Susan Gitlin

The origin, mechanisms, incidence and clinical consequences of chromosomal mosaicism in humans

BACKGROUND Chromosomal mosaicism, the presence of two or more distinct cell lines, is prevalent throughout human pre- and post-implantation development and can lead to genetic abnormalities, miscarriages, stillbirths or live births. Due to the prevalence and significance of mosaicism in the human species, it is important to understand the origins, mechanisms and incidence of mosaicism throughout development. METHODS Literature searches were conducted utilizing Pubmed, with emphasis on human pre- and post-implantation mosaicism. RESULTS Mosaicism persists in two separate forms: general and confined. General mosaicism is routine during human embryonic growth as detected by preimplantation genetic screening at either the cleavage or blastocyst stage, leading to mosaicism within both the placenta and fetus proper. Confined mosaicism has been reported in the brain, gonads and placenta, amongst other places. Mosaicism is derived from a variety of mechanisms including chromosome non-disjunction, anaphase lagging or endoreplication. Anaphase lagging has been implicated as the main process by which mosaicism arises in the preimplantation embryo. Furthermore, mosaicism can be caused by any one of numerous factors from paternal, maternal or exogenous factors such as culture media or possibly controlled ovarian hyperstimulation during in vitro fertilization (IVF). Mosaicism has been reported in as high as 70 and 90% of cleavage- and blastocyst-stage embryos derived from IVF, respectively. CONCLUSIONS The clinical consequences of mosaicism depend on which chromosome is involved, and when and where an error occurs. Mitotic rescue of a meiotic error or a very early mitotic error will typically lead to general mosaicism while a mitotic error at a specific cell lineage point typically leads to confined mosaicism. The clinical consequences of mosaicism are dependent on numerous aspects, with the consequences being unique for each event.

Preimplantation genetic diagnosis for Tay-Sachs disease: successful pregnancy after pre-embryo biopsy and gene amplification by polymerase chain reaction

OBJECTIVE To determine the ability to apply preimplantation genetic diagnostic techniques to screen for and prevent Tay-Sachs disease (TSD). DESIGN A couple, both carriers for the 4 base pair (bp) insertion in exon 11 of the beta-hexosaminidase A gene, which results in TSD, underwent IVF, pre-embryo biopsy, polymerase chain reaction (PCR) DNA amplification of the biopsied blastomeres, and pre-embryo transfer. One to two blastomeres were aspirated using a biopsy pipette that was inserted through an opening in the zona formed with acidified phosphate buffer. Polymerase chain reaction was performed on the individual blastomeres for 20 cycles followed by an additional 30 cycles using nested primers. This yielded amplified DNA products of 272 and 276 bp for the normal and mutant gene, respectively. Heteroduplex formation was used for identification of normal, homozygous affected, and heterozygous pre-embryos. RESULTS Seven of 13 oocytes fertilized normally and were biopsied at the four- to eight-cell stages. Deoxyribonucleic acid amplification occurred in four of seven pre-embryos (one homozygous affected and three homozygous normal pre-embryos). The three normal pre-embryos that continued to cleave after biopsy were transferred on the evening of day 3 after retrieval. Subsequently, a single gestational sac was observed and the genetic diagnosis was confirmed at amniocentesis. CONCLUSION A successful pregnancy and birth were accomplished after preimplantation genetic diagnostic screening for the prevention of TSD.

Globozoospermia is mainly due to DPY19L2 deletion via non-allelic homologous recombination involving two recombination hotspots

To date, mutations in two genes, SPATA16 and DPY19L2, have been identified as responsible for a severe teratozoospermia, namely globozoospermia. The two initial descriptions of the DPY19L2 deletion lead to a very different rate of occurrence of this mutation among globospermic patients. In order to better estimate the contribution of DPY19L2 in globozoospermia, we screened a larger cohort including 64 globozoospermic patients. Twenty of the new patients were homozygous for the DPY19L2 deletion, and 7 were compound heterozygous for both this deletion and a point mutation. We also identified four additional mutated patients. The final mutation load in our cohort is 66.7% (36 out of 54). Out of 36 mutated patients, 69.4% are homozygous deleted, 19.4% heterozygous composite and 11.1% showed a homozygous point mutation. The mechanism underlying the deletion is a non-allelic homologous recombination (NAHR) between the flanking low-copy repeats. Here, we characterized a total of nine breakpoints for the DPY19L2 NAHR-driven deletion that clustered in two recombination hotspots, both containing direct repeat elements (AluSq2 in hotspot 1, THE1B in hotspot 2). Globozoospermia can be considered as a new genomic disorder. This study confirms that DPY19L2 is the major gene responsible for globozoospermia and enlarges the spectrum of possible mutations in the gene. This is a major finding and should contribute to the development of an efficient molecular diagnosis strategy for globozoospermia.

