Luca Gianaroli

Preimplantation diagnosis for aneuploidies in patients undergoing in vitro fertilization with a poor prognosis: identification of the categories for which it should be proposed

OBJECTIVE To verify whether advantages can derive from the implementation of preimplantation genetic diagnosis for aneuploidy in patients with a poor prognosis of full-term pregnancy, compared with conventional treatment procedures. DESIGN A randomized, controlled study. SETTING Reproductive Medicine Unit of the Società Italiana Studi Medicina della Riproduzione, Bologna, Italy. PATIENT(S) In a total of 262 stimulated cycles, women presented with the following poor-prognosis indications: maternal age of > or =36 years (n = 157), > or =3 previous IVF failures (n = 54), and an altered karyotype (n = 51). After giving consent, 127 patients underwent preimplantation genetic diagnosis for aneuploidy, whereas 135 controls underwent assisted zona hatching. INTERVENTION(S) Analysis of chromosomes XY, 13, 14, 15, 16, 18, 21, and 22 was carried out with the fluorescence in situ hybridization technique in a blastomere biopsied from day 3 embryos. Assisted zona hatching was performed on day 3 embryos from the control group. MAIN OUTCOME MEASURE(S) Embryo morphology and chromosomal status, number of transferred embryos, clinical pregnancies, implantation rates, and abortions. RESULT(S) In the study group, 717 embryos were analyzed by fluorescence in situ hybridization, and 60% were chromosomally abnormal. A mean of 2.3+/-0.9 euploid embryos were transferred in 99 cycles, resulting in 37 clinical pregnancies (37%) and a 22.5% ongoing implantation rate. In the control group, 126 cycles were performed with 3.2+/-1.3 embryos transferred, yielding 34 clinical pregnancies (27%) and a 10.2% ongoing implantation rate. CONCLUSION(S) The advantage of selecting embryos with a normal chromosome complement has an immediate impact on the ongoing implantation rate, especially in patients aged > or =38 years and carriers of an altered karyotype.

Preimplantation diagnosis of the aneuploidies most commonly found in spontaneous abortions and live births: XY, 13, 14, 15, 16, 18, 21, 22

The present preimplantation diagnosis test is able to screen for the most common aneuploidies from single blastomeres in about five hours with a 15 per cent misdiagnosis. This means that the risk of spontaneous abortion and trisomic offspring for women of advanced maternal age could be reduced to the same level as younger women for whom prenatal diagnosis is usually not necessary. Better probes and more fluorochromes could improve the success rate of the test. Copyright

Polar body array CGH for prediction of the status of the corresponding oocyte. Part I: clinical results

BACKGROUND Several randomized controlled trials have not shown a benefit from preimplantation genetic screening (PGS) biopsy of cleavage-stage embryos and assessment of up to 10 chromosomes for aneuploidy. Therefore, a proof-of-principle study was planned to determine the reliability of alternative form of PGS, i.e. PGS by polar body (PB) biopsy, with whole genome amplification and microarray-based comparative genomic hybridization (array CGH) analysis. METHODS In two centres, all mature metaphase II oocytes from patients who consented to the study were fertilized by ICSI. The first and second PBs (PB1and PB2) were biopsied and analysed separately for chromosome copy number by array CGH. If either or both of the PBs were found to be aneuploid, the corresponding zygote was then also processed by array CGH for concordance analysis. RESULTS Both PBs were biopsied from a total of 226 zygotes from 42 cycles (average 5.5 per cycle; range 1–15) in 41 couples with an average maternal age of 40.0 years. Of these, the ploidy status of the zygote could be predicted in 195 (86%): 55 were euploid (28%) and 140 were aneuploid (72%). With only one exception, there was at least one predicted aneuploid zygote in each cycle and in 19 out of 42 cycles (45%), all zygotes were predicted to be aneuploid. Fresh embryos were transferred in the remaining 23 cycles (55%), and one frozen transfer was done. Eight patients had a clinical pregnancy of which seven were evolutive (ongoing pregnancy rates: 17% per cycle and 30% per transfer). The ploidy status of 156 zygotes was successfully analysed by array CGH: 38 (24%) were euploid and 118 (76%) were aneuploid. In 138 cases complete information was available on both PBs and the corresponding zygotes. In 130 (94%), the ploidy status of the zygote was concordant with the ploidy status of the PBs and in 8 (6%), the results were discordant. CONCLUSIONS This proof-of-principle study indicates that the ploidy of the zygote can be predicted with acceptable accuracy by array CGH analysis of both PBs.

