[Spermatozoon mitochondrial DNA]. / ADN mitochondrial du spermatozoïde.

Mitochondria play a primary role in cellular energetic metabolism, homeostasis and death. In spermatozoa, in particular, mitochondria produce the ATP necessary for motility. Mitochondrial functions depend, at least partially, on mitochondrial DNA (mtDNA). The mitochondrial genome, the transmission of which is exclusively maternal contributes to cytoplasmic heredity. Qualitative and quantitative mtDNA abnormalities have been associated with male infertility. This review focuses on the role of mtDNA in human fertility.

Low oocyte mitochondrial DNA content in ovarian insufficiency.

BACKGROUND: Mitochondrial biogenesis and bioenergetics play an important role in oocyte maturation and embryo development. We have investigated the relationship between defective mitochondrial biogenesis and the lack of oocyte maturity observed during IVF procedures with patients suffering from ovarian dystrophy and ovarian insufficiency. METHODS: We used real-time quantitative PCR to quantify mitochondrial DNA (mtDNA) in 116 oocytes obtained from 47 women undergoing the ICSI procedure. We compared the mtDNA content of oocytes from women with a normal ovarian profile with that of oocytes from women with ovarian dystrophy and ovarian insufficiency. RESULTS: We found an average of 256,000 +/- 213,000 mitochondrial genomes per cell. The mean mtDNA copy number was not significantly different in ovarian dystrophy compared with controls, but it was significantly lower in oocytes from women with ovarian insufficiency (100,000 +/- 99,000, P < 0.0001). CONCLUSIONS: Our results suggest that low mtDNA content is associated with the impaired oocyte quality observed in ovarian insufficiency.

Increased sperm mitochondrial DNA content in male infertility.

BACKGROUND: There is increasing evidence that mitochondrial DNA (mtDNA) anomalies in sperm may lead to infertility. Point mutations, deletions and the presence of a specific mtDNA haplogroup have been associated with poor sperm quality, but little attention has been paid to the role of mtDNA content. METHODS: Using density gradient separation and swim-up methods, we selected motile sperm from 32 normal and 35 abnormal sperm samples. The mtDNA/beta-globin gene ratio was determined by real-time quantitative PCR. RESULTS: The average mtDNA/beta-globin ratio of sperm collected from 100% density layers was 1.4 for normal sperm, 6.1 for sperm samples presenting at least one abnormal criterion [among the three criteria established by World Health Organization (1999), i.e. sperm count, motility and morphology], and 9.1 for sperm samples presenting two or more of these abnormal criteria. These differences are very highly significant (P < 0.0001). The mtDNA numbers were also much greater in sperm collected from the 40% density gradient layers (mean: 17.1, P < 0.001), known to contain the most abnormal sperm of the sperm samples, than in those collected from the 100% layers known to contain sperm with the best fertilizing ability. CONCLUSION: Our results showed significant mtDNA amplification in sperm collected from abnormal sperm samples.

[Genetic male infertility and medically assisted reproduction]. / Infertilité masculine d'origine génétique et assistance médicale à la procréation.

About half the cases of infertility have their origin in the male partner. Infertility due to males has several possible aetiologies. In about 30% of cases, genetic disorders are suspected of being the main cause. They could interfere with the development of the male gonads, the urogenital tract or the hypothalamo-hypophyseal axes. Such disorders could also stop germ cell generation and maturation or lead to the production of non-functional spermatozoa. Genetic disorders of chromosomal origin could give rise to abnormal karyotypes or germinal mosic figure. They could involve gene abnormalities affecting numerous genes localized on several chromosomes, in particular the Y chromosome. The physiopathologic identification of male infertility is interesting because of the risk of the genetic factors involved being transmitted to the offspring. The subject is of importance, specially because of the increasing use of intracytoplasmic sperm injections. Couples should therefore be precisely counselled to enable them to make a well-informed choice among various solutions, e.g. ART, with or without sperm donation, or adoption.

Mitochondrial DNA content affects the fertilizability of human oocytes.

