Accuracy of preimplantation genetic screening (PGS) is compromised by degree of mosaicism of human embryos.

BACKGROUND: To preclude transfer of aneuploid embryos, current preimplantation genetic screening (PGS) usually involves one trophectoderm biopsy at blastocyst stage, assumed to represent embryo ploidy. Whether one such biopsy can correctly assess embryo ploidy has recently, however, been questioned. METHODS: This descriptive study investigated accuracy of PGS in two ways. Part I: Two infertile couples donated 11 embryos, previously diagnosed as aneuploid and, therefore, destined to be discarded. They were dissected into 37 anonymized specimens, and sent to another national laboratory for repeat analyses to assess (i) inter-laboratory congruity and (ii) intra-embryo congruity of multiple embryo biopsies in a single laboratory. Part II: Reports on human IVF cycle outcomes after transfer of allegedly aneuploid embryos into 8 infertile patients. RESULTS: Only 2/11 (18.2 %) embryos were identically assessed at two PGS laboratories; 4/11 (36.4 %), on repeat analysis were chromosomally normal, 2 mosaic normal/abnormal, and 5/11 (45.5 %) completely differed in reported aneuploidies. In intra-embryo analyses, 5/10 (50 %) differed between biopsy sites. Eight transfers of previously reported aneuploid embryos resulted in 5 chromosomally normal pregnancies, 4 delivered and 1 ongoing. Three patients did not conceive, though 1 among them experienced a chemical pregnancy. CONCLUSIONS: Though populations of both study parts are too small to draw statistically adequately powered conclusions on specific degrees of inaccuracy of PGS, here presented results do raise concerns especially about false-positive diagnoses. While inter-laboratory variations may at least partially be explained by different diagnostic platforms utilized, they cannot explain observed intra-embryo variations, suggesting more frequent trophectoderm mosiaicsm than previously reported. Together with recentl published mouse studies of lineages-specific degrees of survival of aneuploid cells in early stage embryos, these results call into question the biological basis of PGS, based on the assumption that a single trophectoderm biopsy can reliably determine embryo ploidy.

The status of public reporting of clinical outcomes in assisted reproductive technology.

OBJECTIVE: To assess the transparency of assisted reproductive technology (ART) surveillance reports published by the Centers for Disease Control and Prevention (CDC) and by the Society for Assisted Reproductive Technologies (SART). DESIGN: Retrospective analysis. SETTING: Private clinical ART and research center. PATIENT(S): We analyzed ART data for the years 2005-2010, which were reported under federal mandate to the CDC (818,927 completed cycles) and voluntarily to SART (812,400 initiated cycles). INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Initiated cycles excluded from final outcome reporting were used to evaluate transparency. RESULT(S): Only SART, but not CDC, reported initiated cycles, allowing analysis of excluded cycles. Excluded cycles increased significantly from 3.3% to 7.4% between 2005 and 2010. By 2010, 13/341 (3.8%) ART centers accounted for 50% of excluded cycles, representing an average of 37.3% of their cycles. These 13 clinics reported significantly better pregnancy and cancellations rates than national averages and collectively increased by 19.9% their share of U.S. ART cycles. CONCLUSION(S): Our data indicate decreasing transparency in public ART reporting in the United States, likely due to changes in practice and reporting patterns. A few clinics accounted for the majority of excluded cycles, leading to improved reported clinical outcomes and increasing market share. CDC and SART should ensure that all ART clinics publicly report the outcomes of all initiated cycles including embryo-banking cycles. ART surveillance and quality of care may be improved by prospectively tracking the total reproductive potential of each initiated cycle.

Successful treatment of unresponsive thin endometrium.

OBJECTIVE: To assess whether inadequate, thin endometrium (<7 mm), after failure to expand with standard treatment options, will be responsive to cytokine treatment. DESIGN: Prospective cohort study of four patients. SETTING: Two independent IVF centers in New York City. PATIENT(S): Four consecutive women undergoing IVF who, after standard endometrial preparation, still demonstrated highly inadequate endometrium. INTERVENTION(S): Transvaginal endometrial perfusion with granulocyte colony-stimulating factor (G-CSF). MAIN OUTCOME MEASURE(S): Endometrial thickness on day of ET, with pregnancy as secondary endpoint. RESULT(S): We report successful endometrial expansion to at least minimal thickness of 7 mm after uterine perfusion with G-CSF in four patients previously resistant to treatment with estrogen and vasodilators. All four patients therefore reached ET, and all four also conceived, although one pregnancy required termination because of intramural, corneal ectopic location. Endometrial expansion to minimal thickness occurred within approximately 48 hours from infusion. CONCLUSION(S): Uterine perfusion with G-CSF represents a promising new tool for the currently mostly intractable problem of inadequate, thin endometrium. This treatment also deserves further investigation for its potential to improve implantation chances in association with IVF and, therefore, pregnancy rates.

Human embryo twinning with applications in reproductive medicine.

OBJECTIVE: To assess the efficacy of human embryo twinning by blastomere biopsy at different early embryonic stages (splitting efficiency) and to determine the in vitro developmental capacity of twinned human embryos (developmental efficiency). DESIGN: Randomized comparative study. SETTING: Private IVF centers. PATIENT(S): Couples undergoing IVF donating triploid embryos. INTERVENTION(S): Embryos at the 2- to 5- and 6- to 8-cell stage were split into twin embryos. Half the number of blastomeres from donor embryos were removed and inserted into recipient empty zonae pellucidae. After embryo splitting, donor and recipient embryos were cultured in vitro. MAIN OUTCOME MEASURE(S): Development of twinned embryos to the blastocyst stage. RESULT(S): The number of developing embryos obtained after splitting could be increased in comparison with the number of embryos available before splitting at the 6- to 8-cell stage but not at the 2- to 5-cell stage (splitting efficiency). Splitting of 6- to 8-cell embryos yielded superior rates of twin embryos developing to blastocysts (developmental efficiency). Twinning success was related to the superior morphological quality of embryos used for splitting. CONCLUSION(S): This is the first report on twinned human embryos developing to blastocysts. This study exhibits the potential for novel applications in human assisted reproduction.

