following SCSA testing. Data is grouped in thirty-day intervals from SCSA testing to egg recovery. Men with high DFI’s were consistently able to produce healthy children following in vitro fertilization regardless of the time of testing to egg retrieval. 1: Clinical pregnancy defined as the presence of a fetal sac at 6 weeks. 2: Sacs electively terminated prior to birth. Added note: three patients with DFI’s ≥50%, within 4 and 59 days of egg retrieval produced 7 healthy children following IVF. The data presented is in contrast to reports that a DFI ≥ 30% strongly predicts a male patient’s inability to produce a sustained pregnancy. Fertility Blend™ supplement for men contains L-Carnitine and other antioxidants that may have led to reduced and improved DFI scores by the time of IVF. Men with an initial DFI ≥ 30% should not move immediately to sperm donation when attempting to father a child by IVF.

Tests d'exploration de la qualité nucléaire du spermatozoïde : relations avec la fertilité et la qualité du conceptus

Several assays are available for testing nuclear quality of spermatozoa, many of them allowing to define a DNA fragmentation index (DFI). Numerous recent studies on this subject agree on several points: negative correlations are observed between DFI and sperm characteristics. Concerning the relationships between DFI and artificial reproductive technologies, there are some disagreements about correlations between DFI and fertilization rates; conversely, in case of high DFI, both blastocyst formation rate and pregnancy rate are significantly reduced. Several authors have defined a threshold value for DFI, corresponding to an absence of pregnancy, or a very low pregnancy rate, for samples above this value. Unfortunately, there are no data available concerning the relationships between sperm DNA quality and abnormalities at birth.

Sperm chromatin structure assay (SCSA®) parameters relationship with fertilization rate, blastocyst development and pregnancy outcomes following in vitro fertilization (IVF) and IVF with intracytoplasmic sperm injection (ICSI)

Objective: To determine the prognostic value of the DNA Fragmentation Index (DFI) and High DNA Stainability (HDS) for fertilization rate, blastocyst development, spontaneous abortions, and chemical and ongoing pregnancies following conventional IVF and IVF with ICSI. Design: Two-hundred-forty-nine couples (female age; 34 ± 4, 25 - 48 yrs) undergoing their first IVF and/or ICSI cycle at a private IVF clinic were included in this combined retrospective review and prospective study. Materials and Methods: Conventional semen parameters were evaluated including semen volume and sperm count, motility and morphology by World Health Organization guidelines (WHO, 1992). An aliquot of the pre-wash semen was frozen for the SCSA, a flow cytometric test that identifies sperm with a high susceptibility to acid-induced nuclear DNA denaturation (DFI) and high DNA stainability (HDS) in situ. Following conventional IVF and ICSI (75% of couples), ICSI only (18%) or conventional IVF only (7%), fertilization was assessed at 16–18 hours post insemination or injection. Blastocysts were graded according to Gardner and Schoolcraft’s system and up to 2 transferred on day 5. Pearson’s Chi-Square Test was used to measure the relationship between high and low DFI (≥30% DFI vs. <30% DFI) and HDS groups (≥15% HDS vs. <15% HDS) and the presence of male factors and IVF/ICSI. Results: IVF and ICSI fertilization rates were not statistically different between the high and low DFI groups. However, a significantly higher percentage of men with ≥15% HDS had low (<25%, p < 0.001) and moderately low (<50%, p < 0.01) IVF fertilization rates. HDS was not related to ICSI fertilization rates. HDS did not significantly impact later embryo development or pregnancy outcomes following IVF or ICSI. Men with ≥30% DFI had a higher risk of having male factors (p < 0.001), poor blastocyst rates (<30%; p <0.003) and no ongoing pregnancy >12 weeks (p < 0.01). WHO thresholds for abnormal volume, count, motility and morphology were not predictive of ongoing pregnancy. Conclusion: The relationship between HDS and poor IVF fertilization rates provides preliminary evidence that ICSI may be indicated in men with ≥15% HDS. Men with high (≥30% DFI) levels of DNA fragmentation were at greater risk for low blastocyst rates and failure to initiate an ongoing pregnancy. The SCSA provides valuable prognostic information to physicians counseling couples prior to IVF and/or ICSI cycles.

Apoptosis of malignant cells in Hodgkin's disease is related to expression of the cdk inhibitor p27KIP1.

Previous results from B-cell chronic lymphocytic leukaemia suggest that expression of p27KIP1 might be important in protection from apoptosis. Given the relevance of apoptosis to the pathogenesis of Hodgkin's disease (HD), it was decided to examine the expression of p27KIP1 in relation to apoptosis in these lesions. Paraffin-wax sections from a total of 65 histologically confirmed HD tumours were used to derive apoptotic index (AI) and DNA fragmentation index (DFI) scores, which were compared with the expression of various cell-cycle-regulating proteins, including p27KIP1 (p27), p21WAF1/CIP1 (p21) and cyclin D1, and with Epstein-Barr virus (EBV) status. The DFI was measured by TdT-mediated dUTP-FITC nick end-labelling (TUNEL), and the AI by conventional morphology. Cells showing the typical morphology of apoptosis, together with those resembling so-called 'mummified' Hodgkin/Reed-Sternberg (HRS) cells, were included in AI measurements. Increasing numbers of p27-positive HRS cells were associated with lower levels of apoptosis in these cells, as indicated by significantly lower AI and DFI scores. There was a trend towards poorer survival in those patients with the highest numbers of p27-positive HRS cells and with lower AI and DFI scores, but these differences were not statistically significant. p21-positive HRS cells were significantly more frequent in those cases with lower AI scores. A similar trend was observed for p21 and DFI, although this relationship was not statistically significant. There was also a trend towards higher levels of cyclin D1 protein in HD cases with high AI and DFI values. A tendency for increasing numbers of p27-positive and p21-positive HRS cells in EBV-positive cases was noted, but this relationship was not statistically significant. EBV status did not correlate with either AI or DFI scores. The results of this study suggest that p27, and possibly also p21, may be involved in protection from apoptosis in HD.