[Role of Doppler velocimetry of uterine arteries and ambulatory blood pressure monitoring in detecting pregnancies at risk for preeclampsia]. / Ruolo della Doppler flussimetria delle arterie uterine e del monitoraggio in continuo della pressione arteriosa nell'individuazione di soggetti a rischio per lo sviluppo di preeclampsia.

AIM: The aim of this study is to evaluate the performance of ambulatory blood pressure monitoring (ABPM) in association with Doppler velocimetry of uterine arteries in the detection of preeclampsia (PE). METHODS: In a prospective study, 22 patients at risk for the development of PE have been en-rolled: 17 were nulliparous women with increased resistances in uterine arteries at 24 weeks and 5 had an obstetric history of PE. All patients were tested with ABPM and uterine arteries Doppler velocimetry. A resistance index (RI) higher than 0.62 for the Doppler velocimetry and a diastolic midline estimating statistic of rhythm (MESOR) higher than 68 for the ABPM were considered abnormal. Patients were followed longitudinally until delivery. The occurrence of PE and low birth weight were considered as gestational outcomes endpoints. RESULTS: Overall, the mean gestational age at delivery was 36.3+/-3.4 weeks (range 29.4-41). Six out of 22 (27.2%) patients developed PE; 4 out of 22 (18%) newborns were small for gestational age (SGA) <5 degrees percentile. In the prediction of PE, ABPM in association with Doppler velocimetry showed a positive predictive value (PPV) of 42% and a negative predictive value (NPV) of 90%, for the development of low birth weight the PPV was 33% and the NPV 100%. CONCLUSION: This study shows that ABPM in association with Doppler velocimetry evaluation is a useful test to detect patients at high risk for the development of PE.

Clinical review of 55 cases of malignant ovarian germ cell tumors.

PURPOSE OF INVESTIGATION: A retrospective analysis of 55 cases of malignant germ cell tumors in a 20-year period was done to evaluate the impact of conservative surgery and adjuvant treatment on survival and fertility. METHODS: Fifty-five cases of malignant ovarian germ cell tumors (MOGCTs) were studied. Mean age was 22 years. Dysgerminoma was the most common histotype (45%). RESULTS: Thirty-nine patients (71%) presented with FIGO surgical Stage I disease. Fertility-sparing surgery was performed in 39 (71%) women. Postoperative systemic chemotherapy was administered to 40 women (73%), 27 (68%) had received conservative treatment. One woman developed renal failure after the first cycle of chemotherapy and died a few days thereafter and there was one case of bleomycin-induced death due to pulmonary fibrosis. There were eight (14.5%) clinical recurrences. Overall survival rate for relapsing women was 75% (6/8). The recurrence rate for women treated conservatively was 15%, and it was 13% for those treated radically. With a median follow-up of 129 months the overall survival rate for the entire study-population was 90.9%. Eleven pregnancies occurred in 36 women treated with fertility-sparing surgery who were of child-bearing age. CONCLUSION: The management of MOGCTs with fertility-sparing surgery is a safe, practicable treatment option. The majority of these patients can retain normal ovarian function and reproductive potential after chemotherapy treatment.

Width of the fetal lateral ventricular atrium between 10 and 12 mm: a simple variation of the norm?

OBJECTIVES: The prognosis of borderline forms of anomalies that can be detected by ultrasound is one of the most challenging issues in prenatal diagnosis. The aim of this study was to determine the prognosis for fetuses presenting with isolated mild ventriculomegaly (MVM). METHODS: Fetuses in which the width of the lateral ventricular atria was 10-12 mm and which had no other detectable chromosomal or morphological anomalies were followed by monthly ultrasound examinations until delivery. For the cases identified up to December 1997, postnatal information was gathered retrospectively through interviews. Children born from January 1998 onwards were included in a protocol involving planned neuropsychiatric visits at 12 and 18 months of age in which the Griffith scale was used to assess neurodevelopment. RESULTS: Between September 1992 and January 2001, 60 fetuses with isolated MVM were identified. Ventricular dilatation diminished in 18 cases (and became normal in nine of these) and stabilized in 42 cases. Information was obtained on 38 children born up to December 1997 and their neurodevelopment was found to be completely normal. The 22 children born from January 1998 onwards showed normal development at 12 and 18 months of age. CONCLUSIONS: When MVM is observed on prenatal ultrasound examination it can be very difficult to offer parents appropriate counseling. It is important to exclude aneuploidy or morphological abnormalities but even then there will be anxieties about long-term neurological outcome. Our data, which show normal neurodevelopment between 18 months and 10 years after birth in cases of MVM (10-12 mm), should provide a basis for reassuring counseling.