Efficacy of native and hyperglycosylated follicle-stimulating hormone analogs for promoting fertility in female mice.

OBJECTIVE: To compare the efficacy of recombinant human FSH (rhFSH) with rhFSH with four additional O-linked carbohydrates (rhFSH-CTP), rhFSH with four additional N-linked carbohydrates (rhFSH-N4), and the current gold standard for rodent ovarian stimulation, pregnant mare serum gonadotropin (PMSG), on fertility parameters in mice. DESIGN: Animal study. SETTING: Academic research center. ANIMAL(S): Adult C57Bl/6J female mice. INTERVENTION(S): Ovarian stimulation with 5 IU of rhFSH, rhFSH-CTP, rhFSH-N4, or PMSG. Forty-six hours later, 5 IU of hCG was injected to promote ovulation and females were mated overnight. MAIN OUTCOME MEASURE(S): Eggs retrieved after ovulation, in vitro embryo development, delivery rate, and litter size. RESULT(S): The hyperglycosylated FSH analogs, rhFSH-CTP and rhFSH-N4, enhanced ovulation and embryo maturation significantly better than rhFSH. RhFSH-N4 produced more eggs (28.5 +/- 1.9 per mouse) and embryos (17.8 +/- 1.6) compared with rhFSH-CTP (18.3 +/- 1.2 and 9.0 +/- 1.0, respectively). Treatment with rhFSH, rhFSH-N4, and PMSG produced statistically equivalent delivery rates and litter sizes. The delivery rate was surprisingly lower with rhFSH-CTP (14%) compared with PMSG (33%). CONCLUSION(S): Compared with rhFSH, treatment with hyperglycosylated rhFSH-CTP and rhFSH-N4 led to superior rates of ovulated eggs and subsequent in vitro embryo development. RhFSH-N4 was equivalent to PMSG, while all of the fertility parameters studied were lower with rhFSH-CTP than with PMSG therapy.

Improved multiple displacement amplification with phi29 DNA polymerase for genotyping of single human cells.

The ability to genotype multiple loci of single cells would be of significant benefit to investigations of cellular processes such as oncogenesis, meiosis, fertilization, and embryogenesis. We report a simple two-step, single-tube protocol for whole-genome amplification (WGA) from single human cells using components of the GenomiPhi V2 DNA Amplification kit. For the first time, we demonstrate reliable generation of 4-7 microg amplified DNA from a single human cell within 4 h with a minimum amount of artifactual DNA synthesis. DNA amplified from single cells was genotyped for 13 heterozygous short tandem repeats (STRs) and 7 heterozygous single nucleotide polymorphisms (SNPs), and the genotyping results were compared with purified genomic DNA. Accuracy of genotyping (percent of single-cell amplifications genotyped accurately for any particular STR or SNP) varied from 37% to 100% (with an average of 80%) for STRs and from 89% to 100% (averaging 94%) for SNPs. We suggest that the method described in this report is suitable for WGA from single cells, the product of which can be subsequently used for many applications, such as preimplantation genetic analysis (PGD).

Bye-bye urinary gonadotrophins? Recombinant FSH: a real progress in ovulation induction and IVF?

Whether recombinant gonadotrophin products do, indeed, represent progress for routine ovulation induction and IVF cycles, in comparison with urinary products, has remained controversial. Here we review published data with regard to respective risks, outcomes and cost for both medication options. Safety considerations favour recombinant products, while overall outcome and cost considerations favour urinary gonadotrophins. Outcome, however, appears to differ, based on age and ovarian function, with younger patients benefiting from the FSH/LH combination offered by urinary products, while older women and young women with ovarian resistance, apparently benefiting from pure FSH stimulation. Young women with poor ovarian reserve may be best stimulated with a pure FSH/antagonist protocol. We conclude that under current pricing structures in the United States, recombinant gonadotrophins do not represent a major progress for the treatments of ovulation induction and IVF. They, however, allow for an improved selectivity of stimulation protocols. The creation of recombinant FSH/LH products and cost adjustments for recombinant products, may affect these conclusions in favour of recombinant products.

The immunological "Wars of the Roses": disagreements amongst reproductive immunologists.

The relevance of abnormal autoimmune function to reproductive function in the female has over recent years become an increasingly controversial and contentious issue. Opposing views have led to a polarization of opinions which, at times, resulted in publications of rather vocal opinions by individuals as well as societal committees. This communication is an attempt to reconcile these, at times diametrically opposing opinions, in a concept of (auto)immune-driven reproduction failure, which could explain and unify these opposing opinions and, thus, hopefully end the ongoing "immunological wars of the roses".

An Update on Laparoscopic Hysterectomy and Pelvic Floor Reconstruction.

Laparoscopic hysterectomy and pelvic floor reconstruction have recently undergone some degree of evolution. New instrumentation has appeared, such as the vaginal delineator, but overall most of the instruments used have remained basically unchanged. This is related mostly to the need of keeping costs down by limiting the use of disposable instrumentation. For pelvic reconstruction, the laparoscopic approach is now usually considered the optimal approach, as it allows the surgeon to visualize structures that the vaginal surgeon could in the past only palpate.