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Objective To determine whether in vitro fertilization (IVF) with preimplantation genetic testing for aneuploidy (PGT-A) is cost effective to achieve a live birth compared with IVF alone in fresh donor oocyte cycles. Design Theoretical cost-effectiveness study. Setting Not applicable. Patient(s) None. Intervention(s) Comparison between the cost of IVF with PGT-A vs. IVF alone to achieve a live birth. The model analyzed a hypothetical single fresh oocyte donor IVF cycle with PGT-A vs. IVF alone and followed the progression of a single embryo through the different decision nodes. Cost estimates assigned to each clinical event were based on data obtained from the literature and institutional costs. Main Outcome Measure(s) Cost per live birth. Result(s) In the base-case analysis, IVF with PGT-A was not cost effective in fresh donor oocyte cycles when compared with IVF alone to achieve a live birth. The cycles using PGT-A cost an additional $6,018.66. The incremental cost-effectiveness ratio was found to be $119,606.59 per additional live birth achieved with IVF with PGT-A. Monte Carlo simulations demonstrated that IVF with PGT-A was not cost effective in nearly all iterations. Conclusion(s) PGT-A in fresh donor oocyte IVF cycles is not cost effective compared with IVF alone over a wide range of probabilities and costs.

PURPOSE: The goal is to determine if variations exist between male and female blastocysts in preimplantation measurements of quality and ploidy and in vitro fertilization elective single-embryo transfer (eSET) outcomes. METHODS: A retrospective chart review was conducted from a private fertility center’s database of blastocysts undergoing preimplantation genetic testing for aneuploidy, along with details of eSET from this screened cohort. Main outcomes included preimplantation embryo quality and sex-specific eSET outcomes. RESULTS: A total of 3708 embryos from 578 women were evaluated, with 45.9% male and 54.1% female. The majority were High grade. No difference existed between embryo sex and overall morphological grade, inner cell mass or trophectoderm grade, or blastocyst transformation day. Female blastocysts had a higher aneuploidy rate than male blastocysts (P < 0.001). Five hundred thirty-nine eSETs from 392 women were evaluated, with High grade embryos more likely to have implantation (P < 0.001), clinical pregnancy (P < 0.001), and ongoing pregnancy (P = 0.018) than Mid or Low grade embryos. Day 5 blastocysts were more likely to have implantation (P = 0.018), clinical pregnancy (P = 0.005), and ongoing pregnancy (P = 0.018) than day 6 blastocysts. Male and female embryos had similar transfer outcomes, although female day 5 blastocysts were more likely to result in clinical pregnancy (P = 0.012), but not ongoing pregnancy, than female day 6 blastocysts. Male eSET outcomes did not differ by blastocyst transformation day. CONCLUSION: Male and female embryos have comparable grade and quality; however, female embryos were more likely to be aneuploid. Ongoing pregnancy rates did not differ by embryo sex. Day 5 embryos had more favorable transfer outcomes than day 6 embryos.

PURPOSE: The aim was to study the association between embryonal mitochondrial DNA (mtDNA) content and embryo quality and implantation outcomes. METHODS: A retrospective chart review was performed with data collected from a private IVF center database. The study population included female infertility patients with ages ranging from 31 to 38 years old, and the main outcome measures were embryo quality and transfer outcomes. RESULTS: From a total of 1510 blastocyst biopsies, the majority of embryos consisted of grade 1 (High), followed by grade 2 (mid), and grade 3 (poor). Embryos with higher mtDNA content were found to be of poorer quality (grade 3) relative to grades 1 and 2 (P = 0.003). Using a logistic model, mtDNA best predicted lowest and highest grades, but not mid-grade embryos. There was no correlation between mtDNA content and the subjects’ age (R(2) = 0.0018). In an analysis of only euploid embryos (N = 717), there was no longer an association between mtDNA content and embryo quality (P = 0.834). There was no difference in mtDNA content between groups of embryos that did and did not implant (P = 0.53). There was also no association noted between mtDNA content and ongoing pregnancy. Compared to day 6, day 5 blastocysts contain significantly higher amounts of mtDNA (P = 0.0005), lower rates of aneuploidy (P

