J. Grifo

Clinical utilisation of a rapid low-pass whole genome sequencing technique for the diagnosis of aneuploidy in human embryos prior to implantation

Background The majority of human embryos created using in vitro fertilisation (IVF) techniques are aneuploid. Comprehensive chromosome screening methods, applicable to single cells biopsied from preimplantation embryos, allow reliable identification and transfer of euploid embryos. Recently, randomised trials using such methods have indicated that aneuploidy screening improves IVF success rates. However, the high cost of testing has restricted the availability of this potentially beneficial strategy. This study aimed to harness next-generation sequencing (NGS) technology, with the intention of lowering the costs of preimplantation aneuploidy screening. Methods Embryo biopsy, whole genome amplification and semiconductor sequencing. Results A rapid (<15 h) NGS protocol was developed, with consumable cost only two-thirds that of the most widely used method for embryo aneuploidy detection. Validation involved blinded analysis of 54 cells from cell lines or biopsies from human embryos. Sensitivity and specificity were 100%. The method was applied clinically, assisting in the selection of euploid embryos in two IVF cycles, producing healthy children in both cases. The NGS approach was also able to reveal specified mutations in the nuclear or mitochondrial genomes in parallel with chromosome assessment. Interestingly, elevated mitochondrial DNA content was associated with aneuploidy (p<0.05), a finding suggestive of a link between mitochondria and chromosomal malsegregation. Conclusions This study demonstrates that NGS provides highly accurate, low-cost diagnosis of aneuploidy in cells from human preimplantation embryos and is rapid enough to allow testing without embryo cryopreservation. The method described also has the potential to shed light on other aspects of embryo genetics of relevance to health and viability.

Oral versus intramuscular progesterone for in vitro fertilization: a prospective randomized study

OBJECTIVE To evaluate the efficacy of oral micronized progesterone compared with IM progesterone in oil for luteal support in patients undergoing IVF who are treated with a GnRH agonist. DESIGN Randomized prospective clinical trial. SETTING University-based IVF center. PATIENT(S) Women <40 years of age who were undergoing IVF with luteal GnRH pituitary down-regulation. INTERVENTION(S) Patients were randomized to receive either oral micronized progesterone (200 mg three times daily) or IM progesterone (50 mg daily). MAIN OUTCOME MEASURE(S) Progesterone levels at standardized days 21 and 28, and pregnancy and embryo implantation rates. RESULT(S) Day 21 progesterone levels were 77.6+/-13.2 ng/mL in the IM group and 81.5+/-16.2 ng/mL in the oral group. Day 28 progesterone levels were 76.3+/-15.0 ng/mL in the IM group and 53.6+/-10.1 ng/mL in the oral group. The clinical pregnancy rates were 57.9% and 45.8% for the IM and oral groups, respectively. The implantation rate per embryo was significantly higher in the IM group (40.9%) than in the oral group (18.1%). CONCLUSION(S) When used according to our protocols, oral progesterone and IM progesterone result in comparable levels of circulating progesterone. However, oral progesterone results in a reduced implantation rate per embryo.

Validation of array comparative genome hybridization for diagnosis of translocations in preimplantation human embryos

Fluorescent in-situ hybridization (FISH) for preimplantation genetic diagnosis (PGD) of structural chromosome abnormalities has limitations, including carrier testing, inconclusive results and limited aneuploidy screening. Array comparative genome hybridization (CGH) was used in PGD cases for translocations. Unbalances could be identified if three fragments were detectable. Smallest detectable fragments were ∼6 Mbp and ∼5 Mbp for blastomeres and trophectoderm, respectively. Cases in which three or more fragments were detectable by array CGH underwent PGD by FISH and concordance was obtained in 53/54 (98.1%). The error rate for array CGH was 1.9% (1/54). Of 402 embryos analysed, 81 were normal or balanced, 92 unbalanced but euploid, 123 unbalanced and aneuploid and 106 balanced but aneuploid. FISH with additional probes to detect other aneuploidies would have missed 28 abnormal embryos in the reciprocal group and 10 in the Robertsonian group. PGD cases (926) were retrospectively reviewed for reciprocal translocations performed by FISH to identify which could have been analysed by array CGH. This study validates array CGH in PGD for translocations and shows that it can identify all embryos with unbalanced reciprocal and Robertsonian translocations. Array CGH is a better approach than FISH since it allows simultaneous screening of all chromosomes for aneuploidy.