Assisted oocyte activation overcomes fertilization failure in globozoospermic patients regardless of the DPY19L2 status

STUDY QUESTION Does DPY19L2 status influence intracytoplasmic sperm injection (ICSI) outcomes with or without assisted oocyte activation (AOA)? SUMMARY ANSWER DPY19L2 mutations have no major impact on ICSI outcomes in globozoospermic patients. WHAT IS KNOWN ALREADY Globozoospermia is a rare and severe teratozoospermia characterized by round-headed spermatozoa lacking an acrosome. Recently, it has been shown that DPY19L2 mutations can be found in a vast majority of, but not all, globozoospermic patients (66.7%). These patients suffer from primary infertility due to a sperm-related oocyte activation deficiency secondary to the absence of an acrosome that can be overcome by the application of AOA. STUDY DESIGN, SIZE, DURATION Cohort study, retrospective, 34 patients, 83 cycles. MATERIALS, SETTING, METHODS Clinical and biologic data were collected from 29 patients mutated for DPY19L2 and 5 non-mutated patients. In total, 35 ICSI cycles using AOA and 48 conventional ICSI cycles were included in the analysis. Patients were divided into groups according to whether or not they were mutated for DPY19L2 and whether or not they received AOA. MAIN RESULTS AND THE ROLE OF CHANCE Regardless of the presence of a DPY19L2 mutation, the fertilization rates with AOA are restored to normal when compared with conventional ICSI in our cohort of globozoospermic patients. Also, when performing ICSI plus AOA, both mutated and non-mutated cases have similar positive hCG rates, ongoing pregnancy rates and live birth rates per transfer. On the contrary, the fertilization rate in globozoospermic patients using conventional ICSI is correlated with the presence of a DPY19L2 mutation, with slightly better, although still very low, fertilization rates in patients carrying a DPY19L2 mutation. Nevertheless, when performing conventional ICSI, both mutated and non-mutated cases have similar very low positive hCG rates, ongoing pregnancy rates and live birth rates per transfer. LIMITATIONS A limitation of this study is the low number of included non-mutated cases. WIDER IMPLICATIONS OF THE FINDINGS We propose a pathway for the clinical management of globozoospermic patients depending on the phenotype that includes several diagnostic and therapeutic steps. STUDY FUNDING/COMPETING INTEREST(S) None.

Polymerase chain reaction amplification specificity: Incidence of allele dropout using different DNA preparation methods for heterozygous single cells

AbstractPurpose: The purpose was to evaluate methods of DNA preparation in a single cell to determine the ability to amplify and correctly diagnose a targeted gene. Methods: One- or two-cell lymphoblasts (n=100/group), heterozygous for the normal and 4-base pair insertion on exon 11 of the β-hexosaminidase A gene, were collected and prepared under the following conditions: (1) freeze-thaw liquid nitrogen, then boiling (LN2); (2) potassium hydroxide/dithiothreitol, heated to 65°C, followed by acid neutralization (KOH); (3) boiling only (BI); and (4) water only (H2O). Cells were analyzed by polymerase chain reaction using nested primers. Results: The total number of cells amplifying [in brackets] and the cells with amplification for both alleles (heterozygous), the normal allele, or the mutant allele were as follows, respectively: LN2 [38], 11, 16, 11; KOH [97], 91, 5, 1; BI [41], 17, 13, 11; and H2O [85], 41, 16, 28. With two cells per reaction tube the results were as follows: LN2 [85], 53, 14, 18; and KOH [97], 96, 1, 0. Conclusions: KOH lysis was significantly greater than with all other methods (P<0.006) and should be used for single cells. This study also demonstrates the importance of using heterozygous cells to determine the ability to amplify both alleles as a method of quality control for single-cell analysis.