There are large variations in the number of oocytes within each woman, and biologically, the total quantity is at its maximum before the woman is born. Scientific knowledge is limited about factors controlling the oocyte pool and how to measure it. Within fertility clinics, there is no uniform agreement on the diagnostic criteria for each common measure of ovarian reserve in women, and thus, studies often conflict. While declining oocyte quantity/quality is a normal physiologic occurrence as women age, some women experience diminished ovarian reserve (DOR) much earlier than usual and become prematurely infertile. Key clinical features of DOR are the presence of regular menstrual periods and abnormal-but-not-postmenopausal ovarian reserve test results. A common clinical challenge is counseling patients with conflicting ovarian reserve test results. The clinical diagnosis of DOR and the interpretation of ovarian reserve testing are complicated by changing lab testing options and processing for anti-mullerian hormone since 2010. Further, complicating the diagnostic and research scenario is the existence of other distinct yet related clinical terms, specifically premature ovarian failure, primary ovarian insufficiency, poor ovarian response, and functional ovarian reserve. The similarities and differences between the definitions of DOR with each of these four terms are reviewed. We recommend greater medical community involvement in terminology decisions, and the addition of DOR-specific medical subject-heading search terms.

Conferences serve an essential means of learning and staying up to date in all aspects of medicine. Reproductive endocrinology and infertility is a young and constantly evolving field. The Midwest Reproductive Symposium International (MRSi) is a yearly conference held in Chicago, IL, and is one of the most intimate yet influential conferences in the fertility world. This conference is geared towards all professions and roles in the fertility world such as physicians, geneticists, nurses, allied health professionals, basic scientists, mental health professionals, business administration professionals, reproductive endocrinology and infertility fellows, and obstetrics and gynecology residents alike. The goal of MRSi is to continue to understand this revolutionary field in order to improve patient outcomes while staying up to date with the latest technology.

PURPOSE: Twelve percent of women in the USA will develop invasive breast cancer in their lifetime, and that risk increases to 80% if they carry a BRCA1 or BRCA2 mutation. BRCA1/2 mutations are thought to potentially affect ovarian reserve and/or fertility. METHODS: PubMed and PubMed Central were searched for publications on ovarian reserve–related outcomes (i.e., AMH and response to controlled ovarian hyperstimulation (COH) protocols) that were reported in relation to BRCA1 and/or BRCA2 mutations from 1950 through May 2019. A meta-analysis was conducted to create forest plots and summary effect measures using Review Manager 5.3. RESULTS: This article reviews the 16 qualifying publications. There were several fundamental methodological differences in the study designs and outcome details reported in AMH studies. Summary statistics found no difference in AMH levels between BRCA1/2+ women as compared with controls (Z overall test effects p ≥ 0.45). Regarding responses to COH, there were overall non-significantly fewer total and mature numbers of oocytes retrieved in BRCA1/2+ cases as compared with controls (meta-analysis Z overall test effects p ≥ 0.40). CONCLUSIONS: While the summary measures indicate no significant differences in AMH levels between BRCA1/2+ cases and controls, readers should be aware that there are significant methodological differences in the AMH reports. Additionally, the response to COH protocols does not seem to be significantly lower in BRCA1/2 mutation carriers in the existing literature. Continued research on both of these clinical parameters would be beneficial for patient counseling.

“Mystery, Medicine, and the Magnificent Mile,” the theme for the annual Midwest Reproductive Symposium International (MRSi) in Chicago, IL, captured the attention of reproductive professionals all over the world. Each year, the conference agenda encompasses emerging technologies in assisted reproduction, updates in the management of reproductive diseases, and common challenges encountered in clinical practice. The structure of the meeting, offering a mixture of lectures, panel discussions, and interactive workshops, creates a collaborative environment for physicians, geneticists, embryologists, nurses, mental health professionals, basic scientists, business administrative professionals, reproductive endocrinology and infertility fellows, and obstetrics and gynecology residents. The goal of the MRSi meeting is to provide all reproductive professionals the opportunity to exchange ideas, foster relationships, and deliver quality patient care. As the field continues to evolve, MRSi provides an exciting venue to uncover the mysteries of reproductive medicine with enthusiasm and collaboration.