[Monitoring of pregnancy complicated by maternal-fetal isoimmunization. A comparison between two clinical protocols]. / Monitoraggio della gravidanza complicata da isoimmunizzazione materno-fetale. Due protocolli clinici a confronto.

AIM: Invasive techniques such as amniocentesis and cordocentesis are used for the diagnosis and treatment of fetus at risk for anemia due to maternal red-cell alloimmunization. The purpose of this study was to determine the value of non invasive measurements of the peak velocity middle cerebral artery in the fetus (PVMCA) for the diagnosis of fetal anemia. METHODS: From 1996 to September 2002, we studied 23 pregnancies with anti D title >1:32. In the 1(st) group of 11 women (from 1996 to 1999) fetal anemia was detected by invasive techniques (amniocentesis and cordocentesis). In the 2(nd) group of 12 women (from 1999 to 2002) fetal anemia was suspected on the basis of PVMCA. When PVMCA was significantly increased, cordocentesis was performed in order to rule out fetal anemia and to provide in utero transfusions. RESULTS: In the 1(st) period we performed 23 invasive techniques (7 amniocentesis and 16 cordocentesis) in 11 women, but we identified fetal anemia only in 4 cases. In the 2(nd) period we performed only 2 cordocentesis in women in which PVMCA was increased; the blood sampling confirmed fetal anemia in both cases. CONCLUSION: PVMCA and fetal hematocrit are highly significantly correlated: high values of PVMCA are associated with fetal anemia. Doppler velocity of PVMCA is related to fetal anemia with positive predictive value 100% and negative predictive value 100%. The middle cerebral artery blood velocity is a non invasive technique for detecting anemia in pregnancies complicated by alloimmunization.

Effect of betamethasone on computerized cardiotocographic parameters in preterm growth-restricted fetuses with and without cerebral vasodilation.

OBJECTIVE: To verify the effects of maternal corticosteroid administration on fetal behavior and heart rate variation using computerized cardiotocography (cCTG) in a selected group of growth retarded fetuses. STUDY DESIGN: Fifty singleton pregnancies complicated by fetal growth restriction were enrolled in the study before 34 weeks of gestation. All of them received two intramuscular injections of 12 mg of betamethasone 24 h apart. Fetal heart rate was recorded by cCTG before the first injection, and every 24 h for the 3 days following the end of the treatment. After Doppler evaluation of cerebral circulation, fetuses were divided into a group with and a group without signs of cerebral vasodilation. Basal heart rate, short- and long-term variation, percentage of time spent in high variability, fetal movements and percentage of small accelerations were evaluated. RESULTS: Basal fetal heart rate did not show significant changes. Short-term variation and percentage of time spent in high variability significantly decreased in fetuses with but not in fetuses without vasodilation. Long-term variation and fetal movements significantly decreased in both groups. CONCLUSIONS: Maternal administration of betamethasone in growth-retarded fetuses with cerebral vasodilation is associated with significant but transitory modifications of fetal heart rate variation.

The use of two density gradient centrifugation techniques and the swim-up method to separate spermatozoa with chromatin and nuclear DNA anomalies.

