Fresh Semen Samples Research Articles

Endeometriosis: The influence of peritoneal fluid from patients with minimal stage or treated endometriosis on sperm motility parameters using computer-assisted semen analysis

The aim of this study was to determine the influence of peritoneal fluid from patients with minimal stage or treated endometriosis on sperm motility parameters. Peritoneal fluid aspirated at diagnostic laparoscopy for unexplained infertility from women during the luteal phase of the menstrual cycle (days 20-23) was incubated for 5 h with fresh semen samples obtained from men of recently proven fertility. Spermatozoa were prepared by a swim-up technique from unprocessed semen. Using computer-assisted semen analysis (Hamilton-Thorn Research, MA, USA), sperm motility and motion parameters were observed at 0, 120, 180 and 300 min. Compared with spermatozoa incubated in Earle's balanced salt solution/human serum albumin, the percentage motility, percentage progressive motility and progressive velocity of spermatozoa incubated in peritoneal fluid from patients without visible endometriosis were significantly higher (P < 0.05). Maximal effect was observed at 3 h and maintained until 5 h. We conclude that in an in-vitro study, in contrast to peritoneal fluid from patients with minimal stage endometriosis, peritoneal fluid from patients with unexplained infertility and no visible endometriosis can improve sperm motility when compared with culture medium.

Acrosomal status in fresh and frozen/thawed stallion spermatozoa evaluated by scanning electron microscopy.

Semen from stallions with equal fertility at natural services, but yielding semen with either satisfactory or poor fertilizing capability after freezing and thawing, was processed for scanning electron microscopy before and after freezing and thawing. In all fresh semen samples the following three categories of acrosomal defect were noticed: (1) minor fenestrations of the plasma membrane (PM) and outer acrosomal membrane (OAM), (2) complete vesiculation and loss of PM and OAM and (3) lack of a large circular part of PM and OAM. The frequency of these defects ranged from 15% to 27%. All frozen and thawed samples displayed defects of categories 1-3 at similar frequencies as the fresh ones. However, additional defects categorized as: (4) major fenestrations of PM and OAM and (5) complete vesiculation of PM and OAM without loss of the vesicles were noted upon freezing and thawing. The total frequency of defects categorized as 4 and 5 ranged from 8% to 21%, and they seemed to be more frequent in stallions with poor fertility after freezing and thawing although the difference was not significant. Moreover, a particular defect categorized as (6) loosening of the whole acrosomal cap was found exclusively in stallions yielding semen with poor freezability.

First report on purification of the predominant acidic coagulum protein of human semen prior to liquefaction.

The predominant acidic coagulum protein of freshly ejaculated human semen under reducing sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was purified for the first time from washed seminal coagulum keeping urea throughout the entire procedure. Washed seminal coagulum was isolated from fresh semen sample in acetate buffer at pH 4.5 at 0 degrees C, solubilized rapidly in deionized 4 M urea followed by reduction and carboxymethylation of disulfide bonds. By CM-Sephadex and Sephacryl S-300 high-resolution chromatography in presence of urea at room temperature, the predominant acidic coagulum protein was highly purified (99.1%) with a yield of 2.7% of the coagulum protein. The molecular weight of the purified protein in reducing SDS-PAGE was 162 kD.

Isolation of the predominant coagulum protein of human semen before liquefaction

The predominant protein of freshly ejaculated human semen and seminal coagulum in reducing sodium dodecyl sulphate polyacrylamide gel electrophoresis was purified for the first time prior to cleavage of coagulum proteins during liquefaction. The method involves three steps using semen immediately after ejaculation. Washed seminal coagulum was first collected from fresh semen samples in acetate buffer at pH 4.5 at 0 degrees C, solubilized immediately in deionized urea followed by reduction and carboxymethylation of disulphide bonds among the aggregating coagulum proteins. Then using cation exchange (CM-Sephadex) chromatography, eluted with stepwise ionic strength gradient and finally Sephacryl S-300 high resolution chromatography in the presence of urea, the predominant coagulum protein was highly purified (99%) with a yield of 6.3%. The mol. wt of the purified protein was 57,000 Da. At physiological pH, the protein is rich in positive charge.

