Gene expression in bovine nuclear transfer embryos in relation to donor cell efficiency in producing live offspring.

Developmental abnormalities associated with the cloning process suggest that reprogramming of donor nuclei into an embryonic state may not be fully completed in most of the cloned animals. One of the areas of interest in this regard, is the analysis of gene expression patterns in nuclear transfer (NT) embryos to dissect the processes that failed and develop means to overcome the limitations imposed by these factors. In this study, we investigated expression patterns of histone deacetylase-1, -2, -3 (HDAC-1, -2, -3), DNA methyltransferase-3a (DNMT3A), and octamer binding protein-4 gene (OCT4) in donor cells with different cloning efficiencies and NT embryos derived from these cells employing a real-time RT-PCR assay. All genes investigated followed altered expression patterns in NT embryos when compared to IVF-derived embryos. In general, expression of HDAC genes was elevated especially at the compact morula stage and comparable to in vitro fertilized (IVF) embryos at the hatched blastocyst stage. DNMT3A expression in NT embryos was lower than IVF embryos at all stages. Oct-4 transcript levels were also reduced in cloned compared to IVF embryos at the compact morula and blastocyst stages. This difference disappeared at the hatched blastocyst stage. There was a donor cell effect on the expression patterns of all genes investigated. These results demonstrate altered gene expression patterns for certain genes, in cloned cattle embryos from our donor cells of different efficiency in producing live offspring. Therefore we suggest that differences in expression of developmentally important genes during early embryo development may characterize the efficiency of donor cells in producing live offspring.

Genotyping of Israeli infertile men with idiopathic oligozoospermia.

Microdeletions of the long arm of the Y chromosome involving the azoospermia factor (AZF) region are associated with severe oligo- or azoospermia. Abnormal androgen receptor (AR) structure or function has also been implicated in male infertility. To assess the contribution of these genetic defects to male infertility, 61 Israeli men with severe oligo- (n = 15) or azoospermia (n = 46), were screened for Y chromosome microdeletions, and the AR-(CAG)n repeat length. Fifty fertile Israeli men were similarly analyzed. PCR amplification of 20-54 simple tag sequences (STSs) located at Yq was used to determine the rate and extent of Y chromosome microdeletions. PCR with primers flanking the AR-(CAG)n region and subsequent size fractionation on gradient acrylamide gels were used to determine AR-(CAG)n length. Five azoospermic individuals (5/61-8.2% and 5/46-10.8% of azoospermic patients) displayed Y chromosome microdeletions. The mean CAG repeat number in infertile men was 18.6 +/- 3.0 compared with 16.6 + 2.7 in fertile men (n = 50), a statistically significant difference (p = 0.003). Y chromosome microdeletions contribute to male infertility in our azoospermic population, and the mean length of the AR-CAG is significantly longer in our infertile population than in fertile men.

The critical and expanding role of genetics in assisted reproduction.

With the progress of the human gene mapping initiative, it is expected that the entire genome will be mapped within two years. A significant use for these data will centre on testing for genetic disease. Professionals associated with assisted reproduction are presented with a very special subset of the population, namely, couples suffering from infertility. Infertility may occur in the male, the female or both partners and may be heritable. Infertility, subfertility or recurrent spontaneous miscarriage is associated with chromosomal or genetic anomalies, suggesting that basic developmental genetics should be a part of the education of the physician or clinical embryologist. A review of the most common infertility-associated chromosomal and genetic diseases for which genetic testing has become routine in infertile parents and in the products of assisted reproduction through preimplantation genetic diagnosis (PGD) and prenatal testing follows. Less common genetic diseases that have compromising effects on reproduction and which are likely to be encountered by providers of assisted reproduction are also considered.

The Y chromosome and its role in testis differentiation and spermatogenesis.

The Y chromosome contains genes and gene families that play critical roles in the processes of testis determination and testis differentiation. Great strides have been made toward defining the genetic pathways associated with the determination of gender. The data are summarized and discussed and clinical ramifications are considered.

Defining regions of the Y-chromosome responsible for male infertility and identification of a fourth AZF region (AZFd) by Y-chromosome microdeletion detection.