The purpose of the review was to define the various diagnostic platforms currently available to perform preimplantation genetic testing for aneuploidy and describe in a clear and balanced manner the various strengths and weaknesses of these technologies.A systematic literature review was conducted. We used the terms "preimplantation genetic testing," "preimplantation genetic diagnosis," "preimplantation genetic screening," "preimplantation genetic diagnosis for aneuploidy," "PGD," "PGS," and "PGD-A" to search through PubMed, ScienceDirect, and Google Scholar from the year 2000 to April 2016. Bibliographies of articles were also searched for relevant studies. When possible, larger randomized controlled trials were used. However, for some emerging data, only data from meeting abstracts were available.PGS is emerging as one of the most valuable tools to enhance pregnancy success with assisted reproductive technologies. While all of the current diagnostic platforms currently available have various advantages and disadvantages, some platforms, such as next-generation sequencing (NGS), are capable of evaluating far more data points than has been previously possible. The emerging complexity of different technologies, especially with the utilization of more sophisticated tools such as NGS, requires an understanding by clinicians in order to request the best test for their patients..Ultimately, the choice of which diagnostic platform is utilized should be individualized to the needs of both the clinic and the patient. Such a decision must incorporate the risk tolerance of both the patient and provider, fiscal considerations, and other factors such as the ability to counsel patients on their testing results and how these may or may not impact clinical outcomes.

FMR1 CGG trinucleotide repeat expansions are associated with Fragile X syndrome (full mutations) and primary ovarian insufficiency (premutation range); the effect of FMR1 on the success of fertility treatment is unclear. The effect of FMR1 CGG repeat lengths on IVF outcomes after ovarian stimulation was reviewed. PubMed was searched for studies on IVF-related outcomes reported by FMR1 trinucleotide repeat length published between 2002 and December 2017. For women with CGG repeats in the normal (

To compare single nucleotide polymorphism (SNP) and comparative genomic hybridization (aCGH) microarray platforms to evaluate embryos for parental translocation imbalances and aneuploidy.A retrospective review of preimplantation genetic diagnosis and screening (PGD/PGS) results of 498 embryos from 63 couples undergoing 75 in vitro fertilization cycles due to parental carriers of a reciprocal translocation.There was no significant difference between SNP and aCGH microarrays when comparing the prevalence of embryos that were euploid with no translocation imbalance, euploidy with a parental translocation imbalance or aneuploid with or without the parental chromosome imbalance. The clinical pregnancy rates were also equivalent for SNP (60 %) versus aCGH (65 %) microarrays. Of 498 diagnosed embryos, 45 % (226/498) were chromosomally normal without translocation errors or aneuploidy, 22 % (112/498) were euploid but had a parentally derived unbalanced chromosomal segregant, 8 % (42/498) harbored both a translocation imbalance and aneuploidy and 24 % (118/498) of embryos were genetically balanced for the parental reciprocal translocation but were aneuploid for other chromosomes. The overall clinical pregnancy rate per IVF cycle following SNP or aCGH microarray analysis was 61 % and was higher if the biopsy was done on blastocysts (65 %) versus cleavage stage embryos (59 %), although not statistically significant.SNP or aCGH microarray technologies demonstrate equivalent clinical findings that maximize the pregnancy potential in patients with known parental reciprocal chromosomal translocations.

As medicine has evolved over the last century, medical genetics has grown from nonexistence to one of the most visible aspects of how we understand and treat disease. This increased role of genetics within medicine will only increase in the coming years, and its role in reproductive medicine will be significant. Genetics has emerged as a primary focus of research with translational applications within reproductive medicine. The aim of this article is to outline the applications of genetics currently available, and how these technologies can provide a positive impact on patient care.

We successfully performed preimplantation genetic diagnosis (PGD) and simultaneous preimplantation genetic screening (PGS) using single nucleotide polymorphism (SNP) microarrays for couples with balanced chromosome rearrangements in China. A total of 428 molecular karyotypes were diagnosed from 62 couples undergoing 68 in vitro fertilization (IVF) cycles. Of these, 48.1% of the embryos were chromosomally normal without translocation errors or aneuploidy. Of the 428 total embryos, 18.0% embryos were euploid, but were imbalanced due to the transmission of single translocation chromosome derivatives. A total of 6.5% of the embryos had chromosome abnormalities involving the parental chromosome aberration and other chromosomes aneuploidies. Significantly, 27.4% of the embryos were normal/balanced for the rearranged chromosomes, but were abnormal due to aneuploidy affecting other chromosomes. When evaluated on a per IVF cycle basis, 84% of the cycles had at least one chromosomally normal embryo available for uterine transfer. The clinical pregnancy rate per IVF cycle was 54%. Diagnosing genomically balanced embryos through 24 chromosome SNP microarray PGD/PGS, rather than minimally targeted fluorescence in situ hybridization (FISH), is a promising strategy to maximize the pregnancy potential of patients with known parental chromosomal translocations. Moreover, this is the first study to report the clinical application of SNP arrays to screen all 24 chromosome pairs of blastomeres and trophectoderm cells from patients carrying reciprocal translocations in China.

