Spurious monosomy 7 in leukemia due to centromeric heteromorphism.

We report heteromorphism of the centrometric region of human chromosome 7, which was observed in the cytogenetic assessment of a complete remission of a pre-B acute lymphoblastic leukemia in the bone marrow cells of a 25-year-old woman. Classical cytogenetic study was performed, as well as metaphase and interphase fluorescence in situ hybridization (FISH) carried out with an alphoid DNA probe specific for the chromosome 7 centromere for detection of leukemic clones with monosomy 7 found at the initial diagnosis. We show an important centromeric heteromorphism of this chromosome detected by FISH and clearly visible on all metaphases and nuclei analyzed. This heteromorphism is observable as a fluorescent signal five- or sixfold larger than that on the homologue. To our knowledge, this heteromorphism of chromosome 7 has not been reported in the literature. However, with the use of FISH analysis, it could be easily mistaken for a mosaicism of monosomy 7, which can be misleading in the interpretation of the results.

The reactivation of demembranated human spermatozoa lacking outer dynein arms is independent of pH.

The effect of pH, Mg-ATP, and free calcium on activity of the inner dynein arm was investigated using demembranated human spermatozoa lacking the outer dynein arms (LODA). The results were compared with those obtained for demembranated-reactivated normal spermatozoa to evaluate the functional properties of the inner and outer dynein arms in axonemal motility. The reactivation of Triton X-100-demembranated LODA spermatozoa was analysed at various pHs and concentrations of Mg-ATP and calcium using video recordings. The percentage of reactivated LODA spermatozoa as a function of Mg-ATP concentration was not dependent on pH, whereas reactivation of normal human spermatozoa is pH dependent. This suggests that there may be a pH-dependent regulatory mechanism associated with the outer dynein arms. A delay in the principal bend propagation of normal and LODA reactivated cells was found at pH 7.1. This disappeared at pH 7.8 in normal but not in LODA populations. This suggests a role for outer dynein arms in the initiation of the propagation of flagellar bends at alkaline pH. The level of LODA and normal sperm reactivation both depended on the calcium concentration in the medium. At lower free calcium concentrations, the reactivation level and beat frequency of reactivated cells were higher. Our results suggest a functional difference between outer and inner dynein arms of human spermatozoa based on a differential pH sensitivity. Moreover, calcium seems to exert its regulatory action elsewhere than on the outer dynein arms.

Chromosomal aberrations in hepatocellular carcinomas: relationship with pathological features.

Fluorescence in situ hybridization performed on tissue sections can reveal chromosomal abnormalities related to histopathological features. This technique was performed on serial frozen sections from seven normal livers and 29 hepatocellular carcinomas (HCCs) using pericentromeric repeat-specific probes for chromosomes 1, 4, 6, 7, 8, 16, and 17. For each HCC and each probe, the percentage of cells showing one, two, or more than two signals was counted and compared with the distribution in the normal liver. According to these results, HCCs were categorized as monosomic, disomic, or polysomic (more than two signals) for the chromosome tested. These data were compared with the main histopathological characteristics of HCC. Chromosome gains were very common, preferentially affecting chromosome 1 (23 of 27 cases, 85%), chromosome 16 (16 of 27 cases, 59%), chromosome 7 (16 of 29 cases, 55%), chromosome 6 (15 of 29 cases, 52%) and chromosome 8 (14 of 29 cases, 48%). Monosomy was seen more rarely, affecting preferentially chromosome 16 (19%), chromosome 17 (14%), and chromosome 4 (10%). A significant correlation was observed between aneusomy of chromosome 4 and tumor size (P < .05) or the presence of vascular embolism (P < .05). In conclusion, chromosomal gains are frequent genetic events in human HCC. A significant association between a gain in chromosome 4 and large tumor size or vascular embolism suggests that this genetic abnormality is a late event in liver carcinogenesis.

