In vitro fertilization with preimplantation genetic screening.

BACKGROUND: Pregnancy rates in women of advanced maternal age undergoing in vitro fertilization (IVF) are disappointingly low. It has been suggested that the use of preimplantation genetic screening of cleavage-stage embryos for aneuploidies may improve the effectiveness of IVF in these women. METHODS: We conducted a multicenter, randomized, double-blind, controlled trial comparing three cycles of IVF with and without preimplantation genetic screening in women 35 through 41 years of age. The primary outcome measure was ongoing pregnancy at 12 weeks of gestation. The secondary outcome measures were biochemical pregnancy, clinical pregnancy, miscarriage, and live birth. RESULTS: Four hundred eight women (206 assigned to preimplantation genetic screening and 202 assigned to the control group) underwent 836 cycles of IVF (434 cycles with and 402 cycles without preimplantation genetic screening). The ongoing-pregnancy rate was significantly lower in the women assigned to preimplantation genetic screening (52 of 206 women [25%]) than in those not assigned to preimplantation genetic screening (74 of 202 women [37%]; rate ratio, 0.69; 95% confidence interval [CI], 0.51 to 0.93). The women assigned to preimplantation genetic screening also had a significantly lower live-birth rate (49 of 206 women [24%] vs. 71 of 202 women [35%]; rate ratio, 0.68; 95% CI, 0.50 to 0.92). CONCLUSIONS: Preimplantation genetic screening did not increase but instead significantly reduced the rates of ongoing pregnancies and live births after IVF in women of advanced maternal age. (Current Controlled Trials number, ISRCTN76355836 [controlled-trials.com].).

Polymorphism for a 1.6-Mb deletion of the human Y chromosome persists through balance between recurrent mutation and haploid selection.

Many human Y-chromosomal deletions are thought to severely impair reproductive fitness, which precludes their transmission to the next generation and thus ensures their rarity in the population. Here we report a 1.6-Mb deletion that persists over generations and is sufficiently common to be considered a polymorphism. We hypothesized that this deletion might affect spermatogenesis because it removes almost half of the Y chromosome's AZFc region, a gene-rich segment that is critical for sperm production. An association study established that this deletion, called gr/gr, is a significant risk factor for spermatogenic failure. The gr/gr deletion has far lower penetrance with respect to spermatogenic failure than previously characterized Y-chromosomal deletions; it is often transmitted from father to son. By studying the distribution of gr/gr-deleted chromosomes across the branches of the Y chromosome's genealogical tree, we determined that this deletion arose independently at least 14 times in human history. We suggest that the existence of this deletion as a polymorphism reflects a balance between haploid selection, which culls gr/gr-deleted Y chromosomes from the population, and homologous recombination, which continues to generate new gr/gr deletions.

Partial DAZ deletions in a family with five infertile brothers.

OBJECTIVE: To study the genetic cause of infertility in a family with five infertile brothers. DESIGN: Case report. SETTINGS: Center for reproductive medicine at a university medical center. PATIENT(S): Five brothers presenting with primary infertility due to severely impaired spermatogenesis; also, their parents and two other paternally related family members. INTERVENTION(S): Fluorescence in situ hybridization and sequence family variant analysis was performed in leukocyte DNA to determine the number of deleted in azoospermia (DAZ) genes. Linkage analysis was performed for X chromosome inheritance, and mitochondrial DNA (mtDNA) was screened for mutations. MAIN OUTCOME MEASURE(S): DAZ gene copy number, X chromosome linkage, and mtDNA sequence. RESULT(S): With conventional polymerase chain reaction (PCR) analysis, no deletions of the AZFc region were found, but with fluorescence in situ hybridization and sequence family variant analysis, only two DAZ genes instead of four were detected in all individuals tested. The five brothers did not share an identical X chromosomal locus, and no mutations were found in the mtDNA of the index patient. CONCLUSION(S): A reduced copy number of the DAZ genes is found in five infertile brothers with severely impaired spermatogenesis, as well as in their normospermic father and in two other fertile paternally related family members. This illustrates that the phenotype associated with a reduced copy number of the DAZ genes can be extremely variable.

The use of spermHALO-FISH to determine DAZ gene copy number.

