B. Kaplan

Diminished effect of maternal age on implantation after preimplantation genetic diagnosis with array comparative genomic hybridization.

OBJECTIVE To assess the relationship between maternal age, chromosome abnormality, implantation, and pregnancy loss. DESIGN Multicenter retrospective study. SETTING IVF centers in the United States. PATIENT(S) IVF patients undergoing chromosome screening. INTERVENTION(S) Embryo biopsy on day 3 or day 5/6 with preimplantation genetic diagnosis (PGD) by array comparative genomic hybridization. MAIN OUTCOME MEASURE(S) Aneuploidy, implantation, pregnancy, and loss rates. RESULT(S) Aneuploidy rates increased with maternal age from 53% to 93% for day 3 biopsies and from 32% to 85% for blastocyst biopsies. Implantation rates for euploid embryos for ages <35-42 years did not decrease after PGD: ranges 44%-32% for day 3 and 51%-40% for blastocyst. Ongoing pregnancy rates per transfer did not decrease for maternal ages <42 years after PGD with day 3 biopsy (48.5%-38.1%) or blastocyst biopsy (64.4%-54.5%). Patients >42 years old had implantation rates of 23.3% (day 3), 27.7% (day 5/6), and the pregnancy rate with day 3 biopsy was 9.3% and with day 5 biopsy 10.3%. CONCLUSION(S) Selective transfer of euploid embryos showed that implantation and pregnancy rates were not significantly different between reproductively younger and older patients up to age 42 years. Some patients who start an IVF cycle planning to have chromosome screening do not have euploid embryos available for transfer, a situation that increases with advancing maternal age. Mounting data suggests that the dramatic decline in IVF treatment success rates with female age is primarily caused by aneuploidy.

Idiopathic recurrent miscarriage is caused mostly by aneuploid embryos.

OBJECTIVE To determine any beneficial effects of preimplantation genetic screening (PGS) of all chromosomes by array comparative genomic hybridization (aCGH), with either day 3 or blastocyst biopsy, for idiopathic recurrent pregnancy loss (RPL) patients compared with their expected loss rate. DESIGN Case series report. SETTING Multiple fertility centers. PATIENT(S) A total of 287 cycles of couples with idiopathic RPL (defined as two or more losses). INTERVENTION(S) PGS was done with day 3 biopsy (n = 193) or blastocyst biopsy (n = 94), followed by analysis with aCGH. MAIN OUTCOME MEASURE(S) Spontaneous abortion rate, euploidy rate. RESULT(S) A total of 2,282 embryos were analyzed, of which 35% were euploid and 60% were aneuploid. There were 181 embryo transfer cycles, of which 100 (55%) became pregnant with an implantation rate of 45% (136 sacs/299 replaced embryos) and 94 pregnancies (92%) were ongoing (past second trimester) or delivered. The miscarriage rate was found to be only 6.9% (7/102), compared with the expected rate of 33.5% in an RPL control population and 23.7% in an infertile control population. CONCLUSION(S) Current PGS results with aCGH indicate a significant decrease in the miscarriage rate of idiopathic RPL patients and high pregnancy rates. Furthermore, this suggests that idiopathic recurrent miscarriage is mostly caused by chromosomal abnormalities in embryos.

Prevention of Age-Related Aneuploidies by Polar Body Testing of Oocytes

Purpose:We previously demonstrated that aneuploidy-free oocytes may be preselected by testing the first and second polar bodies removed from oocytes following their maturation and fertilization. The present paper describes the results of the application of the method in 659 in vitro fertilization cycles from patients of advanced maternal age.Methods:Using micromanipulation techniques, 3943 oocytes were tested by polar body sampling and fluorescent in situ hybridization analysis using specific probes for chromosomes 13, 18, and 21.Results:Fluorescent in situ hybridization results were available for 3217 (81.6%) of 3943 oocytes studied, of which 1388 (43.1%) had aneuploidies; 35.7% of the aneuploidies were of first meiotic division origin, and 26.1% of second meiotic division origin. Most errors in the first meiotic division were represented by chromatid malsegregation. The transfer of embryos deriving from 1558 of 1829 aneuploidy-free oocytes in 614 treatment cycles resulted in 131 clinical pregnancies and 88 healthy children born after confirmation of the polar body diagnosis.Conclusions:Polar body testing of oocytes provides an accurate and reliable approach for prevention of age-related aneuploidies in in vitro fertilization patients of advanced maternal age.