Effect of storage on sperm membrane integrity and other functional characteristics of canine spermatozoa: In vitro bioassay for canine semen

The effect of storage of canine semen on sperm membrane integrity, as determined by the hypoosmotic swelling test, and on other functional characteristics of the canine spermatozoa was evaluated by established procedures. The results of this study indicated that storage of canine semen at a chilling temperature of 5°C for 24 h did not significantly impair the physical and functional characteristics of the canine spermatozoa. The overall mean percentage of motility, hypo-osmotic swelling response, which assessed sperm membrane integrity, acrosome-reacted spermatozoa, acrosomal defects, and the percentage of live spermatozoa, did not significantly differ between the fresh and chilled semen samples. However, storage altered the rate of motility and acrosome reaction. The percentage of acrosome reaction in the canine capacitating medium peaked earlier in chilled than in fresh semen. It is probable that storing semen at 5°C initiated/triggered the acrosome reaction. This did not amount to impairment of functional properties. Significant correlations were observed between hypo-osmotic swelling vs motility (r=0.98, P<0.002); hypo-osmotic swelling vs acrosome reaction (r=0.83, P<0.08); and acrosome reaction vs motility (R=0.89, P<0.04) in the fresh semen, and between hypo-osmotic swelling vs motility (r=0.87, P<0.05) and hypo-osmotic swelling vs acrosome reaction (r=0.56, P<0.05) in the chilled semen. It was concluded: that 1) storage of canine semen at 5°C for 24 h did not significantly impair the physical and functional integrity of the spermatozoa; 2) the significant association between motility or acrosome reaction vs hypo-osmotic swelling indicates their value in assessing sperm viability; and 3) the hypo-osmotic swelling assay could have predictive value in screening out subfertile males with apparently normal spermiograms.

Comparative auto-controlled study between swim-up and Percoll preparation of fresh semen samples for in-vitro fertilization

This study compared swim-up and Percoll preparation of fresh semen samples for in-vitro fertilization. Sixty trials of in-vitro fertilization (IVF), 38 with normal semen and 22 with abnormal semen, comprising 734 oocytes were included in the study. Each semen sample was prepared by both a swim-up technique and a simplified discontinuous (50%, 70%, 90%) Percoll gradient. The oocytes for each trial were distributed at random between the two sperm preparations and incubated with the same number of motile spermatozoa. Percoll gradient preparation produced a significantly higher final concentration of spermatozoa than swim-up preparation (mean +/- SEM: 6.6 +/- 1.5 x 10(6)/ml versus 1.9 +/- 0.2 x 10(6)/ml; P less than 0.01) but a significantly lower sperm motility (69 +/- 2% versus 94 +/- 1%; P less than 0.001) and a lower number of normal forms (55 +/- 2% versus 64 +/- 2%; P less than 0.01). The ability of the Percoll gradient method to extract motile spermatozoa was higher than that of the swim-up technique (20 +/- 15.6% versus 0.8 +/- 13.6%). Nevertheless, the rates of fertilization (61%), fertilization failure (18%) and polyspermia (9%), embryo quality evaluated by mean embryo scores (3.8 +/- 0.3) and the mean number of spare embryos frozen per trial (1.4 +/- 0.3) were strictly identical in both groups. The 24 pregnancies (including three from frozen--thawed embryos) obtained in these 60 trials (40% per oocyte retrieval) could not be separated according to the sperm preparation method, as embryos from both groups were replaced together.(ABSTRACT TRUNCATED AT 250 WORDS)

Antisperm Antibodies on Epididymal Spermatozoa

Still under debate is the location in the genital tract where antisperm antibodies transuded from serum and locally produced antibodies, respectively, become attached to the surface of the spermatozoa. The mixed antiglobulin reaction (MAR) for immunoglobulin (Ig) G, IgA, and locally produced secretory IgA (sIgA) could be carried out on motile spermatozoa from epididymis from 13 of 34 men undergoing vasovasostomy. Four months and one year after the operation the MAR was repeated on a fresh semen sample from all patients. It was found that transuded as well as locally produced antisperm antibodies were bound to spermatozoa at the epididymal level of the genital tract. The follow-up results on semen were almost identical to the results obtained with epididymal spermatozoa.