Mitochondrial DNA content varies considerably in oocytes, even when collected from the same patient. In the present study, real-time quantitative polymerase chain reaction analysis of 113 unfertilized oocytes obtained from 43 patients revealed an average of 193,000 (range: 20,000 to 598,000) mitochondrial genomes per cell. We compared several groups of oocytes to investigate the relationship between mitochondrial DNA content and fertilizability. The average mitochondrial DNA copy number was significantly lower in cohorts suffering from fertilization failure compared to cohorts with a normal rate of fertilization. In addition, the mitochondrial copy number of oocytes from patients with fertilization failure due to unknown causes was significantly lower than that of oocytes from patients in which IVF failure was due mainly to a severe sperm defect. The lower mtDNA copy number could be due to defective cytoplasmic maturation of oocytes. We conclude that low mitochondrial DNA content, due to inadequate mitochondrial biogenesis or cytoplasmic maturation, may adversely affect oocyte fertilizability.

Cytomorphometric analysis of vaginal cells during normal cycle, under oral contraceptive therapy or in-vitro fertilization stimulation protocol.

A cytomorphometric analysis of superficial vaginal cells in women in three groups of different types of hormonal concentration was made. There were 15 women in each group. Group I was studied during a natural cycle, group II under oral contraceptive therapy and group III during an in-vitro fertilization (IVF) stimulation protocol. Morphometric parameters were measured on an image analyser. The area, perimeter and several form factors were measured separately for nuclei and cytoplasm. The nucleus:cytoplasmic ratio was also determined. The cytoplasmic area was significantly reduced in group II and was associated with a statistically significant reduction of the nuclear area. The nucleus:cytoplasmic ratio appeared significantly increased in group II and reduced in group III. Low oestradiol impregnation obtained with an oral minidosed contraceptive interfered with vaginal cell maturation. High oestradiol concentrations obtained during IVF protocols induced marked nuclear pycnosis but did not induce supra-physiological cell enlargement. Maximal cell size is genetically regulated according to Driesch's law of volume invariance and hormonal over-stimulation has no effect on cell size. The nucleus:cytoplasmic ratio appears to be a powerful parameter reflecting the opposite effects of hormones on cell compartments.

Long PCR analysis of human gamete mtDNA suggests defective mitochondrial maintenance in spermatozoa and supports the bottleneck theory for oocytes.

The long PCR and the Southern blot techniques were used to study mitochondrial DNA (mtDNA) in 94 sperm samples, and in 35 oocytes collected from 12 women. The sperm samples were classified in two sets: 37 samples from normal subjects, and 57 samples from patients with oligoasthenospermia. In both sets, most of the spermatozoan mitochondria had multiple mtDNA deletions. The rate of mtDNA mutation, which appears unexpectedly high, considering the short life span of the spermatozoa, may be due to impaired maintenance during differentiation. In contrast, despite the long life span of oocytes and the extended meiotic period, oocyte mitochondria showed few mtDNA rearrangements. However, mitochondria in oocytes from a given donor revealed considerable mutational heterogeneity. This supports the bottleneck theory of rapid segregation of mtDNA genotypes during early oogenesis. The long PCR technique, which allows analysis of the entire mitochondrial genome, provides new information on mtDNA instability in human gametes. Our findings suggest that mtDNA maintenance differs in the two types of gametes.

Oligoasthenospermia associated with multiple mitochondrial DNA rearrangements.

A patient who wished to be treated for infertility by intracytoplasmic sperm injection (ICSI) was referred to our group for assessment. Upon clinical examination, a ptosis (partial closure of the eyelid) was noted, and histology revealed ragged red fibres in the skeletal muscle. Southern blot analysis of spermatozoa and skeletal muscle revealed the presence of multiple mitochondrial DNA deletions. This kind of rearrangement may be of nuclear origin since three nuclear loci have been ascribed to multiple mitochondrial DNA deletions in humans. Since mitochondrial DNA is maternally transmitted, the use of ICSI was feasible. However, an alteration of nuclear gene product affecting the integrity of mitochondrial DNA, and thus sperm mobility, might be transmitted to the offspring with the risk of developing a mitochondrial DNA disease.

Male infertility associated with multiple mitochondrial DNA rearrangements.