Human semen is heterogeneous in quality, not only between males but also within a single ejaculate. Differences in quality are evident, both when examining the classical parameters of sperm number, motility and morphology and in the integrity of the sperm nucleus. The aim of this study was to determine the efficiency of the PureSperm((R)), Percoll((R)) and swim-up preparation techniques to eliminate spermatozoa with nuclear anomalies. Semen samples were collected, washed and one part of the semen spread on a slide, the remainder was prepared using the swim-up, PureSperm((R)) or Percoll((R)) techniques. Spermatozoa from different fractions were fixed on slides and assessed. Sperm samples (n) from different men were stained using the chromomycin A(3) (CMA(3)) fluorochrome, which indirectly demonstrates a decreased presence of protamine (n = 31 for swim-up; n = 45 for PureSperm((R)); n = 39 for Percoll((R))). Spermatozoa prepared using PureSperm((R)) (n = 35) and Percoll((R)) (n = 37) were also examined for the presence of endogenous DNA nicks. Good quality spermatozoa should not possess DNA nicks and not stain (i.e. fluoresce) with CMA(3). When prepared using the swim-up technique the spermatozoa recovered showed no significant improvement with the CMA(3) staining. When spermatozoa were prepared using the PureSperm((R)) and Percoll((R)) techniques, a significant (P < 0.001) decrease in both CMA(3) positivity and DNA strand breakage was observed. These results indicate that both the PureSperm((R)) and Percoll((R)) techniques can enrich the sperm population by separating out those with nicked DNA and with poorly condensed chromatin.

Cisplatin-epirubicin-paclitaxel weekly administration in advanced breast cancer: a phase I study of the Southern Italy Cooperative Oncology Group.

PURPOSE: Both cisplatin and epirubicin have been shown to enhance the antitumor activity of paclitaxel in vitro. Weekly administration could result in a substantial improvement in the therapeutic index of cisplatin and paclitaxel. This study was aimed at determining the MTDs of epirubicin and paclitaxel given weekly with a fixed dose of cisplatin. PATIENTS AND METHODS: Sixty-three breast cancer patients with advanced disease (24 locally advanced and 39 metastatic), who had not received prior chemotherapy (except adjuvant), received weekly cisplatin (CDDP) doses of 30 mg/m2 together with escalating doses of paclitaxel (PTX) and epirubicin (EPI) for a minimum of six cycles. The dose escalation was stopped if DLT occurred during the first six treatment cycles in > 33% of patients of a given cohort. RESULTS: Nine different dose levels were tested, for a total of 506 weekly cycles delivered. G-CSF support on days 3-5 of each week was also given in the last four cohorts (24 patients). An overall 11 patients showed DLT in the first six cycles. EPI and PTX doses up to 40 and 85 mg/m2/week, respectively, were safely delivered without G-CSF support. However, the actually delivered mean dose intensity was only 64% in this cohort. Therefore, the dose escalation continued with the addition of filgrastim from day 3 to day 5 each week. Doses of EPI and PTX up to 50 and 120 mg/m2/week were administered without observing DLT in the first six cycles in more than one third of the patients enrolled. No toxic deaths were observed. Only two patients had to be hospitalized because of sepsis. Grade 3-4 neutropenia, thrombocytopenia, and anemia occurred in 25, 9, and 16 patients, respectively. Alopecia was almost universal. Other nonhematologic toxicities were generally mild, being of grade 3-4 in only eight patients (fatigue and loss of appetite in two cases, diarrhoea in four cases, peripheral neuropathy and mucositis in one case). Fifteen complete and 37 partial responses have been registered for an 82% (95% CI = 71-91) overall clinical response rate (ORR). Eight complete and 14 partial responses occurred in the 24 patients with locally advanced disease, for a 92% (95% CI = 73-99) ORR, as compared to seven complete and 23 partial responses in the 39 women with metastatic disease, 77% (95% CI = 61-89). A clear dose-response relationship was not observed, since an overall response rate of at least 70% was achieved at all dose levels. However, the ORR increased to 92% in the last four cohorts which included patients who received higher doses of EPI and PTX with G-CSF support. All of the 24 patients with locally advanced disease underwent modified radical mastectomy with axillary dissection. Three of them showed no invasive cancer on pathologic examination, and in another five patients a tumor smaller than 1 cm was found in the surgical specimen of the breast. At a nine-month median follow-up (range 2-14), 11 patients have progressed and three have died. Twenty-three out of 24 patients who underwent surgery are still free from progression. The one-year projected progression-free survival is 77% for the whole population. CONCLUSIONS: The CDDP/EPI/PTX weekly administration is a well tolerated and very effective approach in advanced breast cancer patients. Full doses of all the three drugs can be delivered even in absence of G-CSF support. A very impressive increment of the dose-intensity can be obtained, however, by adding filgrastim. A phase II study is under way to better define the therapeutic efficacy of this regimen in patients with advanced breast cancer.