Oocyte biology and genetics revelations from polar bodies

The enormous volume of the fertilized egg is attributable to the suppression of cleavage during oocyte growth and the unequal cleavages during the first and second meiotic divisions. The two products of these divisions are the diminutive polar bodies (PB), which contain a redundant set of chromosomes/chromatids plus cytoplasmic organelles. The PB have strictly limited but differential life spans; while viable they possess the genetic potential to support normal embryonic development after transfer to a cytoplast. In addition to the theoretical possibility of using this non-cloning technique to generate more embryos, polar bodies can be used for genetic testing. By cytogenetic analysis of both PB using fluorescent in-situ hybridization (FISH) or chromosome painting, partial or full chromosomal status in the oocyte can be predicted; this approach finds particular application for women of advanced reproductive age as well as with maternally inherited translocations and single gene defects. By studying both of the PB, potential problems of interpretation arising from allele dropout can be reduced; a heterozygous first polar body provides the least ambiguous result. Mitochondria segregate randomly during meiotic cleavages providing an opportunity also to use the PB to screen for mitochondrial mutations and deletions. Thus, the PB can serve useful diagnostic purposes, especially where pre-fertilization screening or avoidance of embryo biopsy is desirable.

Strategies to respond to polymerase chain reaction deoxyribonucleic acid amplification failure in a preimplantation genetic diagnosis program

OBJECTIVES Our purpose was to identify and evaluate practical methods within a preimplantation genetic diagnosis program that will increase the percentage of embryos for which a genetic diagnosis can be obtained, including clinical responses after failure of deoxyribonucleic acid amplification has occurred. STUDY DESIGN Known human lymphoblast cell lines and human embryo blastomeres were evaluated in a single-cell, nested primer polymerase chain reaction system with primer sequences for the specific locus surrounding the four base pair insertion mutation on exon 11 of beta-hexosaminidase A-Tay-Sachs disease, the delta F508 mutation of cystic fibrosis, and the sex-determining region on the Y chromosome. Reamplification polymerase chain reaction with standard polymerase chain reaction and primer extension preamplification was performed in deoxyribonucleic acid preparations after previous polymerase chain reaction amplification attempts had resulted in failure of amplification. RESULTS The amplification efficiency of Tay-Sachs disease, 51% (97/187), was significantly lower than that for cystic fibrosis, 85% (87/107), and for the sex-determining region on the Y chromosome, 85% (77/90). Tay-Sachs disease polymerase chain reaction amplification occurred in 51% of one-cell lymphoblasts, 89% of two-cell lymphoblasts, and 94% of samples when more than two cells were processed together. When previous amplification failure had occurred, standard Tay-Sachs disease polymerase chain reaction resulted in an amplification efficiency of 16% (three of 19), whereas primer extension preamplification polymerase chain reaction for Tay-Sachs disease resulted in amplification of 52% (31/59) lymphoblasts and 54% (13/24) of polyspermic human blastomeres. Four of six human blastomeres in which amplification failure occurred in a Tay-Sachs disease preimplantation genetic diagnosis cycle amplified by primer extension preamplification polymerase chain reaction, which increased the diagnostic information obtained from four to six of the seven embryos on which biopsy was performed. CONCLUSIONS We suggest that practical approaches for consideration within a clinical preimplantation genetic diagnosis program to limit the net effect of amplification failure (i.e., reduced embryo transfer number) include increasing the deoxyribonucleic acid content in the polymerase chain reaction tube by using more than one blastomere and by using primer extension preamplification when the initial attempt at amplification fails.

