Childhood outcomes of assisted reproductive technology

There is a large population of children conceived via assisted reproductive technology (ART), which continues to increase worldwide, without a clear understanding of associated long-term outcomes. ART children are more likely to be the result of multiple pregnancies, and thus to be born prematurely or low birthweight. There is growing evidence that ART children are phenotypically and biochemically different from naturally conceived children, but the mechanism(s) leading to these changes have not been elucidated. There is a possible increased risk of rare imprinted gene disorders in these children. However, it remains unclear whether more subtle changes in DNA methylation occur commonly, leading to differences in gene expression and phenotype in ART children. Although an increased risk of cancer among ART children has been reported, the role of ART in the development of cancer has not been demonstrated. Further research and ongoing surveillance of ART children is essential to better understand the possible effects of ART on the long-term health of this population.     

Detection of DNA-crosslinking agents with the alkaline comet assay

The single cell gel electrophoresis, or comet assay, under alkaline conditions is a sensitive, simple and rapid method for the detection of DNA damage at the individual cell level. Its applicability as an indicator for the DNA crosslinking potency of a test substance was investigated in human white blood cells by combined treatment with the DNA damaging agent methyl methanesulphonate (MMS) for 2 hr at 37°C. The known crosslinking agents cisplatinum, mitomycin C and formaldehyde, and the formaldehyde releasers diazolidinyl urea and dimethylol urea, were shown to reduce MMS-induced DNA migration in the comet assay in a concentration-dependent manner. Two other protocols, adding MMS to the cells before or after treatment with a crosslinking agent, were carried out and achieved similar results. The results of this study indicate that the comet assay is a useful tool for the detection of crosslinking agents. Advantages and limitations of this method compared to the alkaline elution technique are discussed. © 1996 Wiley-Liss, Inc.     

Ubiquitin-based sperm assay for the diagnosis of male factor infertility

Sperm morphology is correlated with fertility in men, yet the existing, subjective sperm morphology assays provide only a limited insight into patients' infertility. Here, we provide the experimental background for a new, objective and automated semen assay, based on the cross-reactivity of defective human spermatozoa with antibodies against a proteolytic marker peptide, ubiquitin. Using immunofluorescence and flow cytometry, we screened the spermatozoa from 17 infertility patients and two fertile donors for their cross-reactivity with anti-ubiquitin antibodies. Thirteen out of 17 patients, but neither of two fertile donors, displayed an increased binding of anti-ubiquitin antibodies to sperm surface, that reflected the occurrence of abnormalities in these samples and was corroborated by available clinical data. Highest correlation coefficient (r = -0.432) was obtained with the cleavage rate after IVF. The contribution of male factor was revealed in several couples previously diagnosed with idiopathic infertility. Ubiquitin-cross-reactive sperm-surface proteins thus seem to be a universal marker of semen abnormalities, including sperm head and tail defects and semen contaminants such as spermatids, leukocytes and cellular debris. We propose that the sperm-ubiquitin tag immunoassay (SUTI) may be a valuable diagnostic tool in treatment of male factor and idiopathic infertility.     

Laboratory tests for human male reproductive risk assessment

The criteria for reproductive test selection which were set forth in the beginning of this chapter required that the tests be objective, technically sound, biologically stable, sensitive and feasible. All of the tests which have been discussed can generate objective quantitative data (Table 1). Testicular tonometry appears to be a technically sound procedure which measures a biologically stable parameter, although this remains to be proven. Sperm counts are definitely not a biologically stable parameter. There is insufficient information to judge the biological stability of data obtained from sperm cervical mucus interaction. Data from a number of laboratories suggest that the zona-free hamster egg assay gives stable results when repeated with the same donor, and the tests as performed in specialized laboratories are technically sound at the present time. However, the number of laboratories which can perform the assay is limited. Sensitivity to early toxicity is a very important criterion for test selection. Physical examination does not meet this criterion, endocrine studies do not, and sperm counts do not. Not enough information is currently available to determine the sensitivity of sperm motility assessment. Sperm morphology assessment may be the most sensitive early indicator of reproductive toxicity which is currently available. There is a large body of clinical and basic science literature which suggests that sperm morphology may reflect acute stress effects on the testes. The feasibility of these tests vary. Sperm motility may be feasible only in longitudinal studies in which the video equipment can be set up in a laboratory which is doing repeated assessments. Sperm morphology assessment does not require any specialized equipment in the field. Studies of sperm cervical mucus interaction, for the reasons already stated, remain non-feasible at this time. Tests of sperm-egg interaction are probably feasible if spermatozoa can be shipped to a specialized laboratory for assessment. Thus, there are now a number of new tests for male reproductive function which are available, and which are practical. It is time for this technology to be transferred from the basic science laboratory for application in human reproductive risk assessment.     