Successful application of indirect in-situ polymerase chain reaction to tissues fixed in Bouin's solution.

AIMS: To evaluate the value of polymerase chain reaction-in situ hybridization (PCR-ISH) for the detection of human papillomaviruses (HPV) in paraffin sections of cervical biopsies fixed either in 10% formalin or in Bouin's solution. METHODS AND RESULTS: We analysed 40 biopsies from Italian women infected with the human immunodeficiency virus type 1 (HIV 1). In-situ hybridization techniques were performed with commercial biotinylated probes. The PCR-ISH was carried out by the 'hot start modification'. Cervical intraepithelial neoplasia (CIN) was found in 23 of 40 patients (57. 5%); eight cases showed condylomatous features. Human papillomavirus was detected in 42.5% by ISH and in 65% by PCR-ISH. Sixty-nine per cent of positive biopsies contained HPV 16, 18, 31 and 33. HPV 6 and 11 were found only in condylomata acuminata samples. CONCLUSIONS: The results point to a high incidence of HPV infection as well as of CIN in HIV-positive patients. Human papillomavirus type 16 appears to be most frequently associated with CIN. Polymerase chain reaction-ISH is more sensitive than ISH in the detection and typing of HPV DNA both in clinical and in 'latent' infections. The two techniques yielded the same results with either formalin- or Bouin's-fixed material.

The role of rDNA genes in X chromosome association in the aphid Acyrthosiphon pisum.

Silver staining of mitotic metaphases of the aphid A. pisum reveals the presence of argentophilic bridges connecting the two X chromosomes. The presence of nucleolar material connecting sex chromosomes seems to be quite a common phenomenon in organisms belonging to very different phyla, and suggests a role of nucleolar proteins in chromosome association and disjunction. In somatic cells of A. pisum, bridges connecting X chromosomes are detectable not only after silver staining but also after CMA3 staining. This finding suggests that GC rich DNA is involved in this type of association. Molecular analysis of rDNA intergenic spacers shows several 247 bp repeats containing short sequences having a high level of homology with the chi sequence of Escherichia coli and with the consensus core region of human hypervariable minisatellites. Moreover, each 247 bp repeat presents a perfect copy of a promoter sequence for polymerase I. These aphid repeats show structural homologies with a 240 bp repeat, which is considered to be responsible for sex chromosome pairing in Drosophila, not only in view of their common presence within rDNA spacers but also for their length and structure. The presence of chi sequences in the IGS of A. pisum, by promoting unequal crossing-over between rDNA genes, could thus give rise to the nucleolar organizing region (NOR) heteromorphism described in different aphid species. Although X pairing at NORs is fundamental in aphid male determination, the presence of heteromorphism of rDNA genes does not inhibit male determination in the A. pisum clone utilized for our experiments.

The use of carbon-dioxide laser surgery in the treatment of intraepithelial neoplasia of the uterine cervix.