[Physiology of human gamete interaction]. / Physiologie de l'interaction gamétique dans l'espèce humaine.

Subzonal insemination was the first micromanipulation technique used in the treatment of male factor sterility. Analysis of the sperm pathology prior to patient inclusion in the program allowed us to study gamete interaction in the human and the early embryogenesis. A series of 523 cycles and 3,027 micro-injected oocytes is reported.

Influence of sperm movement parameters on human sperm-oolemma fusion.

Flagellar dyskinesia is characterized by abnormal sperm movement parameters and a negative sperm mucus penetration test. It is associated with structural pathologies of the axonemal complex (lack of outer dynein arms), of the periaxonemal complex (sliding spermatozoa and periaxonemal dyskinesia), or of both structures (short flagella). Even during in vitro fertilization, dyskinesia prevents the spermatozoon from getting through the egg vestment. However, in some cases, fertilization has been achieved using subzonal insemination. Flagellar dyskinesia is therefore an interesting model for investigating the role of sperm movement in the fusion process between the spermatozoon and the oolemma. Thirty-one patients requiring assisted fertilization were included in the study. Fifteen had spermatozoa in which the flagellum lacked outer dynein arms, 11 had anomalies of the periaxonemal complex (five with sliding spermatozoa and six with periaxonemal dyskinesia) and five had spermatozoa with short flagella. Seven men who produced spermatozoa with normal movement were selected as controls. Movement was evaluated using a computer-assisted analyser, and penetration was assessed using zona-free hamster eggs. At 37 degrees C, in semen, the dyskinetic spermatozoa had reduced straight line and curvilinear velocity and lateral head displacement compared with controls (P < 0.01). In the Percoll-selected sperm suspension, the only difference was that spermatozoa with periaxonemal anomalies maintained a narrow lateral head displacement compared with the controls (P < 0.001). After 3 h of incubation at 37 degrees C, the lateral head displacement of dyskinetic spermatozoa had not changed, while that of the controls showed a significant increase (4.5 to 5.6 microns; P < 0.05). The results from the sperm penetration assay for the spermatozoa lacking outer dynein arms were lower than those of the controls (47% versus 77%; P < 0.05) and the results for sliding spermatozoa and spermatozoa with periaxonemal dyskinesia were even lower (25% and 34%, respectively; P < 0.01). The fertilization rates after subzonal insemination were 46.5% for spermatozoa lacking outer dynein arms, 36.1% for spermatozoa with short flagella, 24.8% for sliding spermatozoa and 17.3% for spermatozoa with periaxonemal dyskinesia. There was a significant correlation between the curvilinear velocity of the Percoll-selected sperm suspensions and their fertilization rates after subzonal insemination (r = 0.5; P < 0.05) and their sperm penetration assays (r = 0.7; P < 0.001). The data provide evidence that sperm velocity is correlated with the ability to fuse with the oolemma.

Pregnancy after subzonal insemination with spermatozoa lacking outer dynein arms.

The absence of outer dynein arms in the sperm flagellum induces an abnormal movement pattern associated with male infertility. These spermatozoa can decondense in zona-free hamster oocytes but result in a very low fertilization rate in in vitro fertilization. We hypothesized that subzonal insemination could help achieve fertilization and pregnancy. A randomized prospective trial (five couples, five cycles) comparing subzonal insemination (n = 31 oocytes) and routine IVF (n = 23 oocytes) was carried out. Oocytes were microinjected with 8.5 +/- 3.6 spermatozoa. In a second series (nine cycles), all the oocytes were microinjected with 10.5 +/- 4.3 spermatozoa. In the randomized series, the fertilization rate was 16.1% without polyploidy, whereas no fertilization was obtained after control IVF insemination. In the second series involving nine couples, six of whom were included in the first series, the fertilization rate increased to 57.8% with a 27.8% polyspermic rate. Eighty-eight per cent of the zygotes cleaved normally (29 out of 33). A total of 11 embryo transfers resulted in three pregnancies, one of which terminated one month later, a second being ongoing and the third delivering a healthy girl. A 21.4% pregnancy rate per cycle, with a 37.5% pregnancy rate per couple, justifies the use of subzonal insemination to treat this particular flagellar dyskinesia.