Chromosome abnormalities in human spermatozoa after albumin or TEST-Yolk capacitation

Cytogenetic studies were made on 328 spermatozoa from three individuals using either fresh semen samples capacitated in Biggers, Whitten and Whittingham (BWW) medium plus human serum albumin (BWW + HSA) or semen samples preserved at 4 degrees C in TEST-Yolk buffer. A total of 261 sperm karyotypes were obtained in a series of experiments in which half of each sample was capacitated in BWW + HSA and the other half in TEST-Yolk buffer at 4 degrees C for 2 days; 123 and 138 sperm karyotypes were obtained from the two capacitation methods respectively. Neither the frequency of sperm chromosomal abnormalities nor the sex ratio was significantly different after each capacitation methods. In one individual, however, the sex ratio (19X:32Y in the fresh sample and 49X:28Y in the preserved sample) did show a significant difference. In three experiments with semen samples from a single individual capacitated at 4 degrees C for 1, 2 or 3 days in TEST-Yolk buffer we obtained 33, 30 and 34 sperm karyotypes respectively. No significant differences in the sex ratio was exhibited between these experiments; the number of chromosome anomalies was too low to allow statistical analysis. Our results suggest that TEST-Yolk capacitation for 1, 2 or 3 days does not induce significant variations in the frequency and type of chromosomal abnormalities in human spermatozoa.

Prediction of human sperm penetrating ability using computerized motion parameters

The sperm penetration assay (SPA) is used to assess male fertilizing potential but it is tedious and costly. Computer analysis could replace the need for the SPA in some cases, if computerized sperm motility parameters are highly predictive of SPA performance. The objective of this study was to determine whether computerized motility parameters from fresh semen samples could be used to predict SPA performance. Computer automated semen analysis (CASA; CellSoft, Cryo Resources) was used to quantitate sperm concentration (CONC), percent motility (MOT), curvilinear velocity (VEL), linearity of swimming trajectory (LIN), mean amplitude of lateral head displacement (ALH), and beat/cross frequency (B/CF). The SPA was performed using either Biggers, Whitten, and Whittingham's medium (BWW) or TEST-yolk buffer (TYB). Patients were divided into three groups depending on SPA performance: group 1, BWW-treated, 0% versus greater than 0% penetration; group 2, TYB-treated, 0% versus greater than 0% penetration; and group 3, TYB-treated, less than 20% versus less than or equal to 20% penetration. SPA performance was highly correlated with CONC, MOT, VEL, and B/CF. CONC, MOT, VEL, and B/CF were significantly higher for patients who penetrated in the SPA than for those who failed to penetrate. Discriminant function analysis (DFA) successfully classified 76% of all patients treated with TYB (group 2) who penetrated and 86% of nonpenetrators based on their computerized motility parameters. For group 2 DFA predicted that 93 men would penetrate in the SPA with TYB. Of these, 90 (97%) successfully penetrated at least one egg.(ABSTRACT TRUNCATED AT 250 WORDS)

Acrosome reaction of stallion spermatozoa evaluated with monoclonal antibody and zona‐free hamster eggs

The acrosome of the stallion spermatozoon was visualized by indirect immunofluorescence with monoclonal antibody (18.6) which recognized an integral acrosomal membrane component. Localization was confirmed by electron microscopy using peroxidase labelled antibody. In fresh semen samples (n = 19), 73.9 +/- 9.1% of the spermatozoa from five fertile stallions displayed a uniform bright fluorescence over their acrosome region. In two semen samples from an infertile stallion only 28% and 35% of spermatozoa showed the same pattern of fluorescence. Spermatozoa from fertile stallions incubated for up to 12 hours in TALP medium maintained motility and exhibited a significant progressive loss of acrosomes as detected by immunofluorescence. Alternatively, a similar loss of acrosomes could be induced with calcium ionophore A23187 over a 90 minute incubation. Ultrastructural observations and incubation with zona-free hamster eggs indicated that only with ionophore treatment was immunofluorescent acrosome loss correlated with a physiological acrosome reaction, while prolonged sperm incubation led to degenerative membrane changes. It was concluded that, if carefully validated, immunofluorescent localization of the acrosome of stallion sperm with monoclonal antibody could be used to monitor the acrosome reaction. Furthermore, definitive acrosome visualization would be valuable in assessing semen quality.