From October 1989 to June 1997, 1841 patients (pts) suffering from different diseases of the lower genital tract have been treated with CO2 laser surgery in our Institution: among them, 782 were affected by cervical intraepithelial neoplasia (CIN). All pts underwent CO2 laser procedure for CIN after adequate colposcopic evaluation of the entire lower genital tract, colposcopic guided biopsy of the lesion, adequate pre-surgical work-up for possible infectious and coagulation associated disease. In 736 (94.1%) pts, the procedure was performed on an ambulatory basis while 46 pts (5.9%) were admitted for 1 or 2 days. A CO2 laser Sharplan 55 associated to a Zeiss operative colposcope was employed. The preoperative diagnosis of the 782 pts treated for CIN was 297 CIN1, 255 CIN2, 171 CIN3 and 59 CIS. Mean age was 33.6 years without statistical difference among the grade of disease: 605 pts underwent laser vaporization according to specific selection criteria. The depth of cervical destruction was less than 6 mm in 26 cases, between 6 and 10 mm in 549, between 11 and 15 mm in 157, more than 15 mm in 50 pts. 742 procedures were performed under local anesthesia and pain was absent in 667 pts. (89.9%), mild in 51 (6.8%), moderate in 19 (2.5%) and severe in 5 (0.7%). Intraoperative bleeding was severe in 30 pts. (3.8%), moderate in 77 (9.8%), mild in 204 (26.1%) and absent in 471 (60.2%). The conization procedure was shown to have a higher risk of bleeding but no direct relation was observed with the depth of cervical destruction. Late complications were scarce: 1.3% of late hemorrhages, 1.4% of stenosis of cervical external orifice and cervical endometriosis in 0.3%. In 76 pts (42%) of the 177 conizations the final pathology report was in accordance with the previous biopsy, in 56 (30.9%) a lower grade of CIN was observed, in 53 (29.3%) a worse grade of the lesion was retrieved. Among these latter pts. 10 showed a microinvasive and 2 an invasive cancer: both the invasive but only 3 of the 10 microinvasive cancer pts underwent a surgical procedure (2 radical and 3 extrafascial hysterectomies, respectively). After a mean follow up of 37 months our incidence of recurrence is 2.3% (18 pts): 5 CIN1, 7 CIN2, 3 CIN3, 2 CIS and 1 microinvasive disease. In 78% of the instances the recurrence has occurred within the first year of follow up. All 18 recurrences were successfully treated with further vaporization in 8 cases, conization in 9 and hysterectomy in 1. 93 term pregnancies occurred in 83 pts after CO2 laser treatment of CIN. No cervical incompetence occurred (no cervical cerclage employed) while the incidence of spontaneous abortion was not statistically significant. 82 pregnancies were delivered vaginally without significant variation of labor phase duration. The incidence of caesarian section (11.8%) was lower than the mean incidence in our Institution. These data confirm the successful complete restitutio ad integrum of the cervix after an adequate CO2 laser surgical procedure without any further risk of cervical incompetence, premature delivery or premature rupture of membranes. The use of CO2 laser surgery is recommended as modality treatment of choice in the management of cervical intraepithelial neoplasia.

NOR heteromorphism within a parthenogenetic lineage of the aphid Megoura viciae.

In parthenogenetic females of a clone of the aphid Megoura viciae (Homoptera, Aphididae), more than 50% of the cells show heteromorphism between homologous NORs which are located on one telomeric region of the two X chromosomes. Using different techniques, such as staining with the CG-specific fluorochrome chromomycin A3, silver staining and in-situ hybridization with an rDNA probe, we have shown that the observed heteromorphism is due to an unequal distribution of ribosomal genes between homologous NOR regions. The total number of rDNA genes per individual aphid remained constant. Moreover, the analysis of cells from single embroys has shown that the observed heteromorphism is not only intraclonal but also intraindividual. These data, together with the finding of X chromosomes connected by chromatin bridges between their NORs, allow us to suggest that mitotic unequal crossing over could be the main cause of NOR heteomorphism in this taxon.

Origin of DNA damage in ejaculated human spermatozoa.

The molecular basis of many forms of male infertility is poorly defined. One area of research that has been studied intensely is the integrity of the DNA in the nucleus of mature ejaculated spermatozoa. It has been shown that, in men with abnormal sperm parameters, the DNA is more likely to possess strand breaks. However, how and why this DNA damage originates in certain males and how it may influence the genetic project of a mature spermatozoon is unknown. Two theories have been proposed to describe the origin of this DNA damage in mature spermatozoa. The first arises from studies performed in animal models and is linked to the unique manner in which mammalian sperm chromatin is packaged, while the second attributes the nuclear DNA damage in mature spermatozoa to apoptosis. One of the factors implicated in sperm apoptosis is the cell surface protein, Fas. In this review, we discuss the possible origins of DNA damage in ejaculated human spermatozoa, how these spermatozoa arrive in the ejaculate of some men, and what consequences they may have if they succeed in their genetic project.

Cytogenetic and molecular characterization of a highly repeated DNA sequence in the peach potato aphid Myzus persicae.