The application of genetic technologies to the field of reproductive medicine has ushered in a new era of medicine that is likely to greatly expand in the coming years. Concurrent with an in vitro fertilization (IVF) cycle, it is now possible to obtain a cellular biopsy from a developing embryo and genetically evaluate this sample with increasing sophistication and detail. Information obtained from this analyzed sample may then guide which embryos are selected for uterine transfer. Applications for this technology are currently being employed to avoid the propagation of certain genetically based diseases and to increase the efficiency of assisted reproductive technologies in general. The way such technologies are implemented, however, has caused a continuous and ever evolving debate among reproductive medicine physicians and geneticists. This article will define the current uses of preimplantation genetic testing and discuss the benefits and limitations of this technology.

Unusual and consistent defects in infertility patients merit attention as these may indicate an underlying genetic abnormality, in turn necessitating tailored management strategies. We describe a case of repeated early pregnancy loss from in vivo conceptions, followed by cancelled embryo transfers after one IVF and one ICSI/PGD cycle. Following the unexpected presence of cleaved embryos at the ferti- lization check in the first IVF attempt, oocytes and embryos were subsequently analyzed in an ICSI/PGD case. Part of the oocyte cohort was fixed at retrieval for a cellular evaluation of microtubules, microfilaments and chromatin. The remaining oocytes were injected with sperm, and resultant embryos were biopsied for genetic analysis by fluorescence in situ hybridization (FISH), single-nucleotide polymorphism (SNP) microarray for 23 chromosome pairs, as well as with PCR for sex chromosomes. The presence of interphase microtubule networks and pronuclear structures indicated that oocytes were spontaneously activated by the time of retrieval. FISH revealed aneuploidy in all seven blastomeres analyzed, with all but two lacking Y chromosomes. Microarray SNP analysis showed an exclusively maternal origin of all blas- tomeres analyzed, which was further confirmed by PCR. From our multi-faceted analyses, we conclude that spontaneous activation, or parthenogenesis, was probably the pathology underlying our patient's recurrent inability to maintain a normal pregnancy. Such analyses may prove beneficial not only in diagnosing case-specific aberrations for other patients with similar or related failures, but also for furthering our general understanding of oocyte activation.

Objective To obtain embryonic molecular karyotypes from genomic DNA (deoxyribonucleic acid) isolated from blastocoel fluid (BF) and to compare these karyotypes with the karyotypes from the remaining inner cell mass (ICM) and trophectoderm (TE) of the blastocyst. Design Prospective cohort study. Setting Academic center and preimplantation genetics laboratory. Patient(s) Ninety-six donated cryopreserved embryos. Intervention(s) Embryo biopsy, BF aspiration, DNA analysis using a comparative genomic hybridization microarray (aCGH). Main Outcome Measure(s) The aCGH of a single blastomere, BF-DNA, and ICM-TE. Result(s) The BF-DNA samples resulted in a successful aCGH in 63% of cases. Discordance in karyotypes was found between the BF-DNA and the ICM-TE in 52% of cases. A total of 70% of aneusomic (mosaicism), cleavage-stage embryos differentiated into euploid blastocysts. Probabilities for diagnostic accuracy were calculated and demonstrated the following: sensitivity of 0.88 (95% confidence interval [CI]: 0.62–0.98); specificity of 0.55 (95% CI: 0.39–0.70); positive predictive value of 0.41 (95% CI: 0.25–0.60); negative predictive value of 0.92 (95% CI: 0.75–0.99). Conclusion(s) Genomic DNA from the BF can be amplified and characterized by comprehensive chromosome microarrays. The results demonstrated that aneusomic cleavage-stage embryos differentiated into euploid blastocysts, possibly using a mechanism that marginalizes aneuploid nuclei into the blastocoel cavity. In addition, owing to the high discordance between the karyotypes obtained from the BF-DNA and the ICM-TE, using BF-DNA for preimplantation genetic testing is not yet advised.