Preimplantation Diagnosis of Common Aneuploidies by the First- and Second-Polar Body FISH Analysis

Purpose:A low pregnancy rate in in vitro fertilization (IVF) patients of advanced maternal age may be caused by aneuploidies originating from non disjunction in the first or second meiotic divisions. We introduced genetic testing of oocytes by sampling and fluorescent in situ hybridization (FISH) analysis of the first and second polar bodies, to avoid fertilization and transfer of aneuploid oocytes in IVF patients of advanced maternal age.Methods:Three hundred and sixty-three IVF patients 34 years and older participated in the study. Using micromanipulation procedures, the first and second polar bodies were removed following their extrusion from the oocytes and studied by FISH, using probes specific for chromosomes 13, 18, and 21 to detect oocytes with common aneuploidies.Results:Of a total of 538 IVF cycles, 3250 oocytes were available for FISH analysis, with conclusive FISH results in 2742 oocytes (84.3%). As many as 1102 (40%) of oocytes were predicted to be aneuploid and not transferred. Of 1640 embryos predicted to be normal, 1145 were transferred in 467 treatment cycles, resulting in 107 pregnancies (23%), from which 67 healthy children have been born, 32 pregnancies spontaneously aborted, and 15pregnancies are ongoing after being confirmed normal by prenatal diagnosis.Conclusions:Preimplantation diagnosis by first- and second-polar body FISH analysis allows us to avoid the age-related risk of common aneuploidies in IVF patients of advanced maternal age.

Array CGH analysis shows that aneuploidy is not related to the number of embryos generated

This study retrospectively analysed array comparative genomic hybridization (CGH) results of 7753 embryos from 990 patients to determine the frequency of embryonic euploidy and its relationship with the cohort size (i.e. the number of embryos available for biopsy and array CGH analysis). Linear regression analysis was performed to assess the effect of cohort size on euploidy rate adjusted for the effect of female age. While increasing female age was associated with a significant decrease in euploidy rate of day-3 and day-5 embryos (P<0.001 for both groups), cohort size was not significantly associated with euploidy rate. Logistic regression analysis was performed to assess the effect of cohort size, adjusted for maternal age, on the likelihood of having at least one euploid embryo available for transfer. The odds of having at least one euploid embryo in an assisted cycle was significantly decreased by increasing female age (P<0.01 for both day-3 and day-5 embryos) and was significantly increased by every additional embryo available for analysis (P<0.001 for both day-3 and day-5 embryos).

Polar body diagnosis of common aneuploidies by FISH

AbstractPurpose: The purpose of this work was to investigate the reliability and accuracy of polar body analysis for preimplantation diagnosis of common aneuploidies in IVF patients of advanced maternal age. Design: We have previously introduced polar body analysis as an approach for nondestractive evaluation of the genotype of human oocytes. The method has recently been applied in a clinical trial involving 45 infertile patients, demonstrating the feasibility of preconception diagnosis of common aneuploidies by fluorescent in situ hybridization (FISH). The present paper describes the experience of polar body diagnosis in 135 IVF patients (161 cycles) of advanced maternal age. Results: FISH results of the first and/or second polar bodies were available in 648 (72.4%) of 895 biopsied oocytes subjected to FISH analysis. Of 648 oocytes with FISH results, 208 demonstrated chromosomal abnormalities. Of 440 oocytes predicted to be free from monosomy or trisomy of chromosomes X, 18, and/or 13/21, 314 were normally fertilized, cleaved, and transferred in 122 treatment cycles, resulting in 6 healthy deliveries and 12 ongoing pregnancies following confirmation of the polar body diagnosis by CVS or amniocentesis. Conclusion: The method may be useful for detection of oocytes with common chromosomal trisomies in IVF patients of advanced maternal age.