Cytogenetic and molecular deletion analyses of azoospermic and oligozoospermic males have suggested the existence of AZoospermia Factor(s) (AZF) residing in deletion intervals 5 and 6 of the human Y-chromosome and coinciding with three functional regions associated with spermatogenic failure. Nonpolymorphic microdeletions in AZF are associated with a broad spectrum of testicular phenotypes. Unfortunately, Sequence Tagged Sites (STSs) employed in screening protocols range broadly in number and mapsite and may be polymorphic. To thoroughly analyze the AZF region(s) and any correlations that may be drawn between genotype and phenotype, we describe the design of nine multiplex PCR reactions derived from analysis of 136 loci. Each multiplex contains 4-8 STS primer pairs, amplifying a total of 48 Y-linked STSs. Each multiplex consists of one positive control: either SMCX or MIC2. We screened four populations of males with these STSs. Population I consisted of 278 patients diagnosed as having significant male factor infertility: either azoospermia, severe oligozoospermia associated with hypogonadism and spermatogenic arrest or normal sperm counts associated with abnormal sperm morphology. Population II consisted of 200 unselected infertile patients. Population III consisted of 36 patients who had previously been shown to have aneuploidy, cytological deletions or translocations involving the Y-chromosome or normal karyotypes associated with severe phenotype abnormalities. Population IV consisted of 920 fertile (control) males. The deletion rates in populations I, II and III were 20.5%, 7% and 58.3%, respectively. A total of 92 patients with deletions were detected. The deletion rate in population IV was 0.87% involving 8 fertile individuals and 4 STSs which were avoided in multiplex panel construction. The ability of the nine multiplexes to detect pathology associated microdeletions is equal to or greater than screening protocols used in other studies. Furthermore, the data suggest a fourth AZF region between AZFb and AZFc, which we have termed AZFd. Patients with microdeletions restricted to AZFd may present with mild oligozoospermia or even normal sperm counts associated with abnormal sperm morphology. Though a definitive genotype/phenotype correlation does not exist, large deletions spanning multiple AZF regions or microdeletions restricted to AZFa usually result in patients with Sertoli Cell Only (SCO) or severe oligozoospermia, whereas microdeletions restricted to AZFb or AZFc can result in patients with phenotypes which range from SCO to moderate oligozoospermia. The panel of nine multiplexed reactions, the Y-deletion Detection System (YDDS), provides a fast, efficient and accurate method of assessing the integrity of the Y-chromosome. To date, this study provides the most extensive screening of a proven fertile male population in tandem with 514 infertile males, derived from three different patient selection protocols.

Panel One: marketing strategies and informing the patient/consumer. Infertility diagnostic techniques: the rush to market.

The human genome project will result in many new diagnostic techniques applicable to assisted reproduction. These may be directed both at infertile patients as well as at patients who seek ART because they carry a deleterious genetic disease. There are many potential advantages to the use of preprocedural testing as a means of obtaining additional preliminary information that might allow patients to make a more informed choice, either when deciding between ART and adoption or when comparing the benefits of various ART procedures. The cost of testing is relatively low when compared with the cost of additional IVF or ICSI cycles. In the case of genetic screening protocols, the results may be useful not only to the patient but also to the next generation. Genetic counseling should be made available in concert with expanded opportunities, both for diagnosing infertility and for making preimplantation genetic diagnoses. Already some of the genetic screening that has occurred because of discoveries made during the human genome initiative has become a regulatory concern with regard to insurance availability and other issues. When counseling patients, it is important to point out that certain tests hold far more prognostic value and/or serious health implications than others. A test to diagnose breast cancer susceptibility, for example, holds more serious ramifications than the less predictive test that detects the phenotype for Klinefelter's syndrome. Presymptomatic prognostic screening systems, such as the tests currently used to detect genetic mutations related to breast and prostate cancer and cystic fibrosis, provide diagnostic clues but also have serious societal implications. We are on a rather slippery slope when we attempt to determine which tests actually might prove beneficial to patients, both individually and collectively. Therefore, as part of patient counseling, cost-benefit ratios, in addition to clinical and preventive implications, should be weighed.

Microdeletions in the Y chromosome of infertile men.

