M. Cristina Magli

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.

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.

The Istanbul consensus workshop on embryo assessment: proceedings of an expert meeting

This paper reports the proceedings of an international consensus meeting on oocyte and embryo morphology assessment. Following background presentations about current practice, the expert panel developed a set of consensus points to define the minimum criteria for oocyte and embryo morphology assessment. It is expected that the definition of common terminology and standardization of laboratory practice related to embryo morphology assessment will result in more effective comparisons of treatment outcomes. This document is intended to be referenced as a global consensus to allow standardized reporting of the minimum dataset required for the accurate description of embryo development. This paper reports the proceedings and outcomes of an international consensus meeting on human oocyte and embryo morphology assessment. An expert panel developed a series of consensus points to define the minimum criteria for such assessments. The definition of common terminology, and standardization of laboratory practices related to these morphological assessments, will permit more effective comparisons of treatment outcomes around the world. This report is intended to be referenced as a global consensus to allow standardized reporting of the minimum descriptive criteria required for routine clinical evaluations of human embryo development in vitro.

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 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.

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.

When and how should new technology be introduced into the IVF laboratory

There are many examples in assisted reproduction technology, where new technology and methods have been introduced into the clinical setting without appropriate development and evidence-based medicine to show that the procedure is safe and beneficial to the patient. Examples include preimplantation genetic screening, assisted hatching, in vitro maturation, blastocyst transfer and vitrification. Changes to culture media composition, stimulation regimes and laboratory protocols are also often established internationally without adequate validation. More recently, novel equipment that needs to be validated before it enters routine clinical use is being developed for IVF. With technologies such as producing gametes from stem cells around the corner, it is vital to ensure that the necessary research and development is conducted before bringing new techniques into clinical practice. Ideally, this should include preliminary work on animal models, such as mice/rats/rabbits/larger mammals, followed by studies on human embryos donated for research and finally well-designed RCTs with a follow up of all children born from the procedure. If such preliminary studies are not performed and published, it is possible that technology bringing no clinical benefit or leading to adverse health outcomes in the children born by these practices may be introduced. All IVF clinics need to consider the safety and efficacy of new technologies before introducing them.

Multiple meiotic errors caused by predivision of chromatids in women of advanced maternal age undergoing in vitro fertilisation.

Chromosome aneuploidy is a major cause of pregnancy loss, abnormal pregnancy and live births following both natural conception and in vitro fertilisation (IVF) and increases exponentially with maternal age in the decade preceding the menopause. Molecular genetic analysis following natural conception and spontaneous miscarriage demonstrates that trisomies arise mainly in female meiosis and particularly in the first meiotic division. Here, we studied copy number gains and losses for all chromosomes in the two by-products of female meiosis, the first and second polar bodies, and the corresponding zygotes in women of advanced maternal age undergoing IVF, using microarray comparative genomic hybridisation (array CGH). Analysis of the segregation patterns underlying the copy number changes reveals that premature predivision of chromatids rather than non-disjunction of whole chromosomes causes almost all errors in the first meiotic division and unlike natural conception, over half of aneuploidies result from errors in the second meiotic division. Furthermore, most abnormal zygotes had multiple aneuploidies. These differences in the aetiology of aneuploidy in IVF compared with natural conception may indicate a role for ovarian stimulation in perturbing meiosis in ageing oocytes.

The role of preimplantation diagnosis for aneuploidies.

The clinical application of preimplantation genetic diagnosis (PGD) for aneuploidy has confirmed the hypothesis that implantation failure and spontaneous abortions are frequently due to aneuploidy. Following PGD, a higher implantation rate and a lower incidence of spontaneous abortions are obtained in patient categories where aneuploidy is the main cause of reproductive failure: women in advanced reproductive age, patients with an altered karyotype due to translocations or gonosomal mosaicism, and patients with recurrent spontaneous abortions. In these cases, the transfer of euploid embryos overcomes the poor prognosis condition in these couples. As expected, aneuploidy increases proportionally with female age; however, not all the chromosomes studied show this trend, suggesting that segregation errors could occur at different rates for each chromosome in relation to maternal age. Furthermore, the retrospective analysis of the results obtained in patients who repeated at least twice a PGD cycle permitted to estimate their chances of reproducing the same pattern of chromosomal abnormalities and consequently evaluating their possibility of a pregnancy: when no euploid embryos are detected at the first attempt, the chance of on-term pregnancy is below 10%; however, this chance is approximately 30% for couples with at least two euploid embryos in the first cycle.

Birefringence characteristics in sperm heads allow for the selection of reacted spermatozoa for intracytoplasmic sperm injection

OBJECTIVE To verify clinical outcome after injection of spermatozoa that have undergone the acrosome reaction (reacted spermatozoa) vs. those still having an intact acrosome (nonreacted spermatozoa). DESIGN Prospective, randomized study. SETTING Reproductive Medicine Unit, Italian Society for the Study of Reproductive Medicine, Bologna, Italy. PATIENT(S) According to a prospective randomization including 71 couples with severe male factor infertility, intracytoplasmic sperm injection (ICSI) was performed under polarized light that permitted analysis of the pattern of birefringence in the sperm head. Twenty-three patients had their oocytes injected with reacted spermatozoa, 26 patients oocytes were injected with nonreacted spermatozoa, and in 22 patients both reacted and nonreacted spermatozoa were injected. INTERVENTION(S) Intracytoplasmic sperm injection was performed under polarized light to selectively inject acrosome-reacted and acrosome-nonreacted spermatozoa. MAIN OUTCOME MEASURE(S) Rates of fertilization, cleavage, pregnancy, implantation, and ongoing implantation. RESULT(S) There was no effect on the fertilizing capacity and embryo development of either type of sperm, whereas the implantation rate was higher in oocytes injected with reacted spermatozoa (39.0%) vs. those injected with nonreacted spermatozoa (8.6%). The implantation rate was 24.4% in the group injected with both reacted and nonreacted spermatozoa. The delivery rate per cycle followed the same trend. CONCLUSION(S) Spermatozoa that have undergone the acrosome reaction seem to be more prone to supporting the development of viable ICSI embryos.