Immediate surgery for acute internal carotid artery dissection and thrombosis during filter deployment prior to stenting: a case report.

Carotid artery stenting (CAS) is a validated option in the treatment of selected extracranial carotid artery stenosis. Carotid artery dissection during CAS is a rare but potentially devastating complication. We report a case of acute dissection and thrombosis of the left internal carotid artery during filter tip wire engaging maneuvers, complicated by intraoperative complete blindness of the left eye. Immediate conversion to carotid endarterectomy was performed under general anesthesia with electroencephalographic monitoring. The patient was discharged home symptomless and remains asymptomatic eight months after the operation, with normal left internal carotid patency and fully recovered eyesight. In conclusion, the management of acute carotid occlusion during CAS requires emergent evaluation and definitive endovascular or open surgical repair to minimize neurologic morbidity. We advocate that all endovascular procedures are carried out in a well-established surgical environment.

Percutaneous transluminal angioplasty improves glucose control and quality of life in patients with critical limb ischemia.

AIM: To evaluate the benefit of endovascular peripheral revascularization on glucose control in patients with chronic limb ischemia. METHODS AND RESULTS: Over a 12 month period, 61 patients (41 male, range 49-88 years of age) presenting with critical limb ischemia (CLI) were treated according to the Trans Atlantic Inter Society Consensus (TASC II) guidelines. After discharge, all patients were asked to measure their glucose level three times daily, and glycated hemoglobin was checked monthly up to 12 months, as well as to fill a questionnaire to assess their Quality of Life (QoL). The revascularization procedure was successful in 90% of cases. Glycemic control and glycated hemoglobin in 22 diabetic patients subgroup were significantly improved after the treatment and remained stable over the follow-up period. There was a significant improvement in QoL that increased steadily from the operation and to reach a plateau after six months. CONCLUSIONS: Peripheral percutaneous angioplasty in subjects with CLI significantly improves glycemic control and ameliorates QoL. Revascularization positively effects also long-term diabetes control as well as QoL.

Endovascular management of symptomatic cerebral malperfusion due to carotid dissection after type A aortic dissection repair.

PURPOSE: Type A acute aortic dissection is a surgical emergency, and supra-aortic trunk involvement may be complicated by stroke in 6% to 20% of cases. A 66-year-old Caucasian female patient underwent a composite repair of the ascending aorta for type A aortic dissection. Postoperative period was complicated by episodes of "drop attack." Doppler ultrasound of supra-aortic trunks revealed an intimal flap occluding right internal carotid artery. TECHNIQUE: Multiple stenting was performed from carotid bifurcation to internal carotid artery in order to exclude the dissection intimal flap. After endovascular procedure physiatrist considered that motor functional improvement was better than expected, and we support that endovascular resolution of carotid malperfusion led to a better outcome. CONCLUSION: According to other experience, endovascular procedure resulted as a safe and effective way. Moreover, ultrasound monitoring of supra-aortic trunks in postoperative period is recommended.

Human cervical mucus can act in vitro as a selective barrier against spermatozoa carrying fragmented DNA and chromatin structural abnormalities.

PURPOSE: We have carried out experiments to determine if human cervical mucus can act as an in vitro selective barrier against spermatozoa morphologically normal that carry genetic structural abnormalities. METHODS: Sperm chromatin abnormalities have been evaluated by Chromomycin A3 and "endogenous" nick translation. RESULTS: The data obtained have shown that spermatozoa possessing higher levels of DNA protamination are more proficient in crossing the cervical mucus barrier. Moreover, the levels of positivity to endogenous nick translation treatment was practically zero in such spermatozoa. CONCLUSIONS: We suggest that sperm penetration of cervical mucus could be used to select sperm preparations free of fragmented DNA or chromatin structural abnormalities for assisted reproduction.

Sexuality, partner relations and contraceptive practice after termination of pregnancy.

The aim of this study was to determine the impact of termination of pregnancy (TOP) on women's sexual well-being, the couple and contraceptive practice. In a prospective qualitative and quantitative study, 103 women undergoing induced abortion by vacuum aspiration were interviewed before the abortion and 6 months later. The interview was performed by means of a questionnaire including open and closed questions, and two psychological tests (Locke-Wallace and Horowitz). After TOP, the majority of women did not report changes in their sexual behavior and satisfaction. Eighteen per cent of women reported a decrease in sexual desire and 17% reported orgasmic disorders. About one-third of women described psychosomatic symptoms, but a minority were traumatized by the event. Ninety-eight per cent of the women were informed about, and had practiced, contraception in the past; 69% had actually used some kind of contraception during the menstrual cycle that had resulted in pregnancy (31% had had unprotected intercourse). Six months later, 83% practiced contraception, and only 17% did not. Fourteen out of 84 couples separated after TOP (one in six). Six months after TOP, the large majority of women interviewed seemed able to cope with TOP. A minority presented some persisting sexual dysfunction and/or some psychosomatic symptoms.