Possibilities and limitations of techniques of assisted reproduction for the treatment of male infertility

Since relatively few spermatozoa are needed for oocyte fertilization during gamete intra-Fallopian transfer (GIFT) or in-vitro fertilization (IVF), these methods have been applied in couples with infertility due to male causes. Forty-six couples with male factor infertility were enrolled in this study and results were compared with those attained in 48 couples treated with the same techniques for other than male causes. Overall, GIFT resulted in 26% ongoing pregnancies. GIFT seems to be particularly successful when the sperm concentration is 20 x 10(6)/ml or more, but sperm motility and/or morphology are poor. Nine pregnancies occurred out of 26 GIFT cycles in 18 cases selected on this basis. The ongoing pregnancy rate after IVF was 16% per patient. The latter treatment should be attempted in male immune infertility and in cases with a low sperm concentration, with or without abnormal sperm motility and/or morphology. In these circumstances, five pregnancies were attained out of 28 cycles in 14 cases. For similar sperm concentrations, the conception rate per cycle attained with techniques of assisted reproduction was more than twice that attained with conventional treatment of male infertility.     

Evaluation of manual and image analysis quantification of DNA damage in the alkaline comet assay

The alkaline comet assay or single cell microgel electrophoresisassay is a sensitive method of detecting DNA strand breaks andalkali labile sites in individual cells. The results of thisassay can be analysed by different methods. In this study wecompared analyses of the same slides by a manual method andby image analysis, post-treatment of clone 707 Friend erythroleukaemiacells with H₂O₂. The parameters which were found to be particularlyuseful were comet area and comet length (measured manually)and percentage tail DNA, tail moment, tail length and tail length/headradius (L/H), measured using image analysis. The manual methodfor comet analysis presented in this paper would appear to providegood and reliable comet data. However, the image analysis cometsystem described offers an alternative analysis method whichavoids the need for photomicrographs and tedious manual analysis.The image analysis parameters: % tail DNA, tail moment, taillength and L/H give good consistent results and for large-scaleanalysis it will, therefore, conceivably be the method of choice. ¹To whom correspondence should be addressed     

特发性男性不育的分子遗传学诊断与生育指导

特发性男性不育是指男性不育症患者除有精子异常外,其余情况均正常,而患者出现精子异常的原因不明.研究证明,Yq11中无精子因子(azoospermia factor,AZF)的缺失,可引起不同程度的生精障碍[1],但随着辅助生殖技术的发展,AZF缺失患者依然可以生育后代,并且不同缺失类型辅助治疗对策各异.因此,有必要对特发性男性不育患者进行Y染色体AZF区域微缺失研究,从而对该类患者的诊断和生育指导提供依据.     

雄激素在辅助生殖技术中的应用

随着女性体内雄激素的生成和代谢途径被逐步了解,越来越多学者关注雄激素在女性体内所发挥的作用。在动物研究中,睾酮通过提高FSH受体活性和刺激IGF-1分泌而促进卵泡发育成熟。在辅助生殖技术中,已有一些通过适当补充雄激素来提高卵巢反应性,以期改善卵巢低反应患者IVF助孕结局的临床数据;然而因为雄激素在卵巢的具体作用机制仍未清楚,部分学者持谨慎态度。近年来,雄激素在评价卵巢储备、预测卵巢反应性方面的作用,已经引起了部分生殖医学临床学者的关注。但是,由于目前展开的研究为数尚少,所以这方面存在较大争议。现对雄激素在辅助生殖技术中的应用作一综述。     