[A new entity of sperm pathology: peri-axonemal flagellar dyskinesia]. / Une nouvelle entité pathologique du spermatozoïde: la dyskinésie flagellaire péri-axonémale.

The study of 17 infertile men has led to define a new entity of sperm pathology as part of the more general field of flagellar dyskinesias. Sperm parameters of the studied patients and a control series have been first estimated by routine analysis (concentration, motility, morphology). To precise their characteristics, kinetic and ultrastructural investigations, as the zona-free hamster oocyte penetration test, have been performed. Sperm parameters of the studied cases, as revealed by routine analysis, were close to the control group. However, a major kinetic anomaly was found which was characterized by an important decrease of the amplitude of lateral head displacement (1.6 microns vs 5.3 microns, p < 0.001), although the progressive velocity was only slightly impaired (20.3 microns vs 24.9 microns, p < 0.05). Electron microscopy revealed anomalies limited to the peri-axonemal structures such as the outer dense fibers and the fibrous sheath. Rates of sperm-oocyte attachment were normal but rates of oocyte penetration were low (27.7% of decondensed sperm heads vs 85.6%, p < 0.001). Attempts to assisted fertilization with the studied patients (51 cycles of insemination, 8 cycles of in vitro fertilization) were unsuccessful. All these data suggest that the infertility can be attributed to the movement disturbances which should impair sperm propulsion throughout the cervical mucus and the zona pellucida.

Effect of sperm-associated antibodies on the dynamics of sperm movement and on the acrosome reaction of human spermatozoa.

Anti-sperm antibodies were eluted from the sperm cell fraction of autoimmune human ejaculates and transferred onto normal motile spermatozoa. The movement and the acrosomal status of these antibody-coated spermatozoa were evaluated after incubation in a capacitating medium. The amplitude of lateral head displacement (ALH) and the straight line velocity (VSL) were analyzed using an HTM automated motility analyser. Acrosomal loss was monitored by an FITC-conjugated lectin binding technique. During the 6-h incubation in BWW-BSA medium, antibody-free and antibody-coated spermatozoa exhibited significant changes of ALH and VSL distribution that evolved differently in the two populations. The dynamics of sperm movement in control spermatozoa were apparently modified by the presence of antibodies on the sperm membrane. The low percentage of spontaneous acrosomal loss obtained in control populations, even after 20 h of incubation, was not modified by the presence of antibodies on spermatozoa. However, the same antibodies decreased the acrosomal loss induced by a calcium ionophore after 3 h of incubation in capacitating conditions. These results suggest that sperm capacitation and acrosome reaction, considered as essential for successful fertilization, can be altered by antisperm antibodies present on human ejaculated spermatozoa.

Effects of pH on the reactivation of human spermatozoa demembranated with Triton X-100.

The aim of this work was to study the role of different parameters involved in the motility of human spermatozoa. Human spermatozoa were totally demembranated with 0.05% Triton X-100, and the demembranation was checked using electron microscopy. We have shown that, with a concentration of ATP-Mg lower than 2 mM, a pH effect was observed with a dose-dependent motility reactivation at pH 7.1, with 14% +/- 2.0% motile cells at 1 mM ATP-Mg and a straight line velocity (VSL) of 12.0 +/- 1.4 microns/sec. However, at pH 7.8, more than 65% of the spermatozoa were reactivated with as low as 0.02 mM ATP-Mg and 77.8% +/- 2.5% of them were motile at 1 mM ATP-Mg and had a VSL of 23.4 +/- 3.9 microns/sec. The depletion of free calcium by the addition of 0.5 mM EGTA in the reactivation medium (RM) improved the percentage of motile cells and the VSL most markedly at low ATP-Mg and low pH. If no MgSO4 was added in RM, cells were not motile at pH 7.8, but 30-40% reactivated at pH 7.1. If 5 mM Ca2+ was added to the RM, up to 88% of the cells became reactivated at both pHs, but the beat frequencies were very low, suggesting different mechanisms of reactivation when Mg2+ or when Ca2+ is present in the RM.