Sperm decondensation during fertilisation in the mouse: presence of DNase I hypersensitive sites in situ and a putative role for topoisomerase II.

In this study our aim was to characterise the presence and the role of DNA alterations during sperm decondensation in the mouse. To visualise the changes during decondensation we investigated for the presence of DNase I hypersensitive sites in situ and for a putative role for topoisomerase II by examining the effect of teniposide, a topoisomerase II inhibitor, during fertilisation. In situ nick translation without the previous addition of DNase I failed to reveal the presence of endogenous nicks in decondensing sperm and pronuclei whereas preincubation of fixed oocytes with DNase I indicated that decondensing sperm were sensitive to this enzyme. Addition of 100 microM teniposide did not completely inhibit pronuclei formation but its addition to the fertilisation medium did lead to the presence of endogenous DNA nicks in decondensing sperm. These observations suggest that DNase I hypersensitivity during sperm decondensation is related to the dramatic conformational changes that the chromatin undergoes during the decondensation process, in which topoisomerase II may be implicated.

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.

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.

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.

Early cleavage of human embryos to the two-cell stage after intracytoplasmic sperm injection as an indicator of embryo viability.

In-vitro fertilization (IVF) embryos are selected for transfer on the basis of morphology and rate of development. However, when a number of embryos have similar characteristics, the selection of the best embryos is left to chance. Recently, we proposed a simple, novel method to overcome this problem, based on pre-selection of embryos cleaving early to the two-cell stage. In this study we have adopted the same method to choose embryos fertilized after intracytoplasmic sperm injection (ICSI). Fertilized embryos that had cleaved to the two-cell stage by 27 h post-injection were designated as 'early cleavage' embryos, while those that had not yet reached the two-cell stage were designated as 'no early cleavage'. In all cases, the early cleavage embryos were transferred when available. Early cleavage was observed in 54 (61.4%) of the 88 cycles assessed. There were significantly (P = 0.04) more clinical pregnancies in the early cleavage group, 14/54 (25.9%), compared with the no early cleavage group 2/34 (3.2%). No differences between the groups were found when comparing key parameters (age, stimulation protocol and semen characteristics) of the couples. Using the ICSI technique, we have shown that early cleavage to the two-cell stage is not influenced by the timing of fertilization, and is more likely due to intrinsic factors within the oocyte or embryo that promote embryo cleavage after fertilization.

Sperm nuclear DNA damage and altered chromatin structure: effect on fertilization and embryo development.

In the first part of this report we investigate whether chromatin anomalies in human spermatozoa can influence fertilization after intracytoplasmic sperm injection (ICSI). We have examined the sperm chromatin packaging quality using the chromomycin A3 (CMA3) fluorochrome and the presence of DNA damage in spermatozoa using in-situ nick translation. When comparing the spermatozoa of patients undergoing in-vitro fertilization (IVF) and ICSI distinct differences are evident in that ICSI males have a higher CMA3 fluorescence, indicating spermatozoa with loosely packed chromatin, and more spermatozoa containing endogenous DNA nicks. When examining the unfertilized oocytes of ICSI patients we found that men who had a high percentage of anomalies in their chromatin, i.e. > 30% CMA3 fluorescence and > 10% nicks, had more than double the number of unfertilized oocytes containing spermatozoa that had remained condensed. The observation that failed fertilized oocytes, injected with spermatozoa from patients with a higher percentage of sperm nuclear anomalies, contain more condensed spermatozoa indicates that a selection process against these spermatozoa may be in place at the time of fertilization. In the second part of the study we show that spare ICSI embryos have significantly lower rates of development to the blastocyst stage compared with those developed after routine IVF. These results show that a greater understanding of the molecular basis of male infertility is therefore needed to broaden our knowledge on the effect that abnormal spermatozoa have on fertilization and embryo development.

Hoechst staining and exposure to UV laser during flow cytometric sorting does not affect the frequency of detected endogenous DNA nicks in abnormal and normal human spermatozoa.