The cryopreservation of donor semen by a simplified method: use in an IVF and GIFT programme

The cryopreservation of semen used in assisted reproduction procedures was carried out exclusively by a simplified method in which a mixture of semen and cryoprotectant was contained in 1-ml tuberculin syringes and plunged directly into liquid nitrogen. Donor semen samples halved and frozen in syringes and in straws in a controlled-rate freezer showed no significant difference in post-thaw motility (P = 0.217) or survival (P = 0.217) after 30 min. However, after 180 min the survival rate showed a significant reduction in syringes (P = 0.045). A significant difference (P less than 0.00008) in the rate of fertilization of oocytes was seen in IVF cycles using frozen-thawed donor sperm (58/142, 42%) when compared to fresh sperm from husbands (2315/3926, 59%). A significant reduction (P less than 0.00005) in fertilization rate was also observed in the case of supernumerary oocytes in GIFT cycles with the cryopreserved donor sperm (29/132, 22%) compared to the husbands' sperm (239/514, 46%). However, the pregnancy rate following IVF and embryo replacement was the same after fertilization with fresh sperm (75/351, 21%) as opposed to frozen sperm (3/14, 21%). Furthermore, a higher pregnancy rate was observed in GIFT with frozen donor sperm (9/19, 47%) than with fresh sperm from husbands (28/103, 27%), though this was not statistically significant (P = 0.079). These results show this simplified methods of semen cryopreservation to be effective when used in an IVF and GIFT programme, giving pregnancy rates comparable to fresh normospermic semen samples. The method is simple, quick and inexpensive.

Investigations on the Control of Bacteria in Boar Semen

Artificial insemination (AI) of frozen boar spermatozoa results in lowered fertility and a reduction in litter size. Therefore, liquid boar semen are usually used. But 23 days are limit for keeping non-frogen semen without decreasing its fertillizing capacity. The purpose of my investigations were: (1) to determine the number and types of bacteria in boar semen, (2) to investigate the effects of bacteria on the survival of boar spermatozoa, (3) to evaluate the effects of bacteria-contaminated boar semen on the reproductive performance, (4) to find effective antibiotics on bacterial growth during storage, (5) to remove waste products in stored boar semen and prolong the storage period boar semen at 15°C, for improving the conception rate and increasing the litter size. These investigations were carried out from September 1978 to March 1988 at the Shizuoka Prefectural Swine Experimental Station (present name; Swine and Poultry Experimental Station). Total of 124 boars and 99 sows were used.(1) Bacterial counts per ml of fresh boar semen ranged from 5, 500 to 48, 000, averaging 27, 000; collected by the usual hand massage method where only evacuating the urine which had accumulated in the diverticulum prepuce. However, if sterile physiological saline was poured on the penis and collector's hand before semen collection, bacterial counts per ml of fresh boar semen ranged from 0 to 5, 100, averaging 611.(2) in order to reduce the bacterial counts contaminating boar semen, the percoll density gradient centrifugation technic was applied to fresh semen of which bacterial counts were low, (104/ml), no bacteria could be detected in the semen after centrifugation.(3) Thirteen bacterial genera were identified from 46 fresh semen samples, but anaerobic-bacteria were not isolated. Following species were found: Pseudomonas sp. were most frequent at 80.4%, Micrococcus sp., 63.0%, Staphylococcus sp., 56.5%, Klebsiella sp., 52.2%, E. coli., 41.3%, Citrobacter sp., 30.4%. Proteus sp., 21.7%, Actinomyces sp., 15.2%, Serratia sp., 8.7%, Enterobacter sp., 6.5%, Bacillus sp., 6.5%, Streptococcus sp., 4.3%.(4) The survival of spermatozoa was remarkably affected by the presence of 5 species of enteric bacteria including E. coli, and fertility was lost in 12 days when pH decreased to 5.25.7. But, it was affected to a lesser extent by the presence of Pseudomonas sp. Almost no effect on the survival of spermatozoa with 4 species of bacteria, Alcaligenes sp., Actinomyces sp., Streptococcus sp., and Staphylococcus sp. even when the number of bacteria increased to 10101012/ml, and the pH averaged at 6.36.5.(5) Three types of bacteria (E. coli, Staphlococcus sp. and Pseudomonas sp.) in boar semen were inseminated into the uterus with fresh semen to observe their effect on the endometrium and conception of the inseminated sows at estrus and luteal phase. No bacteria were detected from the uteri of all sows if inseminated at estrus. The endometrial morphology of these sows appeared to be normal. The conception rate, number of embryos or fetuses and the litter size at parturition were within normal ranges. On the other hand, a large number of bacteria were isolated from the uteri if inseminated during the luteal phase. Their uteri became edematous or pyometra. However the inflammatory changes disappeared at the subsequent estrus.