In-vitro effects of anti-sperm antibodies on human sperm movement.

The present study was designed to investigate whether autoantibodies to external domains of the sperm plasma membrane affect the movement of normal motile spermatozoa. Eight sera and 20 seminal plasma samples containing high levels of anti-sperm antibodies as well as antibodies eluted from the sperm fraction of 19 autoimmune ejaculates were incubated with donor's motile spermatozoa, obtained by swim-up migration in Tyrode's solution. Sperm movement was analysed using 1 s exposure microphotography when greater than 70% of the spermatozoa were coated with antibodies (after 30-90 min of incubation). At least 50 tracks of progressively motile spermatozoa were analysed in order to obtain the mean values of the amplitude of lateral head displacement (ALH) and the velocity of progression (VSL). Serum antibodies and sperm eluted antibodies had quite consistent but opposite effects on sperm movement; serum antibodies increased ALH and decreased VSL whereas eluted antibodies decreased ALH and increased VSL. Seminal antibodies did not affect these two parameters significantly. Furthermore, seminal antibodies and sperm eluted antibodies obtained from the same ejaculates had distinct effects on ALH and/or VSL. This diversity was apparently not linked to antibody isotype or localization on the sperm membrane; it might be due to differences in the composition of the extracellular media. These results suggest a dynamic effect of anti-sperm antibodies on sperm movement, a possibility that merits further investigation.

Comparative kinetics of short and long sperm in sperm dimorphic Drosophila species.

All species of the Drosophila obscura group exhibit within-ejaculate sperm length dimorphism. The present work is a contribution to the understanding of sperm competition through a comparative study of sperm kinetic parameters in four of these species. Videomicrographic observations at 200 frames per second of sperm from males and females, out of the storage organ, prior or after storage were made. Drosophila sperm display both major and minor waves. The former is analysed by measuring coiling diameter (micron) and the latter by recording both beat frequency (s-1) and wave propagation velocity (micron.s-1). Results show that the 'behaviour' of short and long spermatozoa noticeably differ: short sperm kinetics remains unaltered after storage while both major and minor waves of long spermatozoa are markedly modified. Thus, evidence is provided here of a sort of "differential activation" which is assumed to result in different survival abilities of short and long sperm within the storage organ of females.

Decrease of internal free calcium and human sperm movement.

In order to elucidate the effects of calcium on the movement of human spermatozoa, studies were conducted using motile cells selected by swim-up migration at 37 degrees C in 5% CO2 in air in a synthetic BWW medium containing 1.7 x 10(-3) M CaCl2 or BWW without added calcium (BWW-Ca). Preliminary experiments have confirmed that the addition of EGTA (5 x 10(-3); 10(-2) M) to BWW medium decreased the intracellular calcium concentration ((Ca++)i) of spermatozoa, as measured in cells loaded with a fluorescent Ca++ indicator, Quin-2. Concomitant measurements of (Ca++)i and sperm movement (analysed by videomicrography at 200 f/s at room temperature) were carried out on Quin-2 loaded cells incubated in BWW-Ca medium plus EGTA (10(-5) M; 10(-4) M; 10(-3) M). Under these conditions a decrease in (Ca++)i was observed and associated with a decrease in mean amplitude of lateral head displacement (ALH). Analysis using an automatic analyser (Hamilton Thorn at 37 degrees C) confirmed these results: the percentage of spermatozoa swimming with ALH greater than or equal to 6 microns is decreased when the external free calcium in BWW-Ca is decreased by the addition of 10(-5) M, 10(-4) M, or 10(-3) M EGTA. Flagellar analysis of the sperm population characterized by ALH greater than or equal to 6 microns showed a large proximal curvature of the tail associated with a low propagation wave velocity and a low beat frequency as compared to the spermatozoa with ALH less than 6 microns with similar progressive velocities. These characteristics result in a high flagellar beat efficiency (in terms of head displacement per beat). The disappearance of this pattern of movement when intracellular calcium is lowered indicates that calcium plays a complex role in the relationship between curvature and wave propagation. The ability of spermatozoa to modulate their movement in response to an alteration in the intracellular calcium level confirms the role of calcium in controlling flagellar movement in intact cells.