Controlling the sex of offspring by the separation of X and Y chromosome-bearing spermatozoa using flow cytometry has been reported as a clinical technique aiding prevention of X-linked diseases. Although this technique has resulted in several hundred normal births in animals and at least one human birth, there is still concern over its genetic safety due to the involvement of two potentially mutagenic agents: UV light and the fluorochrome dye, Hoechst 33342 (H33342). Human spermatozoa, particularly those considered abnormal, may be more likely to suffer DNA damage following exposure to mutagenic agents, compared with other mammalian species. The stability of normal fresh and decondensed human spermatozoa were examined after exposure to a range of levels of UV and H33342 staining, using an assay that detects endogenous nicks in the DNA of spermatozoa. The stability of abnormal and normal, fresh and frozen-thawed human spermatozoa was examined following UV laser, H33342 staining and flow cytometry treatments utilizing the same assay. There was an increase in the presence of endogenous nicks when spermatozoa were decondensed compared with fresh spermatozoa. There was no increase in the incidence of nicks in any group of spermatozoa after UV and fluorochrome exposure compared with controls without exposure.

Intrauterine insemination: evaluation of the results according to the woman's age, sperm quality, total sperm count per insemination and life table analysis.

We report on 332 infertile couples who underwent 1115 cycles of intrauterine insemination (IUI) with washed husband's semen. The indication for IUI was an abnormal post-coital test due to either a male or cervical infertility factor. The mean number of IUI cycles per patients was 3.4, the overall pregnancy rate 18.7%, and the pregnancy rate per cycle 5.6%. The cumulative pregnancy rate calculated by life table analysis showed that 16.0% of pregnancies occurred in the first three treatment cycles, while the cumulative pregnancy rate was 26.9% by the sixth cycle. The outcome of the therapy was adversely affected if the woman's age was > 39 years and/or total motile sperm count per insemination was < 1 x 10(6). No pregnancy occurred in women older than 44 years or in cases with a total motile sperm count before semen preparation of < 1 x 10(6).

Sperm chromatin anomalies can influence decondensation after intracytoplasmic sperm injection.

In this study we investigated whether morphology and chromatin anomalies in human spermatozoa can influence fertilization after intracytoplasmic sperm injection (ICSI). We examined unfertilized oocytes, using the fluorochrome Hoechst 33342, to determine whether a relationship exists between failure of fertilization and sperm chromatin quality. Sperm chromatin packaging quality was assessed using the chromomycin A3 (CMA3) fluorochrome, and the presence of DNA damage in spermatozoa, using in-situ nick translation, Normal males present sperm parameters with a normal morphology of > 20%, CMA3 fluorescence of < 30% and exhibit endogenous nicks in < 10% of their spermatozoa. When patients were separated according to these values no difference was observed in their fertilization rates after ICSI. When the unfertilized ICSI oocytes were examined, we found that patients with CMA3 fluorescence of <30% and nicks in < 10% of their spermatozoa had only 17.5 and 21.6% respectively of their unfertilized oocytes containing spermatozoa that remained condensed. In contrast, patients with higher CMA3 and nick values had a significantly higher number, 41.2 and 48.9%, of their unfertilized oocytes containing condensed spermatozoa. sperm morphology did not show any such pattern. The percentage of spermatozoa which had initiated decondensation in unfertilized oocytes was not influenced by morphology, CMA3 fluorescence or nicks. In light of these results we postulate that poor chromatin packaging and/or damaged DNA may contribute to failure of sperm decondensation after ICSI and result in failure of fertilization.

Use of the guanine-cytosine (GC) specific fluorochrome, chromomycin A3, as an indicator of poor sperm morphology.

PURPOSE: We have previously postulated that the chromomycin A3 (CMA3) fluorochrome allows an indirect visualization of sperm chromatin packaging quality and partially denatured sperm DNA. In this study we investigate the relationship between CMA3 positivity and sperm morphology. We also present data on the association between sperm morphology and the presence of endogenous nicks in sperm DNA. METHODS: Semen samples were examined from 81 males of the couples who were consulting for infertility treatment. CMA3 fluorescence was assessed for all samples, while in 24 sperm samples we also examined for the presence of endogenous nicks in the sperm DNA. RESULTS: When sperm morphology was less than 20% normal in a patient, the level of CMA3 fluorescence and presence of endogenous nicks were significantly higher than in patients with a higher incidence of morphologically normal sperm. CONCLUSIONS: CMA3 could be used as an adjunct to the assessment of morphology as an evaluation method for poor sperm. Its value in predicting fertilizing ability when using either SUZI or ICSI awaits to be answered.

Chromatin packaging and morphology in ejaculated human spermatozoa: evidence of hidden anomalies in normal spermatozoa.