BACKGROUND: Some infertile men with azoospermia or severe oligospermia have small deletions in regions of the Y chromosome. However, the frequency of such microdeletions among men with infertility in general is unknown. We sought to determine the prevalence of Y-chromosome microdeletions among infertile men and to correlate the clinical presentation of the men with specific deletions. METHODS: We studied 200 consecutive infertile men. Each man was evaluated comprehensively for known causes of infertility, and Y-chromosome microdeletions were studied with use of the polymerase chain reaction to amplify specific regions of the chromosome. The Y chromosomes of 200 normal men were also analyzed. RESULTS: Fourteen infertile men (7 percent) and four normal men (2 percent) had microdeletions of the Y chromosome. Nine of the infertile men had azoospermia or severe oligospermia (sperm concentration, <5 million per milliliter), four had oligospermia (sperm concentration, 5 million to <20 million per milliliter), and one had normospermia (sperm concentration, > or = 20 million per milliliter). The size and location of the deletions varied and did not correlate with the severity of spermatogenic failure. The fathers of six infertile men with microdeletions were studied; two had the same deletions as their sons, and four had no deletions. CONCLUSIONS: A small proportion of men with infertility have Y-chromosome microdeletions, but the size and position of the deletions correlate poorly with the severity of spermatogenic failure, and a deletion does not preclude the presence of viable sperm and possible conception.

The incidence and possible relevance of Y-linked microdeletions in babies born after intracytoplasmic sperm injection and their infertile fathers.

Microdeletions linked to deletion intervals 5 and 6 of the Y chromosome have been associated with male factor infertility. Members from at least two gene families lie in the region containing azoospermia factor (AZF), namely YRRM and DAZ. With the advent of intracytoplasmic sperm injection (ICSI), it is possible for men with severe male factor infertility to produce a child. The genetic consequences of such a procedure have been questioned. This report describes the first study of a population (32 couples) of infertile fathers and their sons born after ICSI. The objectives were firstly to determine the incidence and map location of Y chromosome microdeletions and to compare the frequencies with other population studies involving severe male factor infertility, and secondly to formulate a working hypothesis concerning developmental aetiology of Y chromosome microdeletions. The incidence of microdeletions in the ICSI population was shown to be 9.4% (within the range 9-18% reported for populations of severe male factor infertility patients). Microdeletions in two out of three affected father/son pairs mapped in the region between AZFb and AZFc and the third involved a large microdeletion in AZFb and AZFc. Of three affected father/son pairs, microdeletions were detected in the blood of one infertile propositus father and three babies. Assuming that the gonomes of the ICSI-derived babies are direct reflections of those of their fathers germ lines, it is possible that two of three infertile fathers were mosaic for intact Y and microdeleted Y chromosomes. In such cases, the developmental aetiology of the microdeletion may be due to a de-novo microdeletion arising as a post-zygotic mitotic error in the infertile propositus father, thus producing a mosaic individual who may or may not transmit the deletion to his ICSI-derived sons depending on the extent of primordial germ cell mosaicism. In one of three affected fathers, the microdeletion detected in his blood was also detected in his ICSI-derived son. In this case the de-novo event giving rise to the microdeletion may have occurred due to a post- (or pre-) meiotic error in the germ line of this father's normally fertile father (i.e. the ICSI-derived baby's grandfather).

Systems for production of calves from cultured bovine embryonic cells.

The development of totipotent bovine embryonic cell cultures has great value in cattle breeding. They provide: (1) a mechanism for making large numbers of clonal offspring by nuclear transfer; (2) an efficient gene transfer system through the use of selectable markers to select transgenic cells; and (3) a mechanism for site-specific gene transfer or deletion by homologous DNA sequence recombination. Bovine embryonic cell cultures have been established from blastocyst inner cell mass (ICM) cells, morulae and the precompaction 16-20-cell stage. All have exhibited similar morphology to mouse embryonic stem (ES) cells, pluripotency on differentiation and proliferation in culture. Culture systems have consisted of microdrop loose suspension short-term cultures or long-term cultures on bovine or murine fibroblast feeder layers, in either a microdrop or a culture dish. The relative merit of culture systems or media requirements for mitosis and prevention of differentiation have not been determined. At present, totipotency is also unknown for cultured cells of the 16-20-cell stage. For cultured ICM cells, totipotency was demonstrated by the birth of four calves from ICM cells cultured 27 days or less in a loose suspension microdrop. Advanced pluripotency and perhaps totipotency was demonstrated in one fetus in a recently reported study where morulae cells cultured in vitro were chimaerized with non-cultured cells. DNA fingerprinting to associate cell lines with offspring and karyotyping to ascertain chromatin normalcy is important in ES cell research. Data pertaining to the use of each are presented.