The adeno-associated virus 2 (AAV)-based vector system has been suggested for its potential use in human gene therapy because the wild-type (wt) AAV genome appears to integrate into the human chromosomal DNA in a site-specific manner. We systematically investigated the integration patterns of the recombinant AAV genomes lacking one or both the viral coding sequences. Four recombinant AAV genomes were constructed containing the genes for resistance to tetracycline (TcR) and the herpesvirus thymidine kinase (TK) promoter-driven gene for resistance to neomycin (neoR; vTc.Neo), the genes for resistance to ampicillin (ApR) and TK-neoR (vAp.Neo), the genes for AAV replication (rep) genes and TK-neoR (vRep.Neo), and the AAV capsid (cap) genes and TK-neoR (vCap.Neo). The integration pattern of each of the recombinant AAV genomes in individual clonal isolates of the human nasopharyngeal carcinoma cell line (KB) analyzed on Southern blots using a neo-specific DNA probe was distinctly different. In addition, in none of the clones examined was the proviral genome covalently linked to the previously described AAV right-junction (Rt.Jn.) human chromosomal DNA fragment, the putative specific-site of integration for the wt AAV genome. Furthermore, whereas a 276-bp DNA fragment could be readily amplified from each of these clones, using a neo-specific primer-pair by polymerase chain reaction (PCR), no amplified DNA product was obtained using the neo- and the Rt.Jn. primer-pair under identical conditions. Fluorescence in situ hybridization (FISH) analyses further revealed the lack of integration of the recombinant AAV into human chromosome 19, even in the presence of a functional rep gene as determined by rescue of the recombinant AAV genome in the presence of adenovirus. These data suggest that the recombinant AAV genomes integrate at sites that are different from that characterized for the wt AAV genome. These studies may have implications in the development of the AAV-based vector system for its potential use in human gene therapy.

Since the first successful in vitro fertilization (IVF) cycle, there have been tremendous advancements in the technologies surrounding IVF. Specific genetic disorders such as parental chromosomal translocations or inversions and single gene mutations such as cystic fibrosis or fragile X, as well as abnormalities in the number of chromosomes, or aneuploidy, are major barriers to achieving a healthy live born infant. Preimplantation genetic diagnosis (PGD) for specific chromosomal or genetic disorders and preimplantation genetic screening (PGS) for determination of the number of chromosomes, are techniques that have been developed to prevent the uterine transfer of genetically abnormal embryos during IVF cycles. The purpose of preimplantation genetic testing (PGT), that is PGD or PGS, is to improve the likelihood of giving birth to a healthy baby.

Preimplantation Genetic Screening (PGS) has been used for some time with fluorescence in situ hybridization (FISH) technology with marginal success. Newer modalities capable of evaluating all 23 pairs of chromosomes are now available for PGS and are superior to PGS with FISH.

Early embryogenesis involves a series of dynamic processes, many of which are currently not well described or understood. Aneuploidy and aneuploid mosaicism, a mixture of aneuploid and euploid cells within one embryo, in early embryonic development are principal causes of developmental failure.^1,2^ Here we show that human embryos demonstrate a significant rate of genetic correction of aneuploidy, or "genetic normalization" when cultured from the cleavage stage on day 3 (Cleavage) to the blastocyst stage on day 5 (Blastocyst) using routine in vitro fertilization (IVF) laboratory conditions. One hundred and twenty-six human Cleavage stage embryos were evaluated for clinically indicated preimplantation genetic screening (PGS). Sixty-four of these embryos were found to be aneuploid following Cleavage stage embryo biopsy and single nucleotide polymorphism (SNP) 23 chromosome molecular karyotype (microarray). Of these, 25 survived to the Blastocyst stage of development and repeat microarray evaluation was performed. The inner cell mass (ICM), containing cells destined to form the fetus, and the trophectoderm (TE), containing cells destined to form the placenta were evaluated. Sixteen of 25 embryos (64%) [95% CI: 44-80%] possessed diploid karyotypes in both the ICM and TE cell populations. An additional three Blastocyst stage embryos showed genetic correction of the TE but not the ICM and one Blastocyst stage embryo showed the reverse. Mosaicism (exceeding 5%), was not detected in any of the ICM and TE samples analyzed. Recognizing that genetic normalization may occur in developing human embryos has important implications for stem cell biology, preimplantation and developmental genetics, embryology, and reproductive medicine.1)Hassold, T. et al. A cytogenetic study of 1000 spontaneous abortions. Ann Hum Genet. 44, 151-78 (1980).2)Menasha, J., Levy, B., Hirschhorn, K., & Kardon, N.B. Incidence and spectrum of chromosome abnormalities in spontaneous abortions: new insights from a 12-year study. Genet Med. 7, 251-63 (2005).