Oocyte cryopreservation outcomes including pre-cryopreservation and post-thaw meiotic spindle evaluation following slow cooling and vitrification of human oocytes

OBJECTIVE To report our oocyte cryopreservation (OC) outcomes including meiotic spindle (MS) evaluation of metaphase II (MII) oocytes destined for OC and thaw. DESIGN Retrospective. SETTING University-based infertility center. PATIENT(S) Women attempting pregnancy using cryopreserved oocytes. INTERVENTION(S) OC, MS evaluation. MAIN OUTCOME MEASURE(S) Survival, two pronuclear (2PN) fertilization, achieving embryo quality suitable for transfer or refreezing, blastocyst formation. RESULT(S) Thirty-two OC-thaw cycles resulted in 20 pregnancies, 18 either ongoing or delivered. In 26 cycles, MS evaluation was performed: 262/303 (86%) thawed/recovered oocytes survived, 218/262 (83%) achieved 2PN fertilization, 133/218 (61%) became suitable for day-3 and 122/218 (56%) for day-5 transfer. In total, 58 embryos were transferred resulting in a 62% pregnancy and a 41% implantation rate. Of oocytes evaluated before cryopreservation, 247 (82%) were spindle-positive; 96% of these were also spindle-positive after thawing. Blastocyst formation and suitability for day-5 transfer was achieved more often if a post-thaw spindle was visualized. Of all slow-cooled and vitrified oocytes, a higher percentage of those slow-cooled achieved 2PN fertilization and usability. MS evaluation of oocytes cryopreserved by either method was associated with similar outcomes. CONCLUSION(S) OC outcomes are improving. An MS was almost always exhibited both before cryopreservation and after thawing, suggesting that, with appropriate technique, OC presents minimal harm to the MII oocyte. A meiotic spindle evaluation might help to further OC technology.

Preimplantation genetic diagnosis in two families at risk for recurrence of Herlitz junctional epidermolysis bullosa

Abstract: The Herlitz type of junctional epidermolysis bullosa (H‐JEB) is a severe inherited bullous disease which leads to the early demise of the affected newborn. Mutations in the genes encoding the 3 polypeptides of the anchoring filament protein laminin 5 underlie this condition. We studied 2 families with affected children who previously died from H‐JEB. Mutation screening using heteroduplex analysis and direct sequencing of the PCR products revealed a previously described hotspot mutation in LAMB3 (R635X), and a novel delayed termination codon in LAMB3 in the first proband. In the second proband, we found a novel initiation codon mutation in LAMB3, and a novel 2 bp deletion in LAMB3. For preimplantation genetic diagnosis (PGD) in these families, we developed nested multiplex PCR assays, amplifying the mutations and informative intragenic polymorphisms in the probands. Single embryonic cells were biopsied from 8‐cell embryos using standard techniques, and subjected to the multiplex PCR assay followed by restriction enzyme digestion. Embryos found not to carry either mutation were transferred to the mothers, and a pregnancy was established in the second family as evidenced by the elevated level of HCG, although the pregnancy did not persist. This study illustrates the feasibility of PGD for an inherited skin disorder for the first time.

Preimplantation Diagnosis of Genetic and Chromosomal Disorders

The mean success rate per cycle is about 10%; it decreases with the number of insemination cycles, from 10.3% during the first 6 months to 2.3% after 24 cycles of treatment. This indicates that more fertile women conceive more readily. This success rate is dependent on the womens ages, the AID indication, the semen quality, and especially, the postthaw motility. The cumulative success rate (dropout excluded) for all women is 48% of pregnancies at 6 cycles, 66% at 12 cycles, and 80% at 24 cycles. The mean success rate per cycle is 15% with intrauterine insemination and 23% with IVFD (Table III).

Preimplantation Genetic Diagnosis of Human Embryos for Marfan's Syndrome

Purpose:Single-cell nested polymerase chain reaction (PCR) and Dde1 endonuclease digestion were used to detect the presence of a Marfans syndrome mutation in human preimplantation embryos derived from in vitro fertilization (IVF). These procedures were conducted to eliminate the possibility of transmission of the affected allele from the father to his offspring. The mutation on chromosome 15 is transmitted as an autosomal dominant trait, and the chance of having a child affected with the disease is 50%.Methods:A couple presented to the Program for In Vitro Fertilization, Reproductive Surgery and Infertility for preimplantation genetic diagnosis. IVF was performed and embryo biopsy was done on day 3 embryos, Single blastomeres were removed from embryos and subjected to nested PCR analysis and endonuclease digestion to detect a Marfans syndrome mutation located on chromosome 15 inherited from the father.Results:Thirteen oocytes were injected with spermatozoa using intracytoplasmic sperm injection, and nine fertilized normally. Following embryo biopsy and polymerase chain reaction amplification-Dde1 endonuclease digestion, five embryos were detected that were positive for the mutation. The four non-affected embryos were transferred to the uterus, resulting in a healthy and normal ongoing pregnancy.