Development of a buffer system for dialysis of bovine spermatozoa before freezing. I. Effect of zwitterion buffers

The effect of N-TRIS(hydroxymethyl)methyl-2-aminoetane sulfonic acid (TES); N,N BIS (2 hydroxvethyl)-2 aminoethane sulfonic acid (BES), N-2(hydroxyethyl)piperazine-N-2-ethane sulfonic acid (HEPES), morpholinopropane sulfonic acid (MOPS), and piperazine-N-N-BIS(2-ethane sulfonic acid (PIPES) solutions on dialyzed semen was studied. Each was titrated to pH 7.0 with TRIS-(hydroxymethyl)-amino methane (TRIS) solution and the osmotic pressure was adjusted to between 320 to 325 mOsm/kg. The new solutions were identified as TEST, BEST, HEPEST, MOPST and PIPEST, respectively. The solutions were used 1) alone, 2) in a composite with equal parts (V/V) of each solution and 3) in a 1:1 (V/V) combination with isosmotic trisodium citrate solution. Later, TRIS and sodium hydroxide (NaOH) were compared as titration bases for piperazine-N-N-BIS (2-ethane sulfonic acid) (PIPES) and N-Tris(hydroxymethyl)methyl-2-aminoethane sulfonic acid (TES). Ejaculates were diluted 1:10 (V/V) in extenders containing buffer, 20% egg yolk and 5% glycerol (V/V). The samples were dialyzed (1:50) during cooling for a period of 2 h. Each sample was dialyzed against the same buffer system containing 5% glycerol without egg yolk and later it was frozen in pellets. The treatments were evaluated by observation of sperm motility in fresh and thawed semen samples. The latter were also analyzed by electronic count of cells that passed through the Sephadex column. Sperm survival was higher in PIPEST (PIPES titrated with TRIS) or the composite buffer, and the inclusion of 50% sodium citrate (Na citrate) improved significantly (P<0.05) sperm motility in fresh and frozen-thawed semen samples. There was no difference (P>0.05) between the titration bases. In the second experiment, sperm survival was superior in extenders containing PIPEST (P<0.05) than in those containing TEST independently of the inclusion of Na citrate.

Prognostic value of in vitro sperm penetration into hormonally standardized human cervical mucus

To analyze the prognostic value of the sperm cervical mucus penetration test (SCMPT), fresh semen samples of 99 male patients under infertility investigation were exposed to capillary tubes filled with freshly obtained cervical mucus (CM) of the patients' wives (WCM), fertile donors (DCM), and bovine CM (BCM). The quality of the human CM was standardized by oral administration of estrogens. The overall pregnancy rate after 6 months was 17.2% (17/99), and was significantly different in couples with poor and good SCMPT with WCM (1/44, 2.3% versus 16/55, 29%; P less than 0.001) in a prospective study. Human CM was superior to BCM as a penetration medium in providing more information about sperm function. The results suggest that in vitro sperm penetration testing with hormonally standardized CM of female partners adds an important dimension to sperm analysis with regard to fertility prognosis.

Frequency of semen collection in boars and quality of ejaculates as evaluated by the osmotic resistance of acrosomal membranes

Semen was collected from five adult boars at 2-day intervals subsequent to a longer testing period with regular twice-weekly semen collections. After ten collections at 2-day intervals, the volumes of the ejaculates were reduced on average from 120 ml (ejaculates 1 and 2) to 86 ml (ejaculates 9 and 10), the sperm numbers from 35×10 9 to 16×10 9, motility from 78% to 63% and spermatozoa with normal acrosomal ridge (NAR) from 83% to 70%. The differences are statistically significant ( P<0.01). The differences in preserved semen samples from ejaculates 9 and 10, compared to samples from ejaculates 1 and 2 of this experiment, were even larger, with decreases in motility from 45% to 12% (frozen/thawed semen) and to 26% (72 h stored semen) and in NAR from 54% to 22% (frozen/thawed semen) and to 27% (stored semen). Such semen samples must be rejected and cannot be used for insemination. All five boars reacted similarly. Incubation of fresh samples from the beginning and from the end of the experiment in media with extreme osmolalities resulted in a dramatic decrease of % NAR in the latter. This demonstrates a reduced osmotic resistance in their acrosomal membranes, probably as a consequence of a shortened maturation of the spermatozoa in the epididymis due to frequent ejaculations. The determination of the osmotic resistance value (ORT) in fresh semen samples permits a quantitative and continuous control of semen quality and of overuse of boars by frequent semen collecting systems.