The AZFc region of the human Y chromosome is frequently deleted in men with spermatogenic failure and contains many multicopy genes. The best-characterized gene family within this region is the Deleted in AZoospermia (DAZ) gene family, which is present in four nearly identical copies. Recent reports claim deletions of some but not all DAZ genes. The assays used in these studies, however, are unable to provide conclusive evidence on the number of DAZ genes. In this study we show that with the use of highly decondensed sperm nuclei with large DNA domains (spermHALO) it is possible to determine the number of DAZ genes accurately. Using this fluorescent in-situ hybridization (FISH) technique, which has both high resolution and high range, we show that in 10 normospermic men, in which PCR digest assays indicated a deletion of one or more DAZ genes, all four DAZ genes were present. Also we confirmed previous findings of a deletion of two DAZ genes in two men and identified a man with six DAZ genes. Our results indicate that spermHALO-FISH allows an accurate determination of DAZ gene copy number, while PCR digest assays do not. Therefore, confirmation of positive results from PCR digest assays with spermHALO-FISH is essential. Furthermore, the spermHALO-FISH technique should prove useful as a genetic mapping technique in other regions of the Y chromosome and similar repetitive regions throughout the genome.

Clinical relevance of partial AZFc deletions.

To determine the number of DAZ gene clusters in the Y-bearing spermatozoa of patients who underwent intracytoplasmic sperm injection (ICSI) and to compare the outcome with the number of clusters found in the spermatozoa of normospermic men. Prospective study. Academic hospital.Forty-seven patients with impaired spermatogenesis who were attending our clinic for ICSI and 56 semen donors. Peripheral blood was drawn to obtain somatic DNA for polymerase chain reaction (PCR) analysis and leukocytes for karyotyping and FISH analysis. Three-color FISH was performed on the spermatozoa remaining after ICSI and on the spermatozoa of semen donors to determine the presence of the X and Y chromosome as well as the number of DAZ gene clusters. Number of DAZ gene clusters in Y-bearing spermatozoa. Five patients had only one DAZ gene cluster, one patient had a complete AZFc deletion, and one patient had three clusters on average. One of the semen donors also showed three DAZ gene clusters in his Y-bearing spermatozoa. None of the semen donors had only one DAZ gene cluster. Besides complete AZFc deletions, partial deletions are also associated with impaired spermatogenesis. As a result, these partial deletions that are not recognized by routine PCR are reintroduced into the population by the ICSI technique.

Use of the total motile sperm count to predict total fertilization failure in in vitro fertilization.

OBJECTIVE: To evaluate the capacity of baseline characteristics and total motile sperm count (TMC) to predict total fertilization failure (TFF) in patients undergoing IVF. DESIGN: Retrospective cohort study. SETTING: University hospital. PATIENT(S): Eight hundred ninety-two couples with a total of 1,569 consecutive IVF cycles. INTERVENTION(S): Prewash and postwash TMC during fertility workup and at the time of ovum pickup (OPU). MAIN OUTCOME MEASURE(S): Analysis of logistic regression and the receiver operating characteristic curve were used to determine which variables could be used to predict TFF. RESULT(S): The area under the curve (AUC) for prewash TMC during fertility workup was 0.72, similar to a combination of pre- and postwash TMC. At the time of OPU, both pre- and postwash TMC had an AUC of 0.73. A model based on selected baseline characteristics (male age, number of IVF cycles, indication for IVF, and prewash TMC during fertility workup) had an AUC of 0.75. A model at the time of OPU, including the number of oocytes, had an AUC of 0.80. CONCLUSION(S): The use of both models, one before start of the IVF cycle and one at the time of OPU, allows an accurate prediction of the chance of TFF and is useful in counseling patients on whether to opt for IVF or ICSI.

Reduced copy number of DAZ genes in subfertile and infertile men.

OBJECTIVE(S): To determine the copy number and identity of the DAZ genes on the Y chromosomes of infertile patients. DESIGN: Prospective study. SETTING: University medical center. PATIENT(S): One hundred and thirty-nine patients with male factor infertility. INTERVENTION(S): The separate genes were detected by polymerase chain reaction (PCR) digestion assays of sequence family variants in leukocyte DNA and by fluorescence in situ hybridization of interphase nuclei and chromatin fibers. MAIN OUTCOME MEASURE(S): Number of DAZ genes present. RESULT(S): One hundred twenty-nine patients had four genes, 6 patients had two genes, and 4 patients had none. Three patients had a deletion of the two proximal DAZ genes, and three were missing both distal genes. Semen analysis showed a less severe phenotype in patients with only two DAZ genes compared with patients missing all four genes. CONCLUSION(S): In six patients, two different partial deletions were found that were not detected by PCR with conventional markers. One patient with an AZFb deletion appeared to also have a partial AZFc deletion that was not detected by routine PCR. Phenotypic differences between patients with different deletions suggest a dose effect of the DAZ genes.