Outcomes of blastocysts biopsied and vitrified once versus those cryopreserved twice for euploid blastocyst transfer

Trophectoderm biopsy with comprehensive chromosome screening (CCS) has been shown to increase implantation and pregnancy rates. Some patients desire CCS on previously cryopreserved blastocysts, resulting in blastocysts that are thawed/warmed, biopsied, vitrified and then warmed again. The effect of two cryopreservation procedures and two thawing/warming procedures on outcomes has not been effectively studied. Cycles were divided into two groups: group 1 patients underwent a cryopreserved embryo transfer with euploid blastocysts that were vitrified and warmed once; group 2 patients had a cryopreserved embryo transfer of a euploid blastocyst that was cryopreserved, thawed/warmed, biopsied, vitrified and warmed. Groups 1 and 2 included 85 and 17 women aged 35.6 ± 3.9 and 35.3 ± 4.9 years, respectively (not significantly different). Blastocyst survival in group 1 (114/116, 98.3%) and survival of second warming in group 2 (21/24, 87.5%) was significantly different (P = 0.0354). There was no difference between biochemical (68.2% and 62.5%) and clinical (61.2% and 56.3%) pregnancy rates, implantation rate (58.4% and 52.4%) and live birth/ongoing pregnancy rate (54.0% and 47.6%) between groups 1 and 2, respectively. Although it is unconventional to thaw/warm, biopsy, revitrify and rewarm blastocysts for cryopreserved embryo transfer, the results indicate that outcomes are not compromised. Trophectoderm biopsy and screening the embryos for chromosomal abnormalities has been reported to increase implantation and pregnancy rates. There is a category of patients requesting chromosomal screening on previously cryopreserved blastocysts. This scenario requires blastocysts to be thawed/warmed, biopsied, cryopreserved, and thawed/warmed again. The effect of double cryopreservation procedures and double thawing/warming procedures on pregnancy is unknown. Patients were divided into two groups, group 1 underwent a cryopreserved embryo transfer with a chromosomally normal blastocyst that was vitrified and warmed once and group 2 included patients that had a cryopreserved embryo transfer of a chromosomally normal blastocyst that was cryopreserved, thawed/warmed, biopsied, vitrified, and rewarmed. A total of 85 and 17 women aged 35.6 ± 3.9 and 35.3 ± 4.9 years were included in groups 1 and 2, respectively. The survival rate for group 1 (114 of 116, 98.3%) compared with the second warming for group 2 (21 of 24, 87.5%) was significantly higher. There was no difference between biochemical (68.2% and 62.5%), and clinical pregnancies (61.2% and 56.3%), implantation (58.4% and 52.4%), and live birth/ongoing rates (54.0% and 47.6%) between groups 1 and 2. Although it is unconventional to twice cryopreserve and twice thaw/warm a blastocyst, our results indicate that outcomes are not compromised.

Comparison of aneuploidy, pregnancy and live birth rates between day 5 and day 6 blastocysts

Comprehensive chromosome screening is typically used for aneuploidy analysis of blastocysts. It is believed that either day of blastocyst development is acceptable. Euploidy rates and outcomes were examined between day 5 and day 6 blastocysts in two studies. First, euploidy rates of day 5 and day 6 blastocysts were examined on a per-embryo and per-patient basis. Second, outcomes were compared when only euploid day 5 or day 6 blastocysts were transferred in a cryopreserved embryo transfer cycle. In cycles (n = 70) that had blastocysts biopsied on both day 5 and day 6, day 5 blastocysts had a higher chance of being euploid than day 6 blastocysts (125/229 [54.6%]) and (77/180 [42.8%]), respectively (P = 0.0231). Similarly, euploid rates in blastocysts from patients (n = 193) with day 5 biopsy, day 6 biopsy, or both, were significantly higher in day 5 (235/421 [55.8%]) compared with day 6 (184/413 [44.6%]) blastocysts (P = 0.0014). In the second study, 50 women (36.1 ± 4.3 years) and 39 women (35.1 ± 3.8 years) with only euploid day 5 or euploid day 6 blastocysts transferred during a cryopreserved embryo transfer had similar cycle outcomes. Although underpowered, these data suggest that euploid day 6 blastocysts are as capable of positive outcomes as their euploid day 5 counterparts.

Smoking and infertility: a committee opinion

Approximately 21% of women of reproductive age and 22% of men of reproductive age in the United States smoke cigarettes. Substantial harmful effects of cigarette smoke on fecundity and reproduction have become apparent but are not generally appreciated. This committee opinion reviews the potential deleterious effects of smoking on conception, ovarian follicular dynamics, sperm parameters, gamete mutations, early pregnancy, and assisted reproductive technology (ART) outcomes. It also reviews the current status of smoking cessation strategies. This document replaces the 2012 ASRM Practice Committee document of the same name (Fertil Steril 2012;98:1400-6).