早期补救性ICSI在原发不孕患者中的临床应用

目的探讨早期补救性ICSI在原发不孕患者中的临床应用价值及可行性。方法以行常规IVF-ET治疗原发不孕患者203周期为实验组,进行短时授精结合第二极体观察判断受精情况,对受精失败或受精率低下患者行早期补救性ICSI治疗。并与同期93个直接ICSI周期进行比较。结果 203个周期中行早期补救性ICSI周期17个,占周期总数8.4%;与直接ICSI周期比较,早期补救性ICSI的受精率、多PN（≥3PN）、卵裂率、优质胚胎率、妊娠率、种植率等方面差异无统计学意义。结论早期补救性ICSI在无明确ICSI指证的原发不孕患者中可以获得较好的临床效果。     

鹿胎膏在辅助生殖技术中的应用研究

目的通过鹿胎膏在辅助生殖技术超促排卵过程中的应用研究,确定其在助孕治疗中的作用机制和应用价值。方法采用前瞻性随机对照的方法,将鹿胎膏作为一种辅助用药,在体外受精（IVF／ICSI）和宫腔内人工授精（IUI）的常规超促排卵方案基础上应用,并与单纯常规方案组比较,将达菲林（GnRHa）（先灵）、果纳芬（rFSH）（雪兰诺）、尿促性素（HMG）（丽珠）的用量;HCG目的血雌二醇（E2）值、子宫内膜厚度、获卵数、受精率、优质胚胎率、移植日血孕酮（P）值、临床妊娠率、着床率;克罗米芬（CC）用量、成熟卵泡数及孕早期流产率等指标进行对照和统计学处理。结果在体外受精时,辅助应用鹿胎膏组的孕早期流产率明显低于未用鹿胎膏组,两组间差异有统计学意义（P〈0．05）。IUI时加用鹿胎膏组孕早期流产率的绝对值较低,但无统计学意义。其他项目两组间差异无统计学意义。结论研究结果说明,鹿胎膏在不孕症治疗中的作用环节主要是在维持妊娠过程。该结果提示我们,如果将鹿胎膏应用于复发性早孕期流产病人,可能具有一定的疗效。     

Impact of Italian legislation regulating assisted reproduction techniques on ICSI outcomes in severe male factor infertility: a multicentric survey

BACKGROUND: In 2004, a law regulating assisted reproduction techniques (ART) was passed in Italy. The new rules allow for the formation and transfer of a maximum of three embryos at one time, whereas embryo selection and embryo storage are prohibited. The aim of this study is to evaluate the impact of these restrictions on ICSI outcome in couples affected by severe male factor infertility. METHODS: Thirteen Italian ART Units were involved in this study. Data were collected on ICSI cycles performed during 2 years before (control group) and 2 years after (study group) the enforcement of the law. Only cases of obstructive azoospermia (OA), non-obstructive azoospermia (NOA) and severe oligoastenoteratozoospermia (OAT) (sperm count 相似文献   

Potential significance of genomic imprinting defects for reproduction and assisted reproductive technology

Recent studies suggest a possible link between human assisted reproductive technology and genomic imprinting disorders. Assisted reproductive technology includes the isolation, handling and culture of gametes and early embryos at times when imprinted genes are likely to be particularly vulnerable to external influences. Evidence of sex-specific differences in imprint acquisition suggests that male and female germ cells may be susceptible to perturbations in imprinted genes at specific prenatal and postnatal stages. Imprints acquired first during gametogenesis must be maintained during preimplantation development when reprogramming of the overall genome occurs. In this review, we will discuss both new developments in our understanding of genomic imprinting including the mechanisms and timing of imprint erasure, acquisition and maintenance during germ cell development and early embryogenesis as well as the implications of this research for future epigenetic studies in reproduction and assisted reproductive technology.     

Recommendations for design of the rat comet assay

Although the rodent comet assay is gaining acceptance as a standard technique for evaluating DNA damage in vivo, there is no internationally accepted guideline for its conduct and several aspects of its experimental design have not been optimized. For example, no standard positive control is used, there is no agreement on how tissue toxicity should be measured and sources of experimental variability have not been considered in relation to experimental design. This study showed that methylnitrosourea is a good alternative positive control inducing DNA damage in all tissues examined (stomach, liver, blood and bone marrow) over a dose range of 25-100 mg/kg at both 3 and 24 h after treatment. At the highest dose, significant toxicity was seen in all tissues using the neutral diffusion assay and also by histopathological/haematological analysis, except in the liver where no change was seen even 7 days after dosing. Analyses using control data pooled from several studies showed that, as expected, the greatest variability was seen between tissue preparations from different animals and that different numbers of animals were required to detect the same fold increases in different tissues. Power analyses showed that, preparing three gels for each tissue and scoring 50 nuclei per gel, a group of six animals allows 2-fold increases over control in the liver, bone marrow and stomach and a 3-fold increase in blood to be detected with 80% probability. It is recommended that similar investigations of experimental variability should be performed to determine optimal experimental design in any laboratory using the rodent comet assay.     