Polymorphism of EsD by isoelectric focusing: phenotyping in human tissues, dental pulps, hair roots, and semen.

The polymorphism of EsD was investigated in tissues of various human organs, dental pulps, hair roots, and seminal stains by isoelectric focusing. The method yielded an excellent resolution of the isoenzyme components. The time limits of determination were: in organ tissues 3 weeks, in dental pulps 1 week, and in hair roots several days. The 7-1 type was less stable than the common types. Phenotyping was possible from fresh semen samples, but was unsuccessful from dried seminal stains after storage. The results show that the EsD typing by isoelectric focusing is of practical use for medicolegal individualization of organs, teeth, and hairs.

Effect of caffeine on human sperm penetration into zona-free hamster ova.

The effect of caffeine on spermatozoal ability to penetrate zona-free hamster ova was examined on fresh and frozen-thawed semen samples. The mean motility of 10 fresh semen samples incubated with caffeine significantly increased from 29% to 35%. Sperm penetration into zona-free hamster ova did not differ between the control group and the specimens to which caffeine was added. The same effect of caffeine on sperm motility and hamster ova penetration was noted in the frozen-thawed sperm samples. Motility was enhanced by 21%, but hamster ova penetration did not significantly change. The increase in sperm motility caused by caffeine does not change the fertilizing ability of fresh and frozen-thawed human sperm.

Local anaesthetic effect of antiarrhythmic drugs and human sperm immobilization: mechanism and application of the interrelationship.

Sperm immobilizing activity of three class I antiarrhythmic drugs, quinidine, procainamide, mexiletine, one adrenoceptor blocking drug, labetalol and one calcium channel blocking drug, diltiazem was measured with a trans-membrane migration method. All these drugs immobilized human sperm. Local anaesthetic effect is the underlying mechanism for these drugs to inhibit sperm motility. Sperm immobilization could be a screening test for the local anaesthetic effect of antiarrhythmic drugs.

Relationship of fine structure of sperm head to fertility of frozen human semen

Damage to the plasma membrane and acrosome of human spermatozoa was quantitated following dilution, freezing, and thawing. Semen was obtained from 23 donors of known fertility attending an artificial insemination program. Dilution of semen with the cryoprotective medium containing glycerol significantly altered all the fine-structural and semen parameters studied. Membranes of the sperm head were severely altered after freezing and thawing. The percentage of spermatozoa with intact plasma membrane, with intact acrosomes, or with intact or swollen acrosomes was significantly reduced in frozen/thawed semen, compared with fresh semen samples. The percentage of spermatozoa with an intact plasma membrane and acrosome was significantly and positively correlated with the fertility of frozen donor's semen used for insemination (r = 0.6, P less than 0.001). The deleterious effects observed in the sperm head membranes following dilution and freezing and thawing may explain why generally the fertility of frozen/thawed semen is lower than that of fresh semen and may also explain individual variation in fertility of donors after insemination of frozen semen.

Rapid Analysis of Normal and Abnormal Cell Types in Human Semen and Testis Biopsies by Flow Cytometry

A simple, rapid procedure is described that quantitates RNA content and DNA content/chromatin condensation for each of many possible cell types and differentiation levels of the cells present in human semen. A fresh semen sample (1-6 hr postemission) or frozen sample (allowing samples to be accumulated and sent to a laboratory) is treated with a detergent solution, stained with acridine orange (AO), and measured by flow cytometry (FCM); approximately 10 minutes are required to measure 5,000 cells per sample and analyze the data with computer assistance. The following can be learned from a single measurement: a) the percentage of each cell type in semen including, i) mature sperm, ii) immature sperm precursor cells, representing all stages of development from spermatogonia to mature sperm, iii) somatic cells, e.g., leukocytes; b) normality/abnormality of sperm nuclear chromatin condensation. These measurements can be correlated with cell types in testis biopsies identified by two-parameter FCM measurements (RNA versus DNA) using AO as the fluorescent probe.