Chromosomal abnormalities in embryos

Chromosomal analysis was performed on 1620 embryos generated in vitro by patients with a poor prognosis of pregnancy. A diagnosis was yielded in 1596 embryos: 536 (34%) were euploid and 1060 (66%) carried chromosomal abnormalities. The results revealed a strong association between chromosomal abnormalities, cellular stage and percentage of fragmentation. In addition, 92% of embryos with multinucleated cells were diagnosed mosaics, whereas the presence of cytoplasmic concentration was associated to 86% chromosomal abnormalities. The rate of development to expanded blastocysts was dependent on both the cleavage stage at the time of blastomere biopsy and the chromosomal status of the embryos. The highest percentage of blastocyst growth derived from embryos with 7-8 cells on the morning of day 3: 68% were generated from euploid embryos and 43% from chromosomally abnormal embryos. These findings suggest that morphological criteria alone are not sufficient in poor prognosis patients to guarantee the best embryo selection.

Infertility therapy associated-multiple pregnancies (births): an ongoing epidemic

Multiple gestation is now recognized as a major problem associated with both assisted reproductive technologies (ART) and also with ovulation induction therapies. Although some countries are beginning to adopt measures to address this issue, either through legislation or the development of clinical guidelines, there is a clear need to ensure recognition and a consistent approach to this problem worldwide. In particular, there is a need to educate both healthcare professionals and the lay population that multiple gestations are not a desirable outcome for the infertile couple.

Pronuclear morphology and chromosomal abnormalities as scoring criteria for embryo selection

OBJECTIVE To verify whether a correlation exists between pronuclear zygote morphology and the chromosomal condition of preimplantation embryos. DESIGN Prospective analysis of pronuclear zygote morphology and preimplantation genetic diagnosis (PGD) for aneuploidy of the resulting embryos. SETTING Reproductive medicine unit, day surgery clinic. PATIENT(S) Seventy-seven patients undergoing 107 PGD cycles because of advanced maternal age (77 cycles) or previous IVF failures (30 cycles). INTERVENTION(S) Evaluation of pronuclear zygote morphology and chromosomal condition of the resulting embryos. MAIN OUTCOME MEASURE(S) Rate of embryo development, proportion of euploid embryos, and distribution of chromosomal abnormalities. The position of pronuclei within the ooplasm, the size and distribution of nucleoli, and the orientation of polar bodies with respect to pronuclei were highly predictive for the presence of complex chromosomal abnormalities in the developing embryos; zygotes with juxtaposed pronuclei, large-size nucleoli, and polar bodies with small angles subtended by pronuclei and polar bodies were the configurations associated with the highest rates of euploidy. CONCLUSION(S) The combination of the patterns related to pronuclear zygote morphology indicated four configurations where the proportion of chromosomally normal embryos was significantly higher compared with the other configurations, suggesting the validity of this scoring system for the selection of embryos generated by PGD patients.

The interface between assisted reproductive technologies and genetics: technical, social, ethical and legal issues

The interface between assisted reproductive technologies (ART) and genetics comprises several sensitive and important issues that affect infertile couples, families with severe genetic diseases, potential children, professionals in ART and genetics, health care, researchers and the society in general. Genetic causes have a considerable involvement in infertility. Genetic conditions may also be transmitted to the offspring and hence create transgenerational infertility or other serious health problems. Several studies also suggest a slightly elevated risk of birth defects in children born following ART. Preimplantation genetic diagnosis (PGD) has become widely practiced throughout the world for various medical indications, but its limits are being debated. The attitudes towards ART and PGD vary substantially within Europe. The purpose of the present paper was to outline a framework for development of guidelines to be issued jointly by European Society of Human Genetics and European Society of Human Reproduction and Embryology for the interface between genetics and ART. Technical, social, ethical and legal issues of ART and genetics will be reviewed.