Male sterility results from a number of characterized exogenous or genetic dysfunctions preventing normal differentiation into mobile spermatozoa. This may now be overcome by intra cytoplasmic sperm injection (ICSI). This practice does not require mobile, or even mature spermatozoa for in vitro fecondation. However, a functional respiratory chain, partly encoded by the mitochondrial DNA (mtDNA), is required for the mobility of the spermatozoa. We report the case of an infertile patient who wished to procreate. ICSI was proposed but he displayed multiple mtDNA deletions of possible nuclear origin in the spermatozoa and in the deltoid muscle. Even though mtDNA is maternally inherited, the possibility of a nuclear-driven mutation affecting the integrity of the mtDNA should be taken into account when ICSI is to be performed. Together with recent genetic in vitro manipulations in mammals, our data point to the importance of studying the mtDNA structure in human spermatozoa, and the potential risks of these non-natural practices for procreation.

Inhibin assays of ovarian cyst liquid obtained by needle aspiration may allow differential diagnosis between functional and organic cysts.

Radioimmunoassays of estradiol, CA125 and inhibin were carried out on ovarian cyst fluid samples. The samples were taken from ten women with functional cysts and 15 women with organic cysts. Statistical analysis shows that estradiol and inhibin assays allows satisfactory differential diagnosis between functional and organic cysts. However, the inhibin assay provides more precise results and appears to be a more reliable marker than estradiol. The differential diagnosis between the two types of cyst would indicate the use of coelioscopic surgery especially in the case of organic cysts, avoiding unnecessary risks for patients with functional cysts.

[Prognostic evaluation of small cell lung carcinoma by flow cytometry. A retrospective study from bronchial brushing samples]. / Evaluation pronostique du carcinome bronchique à petites cellules par cytométrie en flux. Etude rétrospective à partir de prélèvements par brossage bronchique.

The authors determined the desoxyribonucleic acid (DNA) content of 45 small-cell lung cancers, by flow-cytometry (FC), from bronchial brushings to asses the prognostic meaning of the DNA index, the percentage of cells in DNA synthesis phase (% S) and the ploidy. Moreover, the value of this tumorous cells brushing was assessed for Flow-cytometric analysis. Bronchial brushing offers some advantages opposite to biopsies, but the low quantity of collected cells reduces the number of tumors analysable by FC. The output is low (39%) for determination of DNA index, and moreover low (22%) for determination of % S. In regard to the flow-cytometry analysis, the DNA content does not show any significant difference of survival where as the % S is related to survival, but it seems to be paradoxical in this study. Indeed, patients with small-cell lung cancer associated to high cells' percentage in DNA synthesis phase have the longest survival.

[IVF in sterility related to ovarian dystrophy]. / La FIV dans les stérilités liées à la dystrophie ovarienne.

Sterility related to ovarian dystrophy is currently treated by means of standard inductors of ovulation such as Clomid and HMG. In case of failure, patients are recommended in vitro fertilization. In our study of 30 cases of ovarian dystrophy, 9 pregnancies occurred after in vitro fertilization. Slow injections of purified FSH appear to produce similar results but the method is still under evaluation. Our results suggest that after say, two years of failure with classical methods in patients with a long history of sterility, in vitro fertilization may be expected to give good results.

Flow cytometric DNA analysis in the diagnosis of lung tumors. A comparison with conventional methods.

The efficiency of flow cytometric (FCM) DNA analysis in the diagnosis of lung carcinoma was compared with that of conventional cytologic techniques on bronchial brushing and fine needle aspiration samples from 461 patients. The main advantage of FCM was the rapid delivery of results. Unfortunately, this was offset by a poor sensitivity in the detection of bronchial tumors. Nevertheless, DNA analysis may still prove useful in determining the prognosis and in evaluating the effects of chemotherapy on known tumor stem lines.

Angiotensin-I-converting enzyme in germinomas.

Angiotensin-I-converting enzyme (ACE) was detected in 18 germinomas, both of testicular or extratesticular localization, and studied by immunohistochemical methods using specific polyclonal antibodies and by enzyme activity measurements. ACE was also detected in normal human germ cells. On the other hand, it was not present in other types of testicular tumors. Biochemical studies and immunohistochemical findings suggest that at least part of the enzyme is membrane bound. Plasma ACE levels appeared to be normal, indicating that measurement of plasma ACE levels in germinomas would be of little help for the diagnosis and follow-up of patients. However, the apparent specificity of ACE detection in germinomas among germ cell tumors might help in histologic diagnosis, especially for tumors of extragonadal localization.