Motion of individual human spermatozoa, both normal and lacking the outer dynein arms, during a continuous temperature rise.

The effect of increasing temperature from 22-25 degrees C to 37 degrees C on various motion characteristics of individual normal human spermatozoa and spermatozoa lacking the outer dynein arms (LODA) was studied by using a new automatic microscopic tracking method. It was found that: 1) The curvilinear velocity (Vc, measured between 1-3 sec) of both normal and LODA spermatozoa, fluctuated more or less intensely between spermatozoa; this fluctuation was not thermodependent. 2) The average Vc in the two groups of spermatozoa increased with the rise in temperature at a similar rate (1 micron/sec/degrees C), but LODA spermatozoa had an initial Vc lower than that of normal spermatozoa (12.5 +/- 5.3 microns/sec and 34.2 +/- 8.2 microns/sec, respectively). 3) The profile of the Vc increase associated with the temperature rise was different for the two groups of spermatozoa: for LODA spermatozoa it was linear between 25-37 degrees C, whereas for normal spermatozoa a plateau was reached at about 31 degrees C. 4) Various patterns of trajectory were found for both normal and LODA spermatozoa; these patterns were unrelated to temperature. However, LODA spermatozoa had more linear trajectories than normal spermatozoa. 5) Plots derived from reaction rate theory showed that the activation enthalpy, delta H was a function of the increase of Vc for both normal and LODA spermatozoa, but that delta H was higher for LODA spermatozoa.

[The role of Chlamydia trachomatis in producing abnormal movements by spermatozoa]. / Le rôle de Chlamydia trachomatis dans la survenue de mouvements anormaux des spermatozoïdes.

The authors analysed 100 sperm samples from asymptomatic patients and compared them with a control series of sperm donors who had been considered fertile by CECOS of Marseille or from men whose partners had conceived. They found Chlamydia trachomatis in 29% of the cases studied (P less than 0.0001). They found an abnormal movement of the sperm which they called the "Jerk Phenomenon" and which was found in 76% of cases (P less than 0.0001), where the culture for Chlamydia trachomatis was positive. This correlation does not change in association with other germs. Micro-cinematographic studies confirm the asthenospermic character of this movement. Since the sperm movements become normal after treatment with tetracycline one must consider that Chlamydia trachomatis can be an etiological agent for infertility in men.

Male factors and the likelihood of pregnancy in infertile couples. I. Study of sperm characteristics.

A prospective study of 394 infertile men was conducted over 3 years following a primary semen analysis. The cumulative pregnancy rate was 43 and 64% after 1 and 3 years, respectively. The pregnancy rate was significantly higher in the secondary infertile group. The study of various sperm factors and the occurrence of pregnancy showed that they were not of equal significance in predicting male fertility potential. The percentage of pregnancies decreased significantly only when the sperm concentration was less than 5 x 10(6)/ml. The pregnancy rate increased significantly with the percentage of motile sperm. The percentage of sperm with normal morphology was also found to be significantly higher when a pregnancy occurred than when the couple remained infertile (43.6% vs 37.7%). In a detailed morphological analysis of the sperm, six abnormalities (microcephaly, double head, amorphous head, cytoplasmic droplet, bent tail and coiled tail) were found to be significantly more frequent when a pregnancy did not occur. The most predictive value was given by the Multiple Anomalies Index (MAI), which is the mean number of abnormalities observed per abnormal sperm. The pregnancy rate was significantly lower after both 1 and 3 years when the MAI was greater than 1.6. Multivariate analysis showed that the best prognostic indicator of fertility was given by the percentage of motile sperm and the MAI, particularly in patients with primary infertility.