Background: The diagnosis of a chromosomally abnormal third trimester fetus prior to birth permits optimal obstetrical management and psychological support for the parents when the child is born. Although ultrasound findings may lead to suspicion of Edward syndrome, confirmation of the diagnosis depends on the cytogenetic detection of trisomy 18. Certain obstetrical situations may limit the choice of diagnostic procedures to amniocentesis when a rapid cytogenetic result in required. Case: Fluorescence in situ hybridization in uncultured amniocytes permitted rapid detection of trisomy 18 in one twin following preterm labor, allowing a definitive diagnosis before birth. Conclusion: The technique described enables a rapid, targeted cytogenetic result using uncultured amniocytes. This is a valuable new tool when a specific fetal aneuploidy is strongly suggested by sonography and obstetrical factors preclude the use of procedures other than amniocentesis.

Objective: To determine aneuploidy frequencies in sperm from a patient with normal phenotype and 46,XY/45,X mosaicism in somatic cells (peripheral lymphocytes). Design: Case report. Setting: Infertility clinic and genetics laboratory. Patient: A 30-year-old male with primary infertility and moderate oligoasthenoteratozoospermia. Intervention(s): Cytogenetic analysis of somatic cells and determination by fluorescence in situ hybridization of aneuploidy frequencies for the gonosomes (sex chromosomes) and chromosome 18 in sperm from whole and Percoll-separated semen. Main Outcome Measure(s): Somatic and gametic aneuploidy were scored. Result(s): Analysis of lymphocyte metaphase cells showed a mosaic 46,XY (90%)/ 45,X (10%) karyotype. Significantly higher frequencies of gonosomal (semen, 1.92% versus 0.70%; Percoll, 1.12% versus 0.46%), and chromosome 18 (semen, 0.89% versus 0.28%; Percoll, 0.26% versus 0.10%) disomy were detected in the sperm of the patient compared with those observed in spermatozoa from a proved fertile control. Conclusion(s): Significantly higher frequencies of aneuploid sperm suggest that the patient is at elevated risk of producing offspring with numerical chromosome abnormalities.

rate, 68%, was significantly (p

OBJECTIVE: The endoplasmic reticulum (ER) stress is activated in response to an accumulation of unfolded protein in the lumen of the ER. ER stress is critical for cell survival, but chronic or excess ER stress can lead to apoptosis in various cell types. ER stress is known to cause various aging related diseases such as diabetics and neurodegenerative diseases. In this study, the participation of the ER stress in the postovulatory aging of oocyte was examined. DESIGN: Animal model study. MATERIALS AND METHODS: In this study, mouse oocytes released from the oviduct at 12.5 hour and 18.5 hour post-hCG were designated as ‘‘fresh’’ and ‘‘aged’’ oocytes, respectively. Experiments were done as follows: (1) Analysis of expression of BiP (chaperone induced at ER stress) level by western blotting with embryo development in the fresh oocytes, (2) Comparison of expression of BiP at protein level in the fresh and the aged oocytes, (3) Analysis of the effects by ER stress inducer, thapsigargin (Tg: calcium pump inhibitor of ER) on BiP expression and on fertilization rate and embryo development after in vitro fertilization. Animals were treated in accordance with the NIH Guide for the Care and Use of Laboratory Animals, as approved by the Animal Care and Use Committee of Yamagata University. RESULTS: (1) Expression of BiP was the highest in the MII oocytes, and decreased with embryo development after in vitro fertilization. (2) Expression of BiP in the aged oocytes was increased compared with that in the fresh oocytes. (3) Tg treated oocytes showed increase in expression of BiP, low fertilization rate and deteriorated embryo development. Furthermore, Tg treatment in the fresh oocytes deteriorated embryo quality. CONCLUSION: Expression of BiP in mouse oocyte/embryo decreased with the embryo development. The result that excessive expression of BiP in the aged oocyte was observed in association with the poor embryo development suggested that aged oocytes were exposed to excess ER stress. Because excess ER stress induced by Tg leaded low fertilization rate and poor embryo development, these data suggested that ER stress might participate in postovulatory aging process of the mouse oocyte. Supported by: This study was Supported by JSPS KAKEN research grants 20591905 to H.I.