Preimplantation diagnosis for neurofibromatosis

Preimplantation genetic diagnosis (PGD) has recently been performed for inherited cancer predisposition determined by p53 tumour suppressor gene mutations, suggesting the usefulness of PGD for late onset disorders with genetic predisposition, including those caused by the germline mutations of other tumour suppressor genes. Here PGD was performed for two couples, one at risk for producing a child with maternally derived neurofibromatosis type I (NF1), and the other with paternally derived neurofibromatosis type II (NF2). The procedure involved a standard IVF protocol, combined with testing of oocytes or embryos prior to their transfer back to the patients. Maternal mutation Trp-->Ter (TGG-->TGA) in exon 29 of the NF1 gene was tested by sequential PCR analysis of the first and second polar bodies, and paternal L141P mutation in exon 4 of the NF2 gene by embryo biopsy at the cleavage stage. In both cases, multiplex nested PCR was applied, involving NF1 and NF2 mutation analysis simultaneously with the 3 and 2 linked markers, respectively. Of 57 oocytes tested in four PGD cycles for NF1 mutation, 26 mutation-free oocytes were detected, from which eight were preselected for transfer, two in each cycle. These produced two clinical pregnancies, one confirmed to be mutation free by chorionic villus sampling but ending in a stillbirth, and the other still ongoing. Of 18 embryos analysed in a cycle performed for NF2 mutation, eight mutation-free embryos were detected, three of which were transferred back to the patient, resulting in a singleton pregnancy and the birth of a mutation-free child. This suggests that PGD is a useful approach for avoiding the birth of children with inherited cancer predisposition, determined by NF1 and NF2 gene mutations.

Nonsurgical treatment of a viable cervical pregnancy with intra-amniotic methotrexate

Although a rare complication of gestation, the possibility of fertility loss is no more serious than with a cervical pregnancy. A case of a viable cervical pregnancy treated with ultrasound guided transabdominal aspiration and intra-amniotic methotrexate administration is presented. When the diagnosis is made early in gestation this method should be considered for the treatment of a cervical pregnancy when future fertility potential is desirable.

Viability of embryos following second polar body removal in a mouse model

PurposeOur purpose was to evaluate the effect of the second polar body removal on the viability and developmental potential of the resulting embryos.MethodThe second polar body was removed in 343 mouse oocytes and the developmental potential of the resulting embryos was compared in vitro to that in 223 intact mouse embryos and 222 controls which were placed under the same experimental conditions as those micromanipulated.ResultsThe proportion of morphologically normal blastocysts formed by polar body biopsied oocytes (70.8%) was not significantly different from that obtained from the control (81.1%) and intact (85%) oocytes. Cell counts of blastocysts obtained from the manipulated and nonmanipulated oocytes were also not statistically different.ConclusionThe second polar body biopsy does not have a significant adverse effect on preimplantation development and may be applied for genetic evaluation of oocytes.

Conversion of Cycles Involving Ovarian Hyperstimlation With Intrauterine Insemination to in Vitro Fertilization

Conversion to in-vitro fertilization (IVF) and embryo transfer as an alternative to cancellation was offered in 27 consecutive cycles of controlled ovarian hyperstimulation and intra-uterine insemination (IUI) cycles with excessive follicular development in patients with idiopathic infertility. IVF and embryo transfer was performed in 25 cycles, resulting in 13 pregnancies (52%), with 22% of couples having at least two embryos cryopreserved. The pregnancies have resulted in one singleton and two twin births, one spontaneous abortion, and nine ongoing pregnancies (including one triplet gestation). Four patients developed severe ovarian hyperstimulation syndrome (OHSS) after IVF and embryo transfer, including two cases requiring paracentesis. Three of four OHSS patients were pregnant, resulting in live births of healthy twins, one spontaneous abortion and one ongoing singleton gestation. In two cycles a spontaneous luteinizing hormone (LH) surge occurred, preventing oocyte retrieval. For these two women, drainage of all follicles except the five most likely to fertilize (18-22 mm diameter) was performed, followed by IUI, with no pregnancies or OHSS observed. Conversion of patients from IUI cycles to IVF/embryo transfer cycles avoids cancellation of the very cycles with the best chance of achieving pregnancy. OHSS remains a problem, necessitating extensive pre-IVF counselling and post-transfer vigilance.