Male factors and the likelihood of pregnancy in infertile couples. II. Study of clinical characteristics--practical consequences.

A prospective study of 394 infertile men was conducted over 3 years following a primary semen analysis. Simple comparisons between the groups of men whose partners conceived and those that did not, showed that the mean duration of infertility in the primary infertile group was significantly shorter when a pregnancy was observed, while age differed significantly between the two populations in the secondary infertile group. Multivariate analyses, taking these variables and sperm characteristics into account, showed that prognostic variables for the occurrence of pregnancy were the duration of infertility, sperm motility and the multiple anomalies index in the primary infertile group, and age of the male partner and percentage of normal sperm in the secondary infertile group. Tables for estimating the probability of observing a pregnancy as a function of these criteria are presented. In this study no positive influence of treatment on the occurrence of a pregnancy was found during the 3 years following the first semen analysis.

[In vivo and in vitro fertilizing ability of immature human epididymal spermatozoa]. / Fécondance in vivo et in vitro de spermatozoïdes épididymaires humains immatures.

In cases of congenital absence of vas deferens (9 patients) or after failure of previous epididymovasostomy (2 patients), in vitro fertilization (IVF) was attempted with spermatozoa surgically obtained at the epididymal caput level. These sperm populations showed little progressive motility (5.9 +/- 6.5%) and an marked necrozoospermia (19.3 +/- 17.4%). Stimulation by caffeine (4.5 mM) alone or associated with heterologue normal seminal fluid resulted in most of the cases in an initiation of motility with an improvement of the progressive velocity. In 9 IVF attempts, 31 mature oocytes were inseminated with 5.10(3) to 1.5.10(6) motile spermatozoa. The dynamic characteristics in 3 inseminated sperm populations were Vp (24.2 +/- 8.3 microns/s), Ah (8.6 +/- 2.0 microns) at room temperature. Sperm binding to zona pellucida was decreased (0 to about 20 spermatozoa per oocyte) and there was no fertilization. In the same period, 21 attempts of intra uterine insemination and 14 attempts of intracervical inseminations were made in 5 couples who remained infertile after patent high epididymovasostomy (4) or vasovasostomy (1) and having immature spermatozoa stimulated as previously described. Antisperm antibodies were detected on the ejaculated spermatozoa in four men. No pregnancy was obtained with these immature stimulated spermatozoa. The fertility of the female partners was confirmed in 3 women after insemination with donor sperm.

Sperm analysis.

Human semen analysis is the first biological step of male fertility assessment. Its performance requires the use of standardized and objective procedures. The results of the tests may allow to conclude to a man's sterility when they reveal e.g. an azoospermia or an alteration of sperm morphology or motility incompatible with fertilization. More often however, it is difficult to precisely determine the relationships between the observed sperm anomalies and infertility. In a follow-up study of 520 infertile men over 3 years, the pregnancy rate was found to be significantly lower only in the cases where sperm concentration values were less than 5.10(6)/ml and unchanged for concentrations above this value. No threshold values were found for sperm motility and morphology, although the chances of conception were closely related to these two parameters for sperm concentration values above 5.10(6)/ml. Various tests have been described to evaluate the human sperm ability to migrate through the female genital tract and fertilize the oocyte. These tests and the human in vitro fertilization assay allow a better understanding of the structural and dynamical parameters mostly involved in sperm function.