This study aimed to investigate the association between anomalies in sperm chromatin packaging, morphology and fertilization in patients undergoing routine in-vitro fertilization (IVF) or subzonal insemination (SUZI). Sperm chromatin packaging was assessed using chromomycin A3 (CMA3), a fluorochrome specific for guanine-cytosine rich sequences of DNA. One hundred to 150 sperm cells were assessed in 55 patients to compare sperm chromatin packaging and morphology to fertilization after IVF or SUZI. When the morphology and CMA3 fluorescence of individual spermatozoa was assessed, > 75% of the macrocephalic sperm fluoresced in all patients. In contrast, a mean of 37% of the spermatozoa with normal morphology fluoresced in IVF patients compared with 58% of the normal spermatozoa in male factor patients treated by SUZI. SUZI patients displaying a high fluorescence (> 70%) in their spermatozoa also had a significantly lower fertilization rate. Lower packaging quality in morphologically normal spermatozoa may represent a major limiting factor in the fertilizing ability of male factor patients. This study confirms that a high percentage of CMA3 positivity is present in certain forms of male factor infertility and that such a test may be used to distinguish separate populations in morphologically normal spermatozoa.

[Treatment of male sterility using intra-oocytic sperm injection: critical evaluation]. / Traitement de la stérilité masculine par injection intraovocytaire de spermatozoïde: évaluation critique.

Intracytoplasmic injection of a single spermatozoon into an oocyte (ICSI) is capable of achieving fertilization of oocytes in cases of severe male sterility resistant to classical andrological treatments. We report 3 cases of long-standing male sterility in which fertilization of oocytes using ICSI in 3 cases resulted in clinical pregnancies in 2. Indications for ICSI are discussed emphasizing the genetic risk in the case of bilateral absence of the vas deferens, frequently associated with mutation of the gene responsible for cystic fibrosis. To date, follow-up of 339 babies has revealed 2.9% congenital abnormalities.

Relationship between the presence of endogenous nicks and sperm chromatin packaging in maturing and fertilizing mouse spermatozoa.

Mammalian spermiogenesis involves the replacement of histones by protamines, resulting in a highly compacted chromatin. Upon fertilization, the reverse process occurs. We have previously shown that the chromomycin A3 (CMA3) fluorochrome represents a useful tool for detecting protamine deficiency in spermatozoa. In this study we investigated CMA3 fluorochrome accessibility and the presence of endogenous nicks in maturing and fertilizing mouse sperm. Testicular sperm of stages 1-7 and 8-14 showed high positivity (> 96%) to CMA3, decreasing to 63% in stage 15-16 spermatids. In situ protamination of stage 15-16 spermatids saw an inhibition of CMA3 accessibility. Only 8% of the mature spermatozoa in the efferent ducts were CMA3-positive; this value decreased to 0% in the caput epididymidis. At fertilization, CMA, fluorescence reappears in decondensing sperm. Fluorescein isothiocyanate (FITC) fluorescence, identifying endogenous nicks, was evident in 6% of stage 1-7 spermatids, increased to 22% in stage 8-14 spermatids, and disappeared in stage 15-16 spermatids. During fertilization, endogenous nicks were not observed in decondensing sperm. We propose that 1) the presence of nicks in mouse testicular spermatids suggests that DNA cutting and ligating occurs prior to completion of protamination and 2) the absence of nicks during fertilization indicates that decondensation is not simply the reversal of the initial chromatin packaging process.

Presence of endogenous nicks in DNA of ejaculated human spermatozoa and its relationship to chromomycin A3 accessibility.

During spermiogenesis, mammalian chromatin undergoes replacement of nuclear histones by protamines, resulting in a DNA that is highly condensed in the mature sperm. We have previously demonstrated that a percentage of human spermatozoa exhibit 1) positivity to the guanine-cylosine-specific chromomycin A3 (CMA3) fluorochrome and 2) the presence of endogenous nicks in their DNA. In situ protamination of mature human sperm limits the percentage of sperm positive to CMA3 and exhibiting endogenous nicks. In this study, we report further investigations that aim to clarify the relationship existing between levels of CMA3 stainability and the presence of endogenous nicks in the DNA of mature human spermatozoa. Human spermatozoa from 25 different samples showed values of sensitivity to the CMA3 fluorochrome ranging from 13% to 75%. The same samples showed a percentage of sensitivity to endogenous nick translation ranging from 1% to 38%. A strong correlation (r = 0.86) was evident between these two parameters. Prior staining of sperm with the CMA3 fluorochrome drastically reduced sensitivity to nick translation. In contrast, previously nick-translated sperm stained with CMA3 showed very little difference from samples that had not been pretreated. The presence of nicked sperm in the ejaculate may indicate anomalies during spermiogenesis and be an indicator of male infertility.