Electrophoresis following digestion of Myzus persicae genomic DNA with HindIII showed the presence of a prominent band of approximately 200 bp whereas a faint electrophoretic band corresponding to DNA fragments of about 3000 bp was observed after digestion with ApaI. In situ digestion with restriction enzymes, followed by in situ nick translation, showed that ApaI targets are localized at the nucleolus organizer-bearing X telomeric region, whereas HindIII restriction sites are clustered in intercalary C-positive areas on the same X chromosome. Fluorescent in situ hybridization (FISH) carried out by using digoxygenin-labeled HindIII repeats as probe fully confirmed overlapping between the hybridization sites of this probe and the AT-rich intercalary heterochromatic bands on the X chromosome. These findings, together with published data, allow us to conclude that the M. persicae genome possesses three classes of C-positive heterochromatin: (i) a GC-rich argentophilic band located on one telomere of the X chromosome that contains ApaI targets; (ii) AT-rich intercalary bands located on the X chromosome containing clustered HindIII fragments; (iii) AT-rich telomeric bands located on autosomes, consisting of HaeIII repeats. Molecular analysis has shown that the length of the HindIII repeat consensus sequence is 189 bp with an AT content of 67%. Southern blotting with HindIII monomers revealed a regular ladder of bands composed of multimers of basic length that are characteristic of satellite DNAs. The HindIII repeat displays other features typical of eukaryotic satellite arrays such as overlapping with heterochromatic bands and a high degree of sequence similarity among monomers (84%-94%). A similarity plot showed that sequences were particularly variable in the 50-100 bp region whereas they proved to be highly conservative in the first 50 bp, thus suggesting that this portion of the repeat might be functionally important.

Chemotherapy in advanced ovarian cancer: four systematic meta-analyses of individual patient data from 37 randomized trials. Advanced Ovarian Cancer Trialists' Group.

The purpose of this systematic study was to provide an up to date and reliable quantitative summary of the relative benefits of various types of chemotherapy (non-platinum vs platinum, single-agent vs combination and carboplatin vs cisplatin) in the treatment of advanced ovarian cancer. Also, to investigate whether well-defined patient subgroups benefit more or less from cisplatin- or carboplatin-based therapy. Meta-analyses were based on updated individual patient data from all available randomized controlled trials (published and unpublished), including 37 trials, 5667 patients and 4664 deaths. The results suggest that platinum-based chemotherapy is better than non-platinum therapy, show a trend in favour of platinum combinations over single-agent platinum, and suggest that cisplatin and carboplatin are equally effective. There is no good evidence that cisplatin is more or less effective than carboplatin in any particular subgroup of patients.

[Ultrasonography of the uterine cervix in pregnancy. Curve of normality in a longitudinal and cross-sectional study]. / Studio ecografico del collo dell'utero in gravidanza. Curva di normalità in uno studio longitudinale e trasversale.

BACKGROUND: The aim of the study was to establish a normality curve of cervical length variations during pregnancy in our pregnant women population, to be compared with pathological cases (risk of preterm labor and cervical incompetence). DESIGN: A prospective longitudinal and cross-sectional study in women with single pregnancy and without risk factors for preterm labor was carried on. SETTING: Obstetric and Gynecology Department, University of Brescia, Italy. POPULATION OR SAMPLE: One hundred and thirty-four pregnant women with single pregnancy, 112 cross-sectionally and 22 longitudinally followed, with labor at term, were selected. Sixty-eight were nulliparous, 66 pluriparous. Multiple pregnancy, previous placental and vaginal bleeding were excluded. METHODS: A transvaginal probe was used to assess uterine cervix and the patients were studied from the 12th to 41st gestational week. Statistical analysis was carried out by Student's "t"-test and Z-test. MEASURES: Cervical longitudinal diameter, internal uterine os and funneling were assessed. RESULTS: No statistically significant differences emerged in the curves with regard to nulliparous and pluriparous patients. The cut-off between normality and pathology, from the 24th to the 32nd gestational week (2ndSD), seems to correspond to a cervical length lower than 25 mm; a significant progressive decrement of the cervical longitudinal diameter begins from the 28th gestational week. CONCLUSIONS: These results allows the gathering of reference data about normality to compare with pathology (cervical incompetence and preterm delivery), which could be useful both for prevention and follow-up of these cases.