We have determined the origin of the extra chromosomal material in the karyotypes of two spontaneously-occurring long-lived human keratinocyte lines, HKC-N2 and HKC-N6. In each case the extra material was derived from the chromosome on which it was located. Possible relationships between the triplication of chromosomal material and the overcoming of cell senescence are discussed.

Adeno-associated virus type 2 (AAV) is a nonpathogenic parvovirus that generally requires coinfection with a helper virus, such as an adenovirus (Ad) or herpesvirus, to undergo productive infection (3, 4, 7). In cultured cells infected with wild-type (wt) AAV in the absence of helper virus, AAV establishes a latent infection (5, 11, 22) and often integrates site specifically into a sequence located on the q arm of human chromosome 19, termed the AAVS1 site (19, 20, 29–31, 35). AAV proviral DNA remains in this latent state until rescued by a helper virus. Studies on the mode of viral integration suggest that tandem copies of the viral DNA are inserted into the host cell chromosome in a head-to-tail orientation via the inverted terminal repeats of the virus (28). Due to its lack of pathogenicity and its ability to establish persistent infections in human cells, AAV has gained acceptance as a potential vector for human gene therapy (18). Recombinant AAV (rAAV) vectors mediate stable in vivo expression in a wide range of different tissues including the lungs (16), muscles (12, 13, 26, 40), brain (2, 24, 27, 39), spinal cord (34), retinas (14, 32, 43), and liver (36). The use of AAV vectors has not been associated with significant toxicity in animal models. In addition, two phase I trials of recombinant AAV vectors have been undertaken in patients with cystic fibrosis (15, 38). Despite the growing body of data regarding the biology of AAV latency in vitro, very few studies have examined this phenomenon in vivo. Epidemiologic studies have shown a high prevalence of AAV2 seropositivity (6, 8, 9). However, it is not known whether seropositivity is indicative of previous productive infection or of latent infection. Although infectious wt AAV has been cultured from samples from the respiratory and gastrointestinal tract in association with productive helper virus infection (9), latent AAV DNA has been found only in peripheral blood mononuclear cells (PBMCs) (21). Since humans and monkeys are the only species known to possess the AAVS1 integration sequence (35), they are the only two in vivo models which would be expected to faithfully reflect the wt AAV latency pathway. One previous study examined the interactions between rAAV vectors, wt-AAV and Ad in a rhesus macaque model and demonstrated that the productive phase of the wt AAV life cycle could be reproduced in that model (1). In this study, we have examined latent wt AAV infection in that same rhesus model. Additional studies investigated the cell specificity of persistence, the integration state of latent viral genomes, and the immune responses to viral infection. In this setting, persistence was observed, although site-specific integration was infrequent. wt AAV genomes were detectable both at the site of administration and in circulating PBMCs. Neutralizing antibodies directed against wt AAV were observed in serum 21 days postinfection in animals infected intramuscularly or intravenously with wt AAV alone, but cell-mediated immune responses were elicited only in the presence of helper Ad.

Recent evidence suggests that infertile males donating semen for intracytoplasmic sperm injection (ICSI) may be at an increased risk of transmitting numerical (predominantly sex chromosome) abnormalities to their offspring. The present study was designed to determine aneuploidy in spermatozoa from oligoasthenoteratozoospermic (OAT) patients undergoing ICSI. Aneuploidy frequencies of 12 autosomes and the sex chromosomes were determined by fluorescence in-situ hybridization (FISH) on spermatozoa from fresh ejaculate of nine severe OAT patients and four proven fertile donors. FISH, using directly labelled (fluorochrome-dUTP) satellite or contig DNA probes specific for chromosomes 4, 6, 7, 8, 9, 10, 11, 12, 13, 17, 18, 21, X, and Y, was performed on decondensed spermatozoa. Per chromosome disomy frequencies for autosomes and sex chomosomes in OAT males were 0-5. 4%. In contrast, the disomy frequencies in controls were 0.05-0.2%. The frequency of diploid spermatozoa in OAT patients was 0.4-9.6%; controls showed a mean of 0.04%. Using recently developed formulae, the total aneuploidy in our OAT patient population was estimated to be 33-74%. In contrast, estimates of mean total aneuploidy in the spermatozoa of controls ranged from 4.1 to 7.7%, depending upon method of calculation. Six series of ICSI were performed on five of the OAT patients. Four resulted in no establishment of pregnancy; the others failed to establish ongoing pregnancies. Our cytogenetic data show significantly elevated frequencies of diploidy, autosomal disomy and nullisomy, sex chromosome aneuploidy, and total aneuploidy in OAT patients, which may contribute to the patients' infertility.