Isolation of osteoclasts from Pagetic bone tissue: morphometry and cytochemistry on isolated cells.

Giant osteoclasts and other cells were isolated from Pagetic bone tissue using 0.5 mM ethylene diamine tetraacetic acid on bone samples from 8 patients with Paget's disease. The cell suspension contained osteoclasts and osteoblasts as well as some mononuclear cells such as monocytes. The number of nuclei in isolated osteoclasts (33.85 +/- 20.92 nuclei/osteoclast) correlates fairly well (p less than 0.02) with the number of nuclei counted on histologic sections (15.88 +/- 11.80 nuclei/osteoclast) for samples from each patient. Enzyme histochemistry demonstrated acid phosphatase activity in isolated osteoclasts and in mononucleated cells, such as monocytes. Alkaline phosphatase was detected only in osteoblasts while succinate dehydrogenase was observed in osteoclasts, osteoblasts and monocytes. Esterases, such as nonspecific aliesterase and specific naphthol AS-D acetate esterase, were identified in osteoclasts and in macrophages. Inhibition of specific naphthol AS-D acetate esterase in osteoclasts by addition of sodium fluoride suggests that the enzyme could be of monocytic origin.

Somatostatin and adrenocorticotrophic hormone like immunoreactivity in small cell carcinoma of the lung.

The immunocytological detection of adrenocorticotrophic hormone (ACTH) and somatotropin release inhibitor factor (SRIF) like immunoreactivity was carried out on tumour cells from bronchial brush smears in 39 cases of lung tumours. Results obtained were compared with the cytological and histological diagnosis and confirmed the high incidence of ACTH synthesis by malignant bronchial carcinoma cells: the same phenomenon also seems to occur for somatostatin. The concomitant detection of ACTH and SRIF like immunoreactivity seems to be highly suggestive of small cell carcinoma and indicates that the immunocytological detection of hormones carried out at the same time as cytological examination can improve the accuracy of the diagnosis.

Flow DNA analysis in human lung cancer: potentiality and limitations due to sampling methods.

Flow DNA analysis was performed on samples from 71 surgically removed lung cancers and from 145 patients undergoing bronchoscopy. Abnormal DNA stem lines, characterized by their DNA index (DI), were frequently observed in operated lung carcinomas (87%). Two or three abnormal DNA stem lines were discovered simultaneously in 10% of the samples. The mean DI of all abnormal tumor stem lines was lowest for rare tumor cell types and highest for adenocarcinomas. Intermediate mean DI values were found for epidermoid and small cell carcinomas, which were among the most proliferative tumors. The high rate of false negative results suggests poor diagnostic reliability of flow DNA analysis on bronchoscopic samples. However, the method appears to provide a promising objective tool capable of evaluating tumor behavior and prognosis.

[Evaluation of the use of bronchial brushings after cytocentrifugation in the diagnosis of bronchial tumors (475 fibroscopies)]. / Bilan de l'utilisation des brossages bronchiques après cytocentrifugation dans le diagnostic des tumeurs bronchiques (475 fibroscopies).

Since 1971, cytodiagnosis of lung tumors by association of the bronchial brushing and cytocentrifugation techniques has been carried out routinely at the University Hospital of Angers. Our study covers 475 cytodiagnoses of bronchial brushings. The results obtained enable an evaluation of the diagnostic efficiency of bronchial brushing in the detection of lung tumors. The technique is convenient for routine screening and in several cases contributes directly to diagnosis.

Transposed ciliary microtubules in Kartagener's syndrome. A case report with electron microscopy of bronchial and nasal brushings.

Report is made of the case of a 44-year-old white woman with Kartagener's syndrome marked by respiratory disorders and repeated serous otitis since infancy. The technique of cell sampling through bronchial and nasal brushings facilitated observation of ciliary structures in electron microscopy. The results revealed a specific anomaly in the organization of the ciliary microtubules. The doublet transposition observed may be associated with ciliary dyskinesia.