Genotoxicity of a polluted river system measured using the alkaline comet assay on fish and earthworm tissues

Monitoring genotoxicity in the environment by using endemic organisms as sentinels requires sensitive assays. In this study the genotoxic properties of water and sediment collected from the Noyyal River, which is polluted with industrial effluent and sewage, was determined in fish (Cyprinus carpio) and earthworms (Eisenia foetida) using the alkaline comet assay. Upon electrophoresis, extensive DNA damage, measured as the DNA length:width ratio of the DNA mass, was observed in erythrocytes, liver, and kidney cells of fish exposed to polluted water samples and the amount of damage increased with the duration of exposure. Similarly, the mean DNA length:width ratio was significantly higher in the coelomocytes of earthworms placed in sediment samples. The highest levels of DNA damage were obtained with samples taken at and immediately downstream of urban centers. The results of this study indicate that the Noyyal River system is contaminated with substances that are genotoxic to fish and earthworms and that the comet assay has sufficient sensitivity to detect the genotoxicity.     

Efficacy of nafarelin in assisted reproductive technology: a meta-analysis

A systematic review identified nine randomized, controlled trials (both published and unpublished) which assessed the efficacy of nafarelin during IVF compared with other gonadotrophin-releasing hormone (GnRH) agonists. The trials included 1,014 women (nafarelin n = 597) in protocols employing three different dosage regimens, long and short stimulation protocols, and three comparative GnRH agonists (buserelin n = 348; triptorelin n = 14, and leuprolide n = 55). The meta-analysis of the data showed that pregnancy rates per embryo transfer with nafarelin were equivalent to those obtained with other GnRH agonists. Nafarelin and other agonists were also comparable in terms of several intermediate IVF outcomes, including fertilization rates, number of oocytes retrieved, peak oestradiol concentrations, and cycle cancellations. Women treated with nafarelin required fewer ampoules of human menopausal gonadotrophin (HMG)/FSH for ovarian stimulation and fewer days of stimulation. Safety results from both the meta-analysis and a qualitative analysis of 12 additional reports suggested that adverse effects were within the accepted tolerance range; the most frequent adverse effects were hypo-oestrogenic symptoms. In conclusion, the overall efficacy of nafarelin was equivalent to that of other GnRH agonists. The possibility that the reduced gonadotrophin requirements in women taking nafarelin will translate into cost savings per IVF treatment cycle requires further study.     

Detection of ghost cells in the standard alkaline comet assay is not a good measure of apoptosis

The single cell gel electrophoresis assay, or Comet assay, is a powerful tool for measurement of DNA strands breaks, oxidative damage, and alkali labile sites, and the assay was recently modified to detect DNA cross-links. It has also been proposed as a measure of apoptosis since apoptotic cells are suspected to result in total migration of the DNA from the nucleus into the tail. Cells with this appearance are called ghost cells, clouds, hedgehogs, or NDCN (nondetectable cell nuclei). The aim of this study was to determine if ghost cells can be used to measure apoptosis in the standard alkaline comet assay. To answer this question, we made use of two cell lines: CTLL-2 cells that can enter apoptosis upon addition of apoptosis stimuli or IL-2 deprivation, and CTLL-2 bcl2 cells that are protected from apoptosis due to the overexpression of the apoptosis inhibitor gene bcl2. The two cell lines were treated with cytotoxins (nongenotoxic apoptosis inducers, nongenotoxic necrotic agents) or genotoxins. They were also subjected to growth factor withdrawal, which induced apoptosis in the CTLL-2 cell line. The level of apoptosis was measured by the Annexin V-FITC method in parallel with performing the Comet assay. The results obtained in the two cell lines suggest that apoptotic or necrotic death does not correlate well with the detection of ghost cells, presumably because these cells are lost upon electrophoresis. A variant of the alkaline Comet assay that was performed without electrophoresis (halo method) was able to efficiently detect cells undergoing apoptosis, but it was unable to clearly distinguish between apoptosis and genotoxic damage.