Comparison of pregnancy outcomes in elective single blastocyst transfer versus double blastocyst transfer stratified by age.

OBJECTIVE To determine whether there is a difference in pregnancy outcomes, stratified by age, between women undergoing elective single blastocyst transfer (eSBT) versus those undergoing double blastocyst transfer (2BT). DESIGN Retrospective analysis. SETTING University IVF center. PATIENT(S) A total of 1,141 nondonor IVF cycles in women aged <40 years from January 2004-March 2007. INTERVENTION(S) None. MAIN OUTCOME MEASURE(S) Eggs retrieved, embryos cryopreserved, implantation rates, clinical pregnancy rates (PR), live birth rates (LBR), spontaneous abortion rates (SAB). RESULT(S) Pregnancy outcomes in 52 cycles of women <40 years of age who underwent eSBT were compared with 1,086 cycles of women who underwent 2BT in fresh IVF cycles from January 2004-March 2007. Overall, the eSBT was associated with a statistically significant 92% reduction in the twinning rate (from 25%-2%) while maintaining a high clinical PR (63% in the eSBT group vs. 61% in the 2BT group). CONCLUSION(S) Women who are <40 years of age undergoing nondonor fresh IVF cycles can electively choose to transfer a single blastocyst for the purpose of significantly reducing their risk of multiples without compromising their PR.

Pregnancy derived from human zygote pronuclear transfer in a patient who had arrested embryos after IVF

Nuclear transfer of an oocyte into the cytoplasm of another enucleated oocyte has shown that embryogenesis and implantation are influenced by cytoplasmic factors. We report a case of a 30-year-old nulligravida woman who had two failed IVF cycles characterized by all her embryos arresting at the two-cell stage and ultimately had pronuclear transfer using donor oocytes. After her third IVF cycle, eight out of 12 patient oocytes and 12 out of 15 donor oocytes were fertilized. The patients pronuclei were transferred subzonally into an enucleated donor cytoplasm resulting in seven reconstructed zygotes. Five viable reconstructed embryos were transferred into the patients uterus resulting in a triplet pregnancy with fetal heartbeats, normal karyotypes and nuclear genetic fingerprinting matching the mothers genetic fingerprinting. Fetal mitochondrial DNA profiles were identical to those from donor cytoplasm with no detection of patients mitochondrial DNA. This report suggests that a potentially viable pregnancy with normal karyotype can be achieved through pronuclear transfer. Ongoing work to establish the efficacy and safety of pronuclear transfer will result in its use as an aid for human reproduction.

Ooplasmic Influence on Nuclear Function During the Metaphase II-Interphase Transition in Mouse Oocytes

Abstract Nuclear and pronuclear transfer procedures were used to assess the functional competence of the nucleus and cytoplasm of mouse germinal vesicle-stage oocytes denuded of granulosa cells and matured in vitro or in vivo before artificial activation using a sequential treatment of A23187 + cycloheximide. Following activation, in vitro-matured oocytes were “fertilized” by inserting a male pronucleus (PN), cultured to the 2-cell stage, and then transferred to the oviducts of foster mothers. No live births were noted, whereas a 17% live birth rate was observed when in vivo-matured oocytes were used. The developmental competency of other zygotes was similarly assessed following the exchange of haploid PN of matured and activated eggs with the female PN of fertilized zygotes. When PN of oocytes subjected to maturation and activation in vitro were transferred, only 1 of 79 reconstructed zygotes developed to term. In contrast, the live birth rate was 21% (11 of 53) for zygotes reconstructed with PN from in vivo-matured oocytes. Moreover, a live birth rate of 23% (8 of 35) was observed for reconstructed zygotes with female PN from “hybrid” oocytes created by transferring the metaphase II nuclei of in vitro-matured oocytes into enucleated, in vivo-matured oocytes before activation. Such results suggest that the nucleus of an in vitro-matured oocyte can support embryonic development, but only when it is activated in the proper ooplasmic milieu. The cellular factors creating this ooplasmic milieu appear to develop normally in vivo during follicle maturation to metaphase II, but they fail to do so when the oocytes are denuded of granulosa cells and cultured in vitro before the final stages of maturation. In parallel studies, male and female PN of in vivo-fertilized zygotes were inserted into oocytes that were activated and enucleated following either in vitro or in vivo maturation. Live birth rates were comparable at 19% (5 of 27) and 18% (9 of 49), respectively, suggesting that, regardless of the environment of the final stages of oocyte maturation, the resultant ooplasm is competent to support all aspects of embryonic development once activation and PN formation has been completed. Such findings only point further toward the importance of the condition of the ooplasmic milieu at the time of chemical activation. Whether a similar situation exists when eggs are activated following sperm penetration remains to be determined.