'Dual positivity' for neural tube defects and down syndrome at maternal serum screening: gestational outcome.

OBJECTIVE: To evaluate the gestational outcome of pregnancies screen-positive for both neural tube defects (NTD) and Down syndrome (DS) ('dual positivity'). METHODS: Among 10,667 mid-trimester women screened for DS and NTD with alpha-fetoprotein (AFP), unconjugated estriol (uE3), and human chorionic gonadotropin (hCG), delivered up to July 1996, we have selected cases with both an unexplained AFP value > or = 2.5 multiples of median (MoM) and a DS risk > or = 1:250. All these pregnant women were managed with amniocentesis and/or CVS, ultrasound scans, and Doppler velocimetry. We have collected all data about the gestations with 'dual positivity' and no obvious explanation for these findings (cases with fetal malformations related to raised AFP). RESULTS: Twelve women (1.1:1,000) showed unexplained 'dual positivity'. Abnormal karyotypes were found in 3 fetuses, and pregnancies were terminated: there were 2 triploidies with partial hydatiform mola, and 1 DS. In 9 cases the fetal karyotype was normal, but a confined placental trisomy 16 was found in 4. Of the 9 continuing gestations, 8 displayed fetal growth retardation (FGR). One gestation ended with fetal death at 27 weeks. All 9 fetuses were morphologically normal, and 8 were small for gestational age. CONCLUSIONS: 'Dual positivity' at NTD/DS screening may anticipate pregnancy complications. The finding of trisomy 16 confined to the placenta and FGR in 4 cases suggests that at least some fetuses with growth restriction may suffer from a distinct placental disease. Maternal serum screening may have implications different from DS and NTD, as demonstrated by the 2 cases with triploidy and incomplete hydatiform mola, the 4 cases with placental trisomy 16, and the 4 cases of FGR of the 5 fetuses without chromosome abnormalities. As the pathologic outcome of these pregnancies is more important than the mere serum screening results, we feel that these cases need a strict work-up, including CVS, amniocentesis and ultrasound studies to better address the obstetrical management.

In-situ competition between protamine and fluorochromes for sperm DNA.

In this study we investigated the relationship between the presence of bound protamine on mouse and human sperm DNA and the level of chromomycin A3 (CMA3) and 4'6-diamidino-2-phenylindole (DAPI) fluorescence. This was accomplished by performing a competition assay between salmon protamine and fluorochromes on decondensed spermatozoa that had their nuclear proteins extracted and were fixed on slides. Various concentrations (0, 0.005, 0.0225, 0.05, 0.225, 0.5 and 5 mg/ml) of salmon protamine were added to either the CMA3 or DAPI staining solutions. Fluorescence emission measurements of stained sperm nuclei were then performed using a microfluorometer. When the treated decondensed sperm heads were stained with either CMA3 or DAPI all spermatozoa were found to fluoresce intensely. The addition of protamines to the spermatozoa led to an elimination of CMA3 fluorescence, while the intensity of DAPI staining was decreased to approximately 50% at the highest concentrations of protamine. The addition of increasing amounts of salmon protamine also induced the sperm nuclei to regain their initial condensed appearance. This study shows that protamine retains a strong affinity for sperm DNA in situ and that CMA3 fluorescence is a strong indicator of the protamination state of spermatozoa.

DNA strand breaks in ejaculated human spermatozoa: comparison of susceptibility to the nick translation and terminal transferase assays.

The nick translation and terminal transferase assays have been compared to test their relative efficiency in detecting DNA breakage in ejaculated human spermatozoa. The results have been correlated with the percentage of chromomycin A3 positive sperm, a fluorochrome that is indicative of the protamination state of sperm. Examination of the ejaculated sperm of 30 subjects revealed that the percentage of positivity to the nick translation and terminal transferase assays did not differ, even when using different fixatives. It is concluded that the inability of the two assays to distinguish the type of DNA damage, as is possible in somatic nuclei, is most probably linked to the unique nature of sperm chromatin. It is proposed that the presence of the damaged DNA may be the remnants of an imperfect spermiogenesis, probably related to an inadequate protamine deposition. This is supported by the strong correlation between the presence of DNA damage and underprotamination as evidenced by chromomycin A3.