The beneficial effects of preimplantation genetic diagnosis for aneuploidy support extensive clinical application.

The aim of this study was to evaluate the clinical impact of preimplantation genetic diagnosis (PGD) for aneuploidy on 193 patients who subsequently achieved 208 clinical pregnancies, in relation to their reproductive history. The 208 clinical pregnancies included in the study resulted from 1029 assisted conception cycles in combination with PGD for aneuploidy in 740 couples with a history of poor reproductive performance. According to the reproductive history of the 193 patients, 61 had previously experienced 112 pregnancies with 105 abortions and seven deliveries, corresponding to 3.6% take-home baby rate and 10.9% implantation rate. During the PGD cycle, preimplantation embryos were analysed for 5-9 chromosomes. The transfer of euploid embryos was performed in 699 cycles (68% of oocyte retrievals), generating 171 term pregnancies with 210 infants born, whereas 34 aborted spontaneously and three were ectopic, giving a take-home baby rate per pregnant patient of 88.6% and an ongoing implantation rate per pregnant patient of 53.2%. According to these data, selection made in preimplantation embryos against chromosomal abnormalities is associated with a significantly higher (P < 0.001) take-home baby rate when compared with the previous reproductive history of the parents.

What next for preimplantation genetic screening? A polar body approach!

Screening of human preimplantation embryos for numerical chromosome abnormalities has been conducted mostly at the preimplantation stage using fluorescence in situ hybridization. However, it is clear that preimplantation genetic screening (PGS) as it is currently practiced does not improve live birth rates. Therefore the ESHRE PGS Task Force has decided to start a proof of principle study with the aim of determining whether biopsy of the first and second polar body followed by subsequent analysis of the complete chromosome complement of these polar bodies using an array based technique enables a timely identification of the chromosomal status of an oocyte. If the principle of this approach can be proven, it is obvious that a multicentre randomized controlled trial should then be started to determine the clinical value of this technique. In this way the ESHRE PGS Task Force hopes to redirect preimplantation screening from the blind alley to the main road of assisted reproduction.

Gene expression profiling of human oocytes following in vivo or in vitro maturation

BACKGROUND Immature human oocytes matured in vitro, particularly those from gonadotrophin stimulated ovaries, are developmentally incompetent when compared with oocytes matured in vivo. This developmental incompetence has been explained as poor oocyte cytoplasmic maturation without any determination of the likely molecular basis of this observation. METHODS Replicate whole human genome arrays were generated for immature and mature oocytes (matured in vivo and in vitro, prior to exposure to sperm) recovered from women undertaking gonadotrophin treatment for assisted reproduction. RESULTS More than 2000 genes were identified as expressed at more than 2-fold higher levels in oocytes matured in vitro than those matured in vivo (P < 0.05, range 4.98 x 10(-2) -2.22 x 10(-4)) and 162 of these are expressed at 10-fold or greater levels (P < 0.05, range 4.98 x 10(-2)-1.38 x 10(-3)). Many of these genes are involved in transcription, the cell cycle and its regulation, transport and cellular protein metabolism. CONCLUSIONS Global gene expression profiling using microarrays and bioinformatics analysis has provided a molecular basis for differences in the developmental competence of oocytes matured in vitro compared with in vivo. The over-abundance of transcripts identified in immature germinal vesicle stage oocytes recovered from gonadotrophin stimulated cycles and matured in vitro is probably due to dysregulation in either gene transcription or post-transcriptional modification of genes. Either mechanism would result in an incorrect temporal utilization of genes which may culminate in developmental incompetence of any embryos derived from these oocytes.