of chromosomal aneuploidy and single gene disorders (SGD) via PGD. This was done via multiplex PCR combined with array CGH (aCGH). DESIGN: Clinical research study. MATERIALS AND METHODS: A total of 47 IVF cycles were included in this study involving 44 couples and 25 different single gene disorders (HLA matching, autosomal dominant, recessive and X-linked), with mean maternal age of 32.9. Patient specific PCR protocols were optimized for simultaneous detection of mutations and polymorphic sites. PGD was performed by blastocyst biopsy, modified whole genome amplification, followed by analysis with aCGH for aneuploidy and by PCR for the SGD. Only euploid and unaffected/carrier embryos were considered for transfer. To confirm the diagnosis, 25 abnormal embryos were re-biopsied and reanalyzed using separate aCGH/molecular tests. RESULTS: From the 329 blastocyst samples tested in this clinical study, full results were obtained for both SGD and aCGH for 302 embryos (91.8%). Only 2 samples gave no results for both SGD and aCGH (0.61%). For the SGD 324/329 (98.5%) samples gave results, whereas 305/329 (92.7%) samples gave aCGH results. Amongst the embryos with full results, 57 (18.9%) were either normal or carriers of a SGD and euploid. If only SGD results were considered, 150 (49.7%) of the samples tested would have been suitable for transfer, but 93 (62%) would have been in fact aneuploid. Reanalysis of aCGH and SGD revealed a 0% error rate. Preliminary data shows improved implantation compared to PGD cases without aCGH. Among patients who previously had SGD PGD only, several have miscarried with aneuploid fetuses. CONCLUSION: The results show high rates of chromosome abnormalities among embryos that otherwise are unaffected by the SGD. This highlights the limitations of replacing embryos based solely on SGD PGD. The process of PGD is long and traumatic for patients. This may allow them to minimize the risk of miscarriage due to chromosome abnormalities.

Gene therapy vectors based on human DNA viruses could be mobilized or rescued from individuals who are subsequently infected with the corresponding wild-type (wt) helper viruses. This phenomenon has been effectively modeled in vitro with both adenovirus (Ad) and adeno-associated virus (AAV) vectors but has not previously been studied in vivo. In the current study, we have developed an in vivo model to study the interactions of a recombinant AAV vector (AAV-CFTR) with wt AAV type 2 (AAV2) and a host range mutant Ad (Ad2HR405) for which monkey cells are permissive (D.E.Brough, S.A.Rice, S.Sell, and D.F.Klessig, J. Virol. 55:206-212, 1985). AAV-CFTR was administered to the respiratory epithelium of the nose or lung of rhesus macaques. Primary cells were harvested from the infusion site at time points up to 3 months after vector administration to confirm vector DNA persistence. Vector DNA was present in episomal form and could be rescued in vitro only by addition of wt AAV2 and Ad. In in vivo rescue studies, vector was administered before or after wt-AAV2 and Ad2HR405 infection, and the shedding of AAV-CFTR was examined. Ad2HR405 and wt-AAV2 infections were established in the nose with concomitant administration. wt-AAV2 replication occurred in the lung when virus was administered directly at a high titer to the lower respiratory tract. AAV-CFTR vector rescue was also observed in the latter setting. Although these studies were performed with small numbers of animals within each group, it appears that AAV-CFTR DNA persists in the primate respiratory tract and that this model may be useful for studies of recombinant AAV vector rescue.

Summary points Preimplantation genetic (PG) testing is the practice of obtaining a cellular biopsy sample from a developing human oocyte or embryo, acquired via a cycle of in vitro fertilisation (IVF); evaluating the genetic composition of this sample; and using this information to determine which embryos will be optimal for subsequent uterine transfer. PG testing was first described in 1990 when the sex of cleavage stage embryos in two couples, both with X linked diseases, was determined.1 The applications and use of various types of PG testing have continued to increase. However, the benefits and limitations of PG testing, both in popular culture and the medical community, are often misunderstood. This article reviews the technologies available in PG testing, and discusses the risks, ethical considerations, appropriateness, and controversy surrounding its use in different clinical situations. #### Sources and selection criteria The information sources used to compile this …