Anna Pia Ferraretti

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.

Assisted reproductive technology in Europe, 2009: results generated from European registers by ESHRE

STUDY QUESTION The 13th European in vitro fertilization (IVF)-monitoring (EIM) report presents the results of treatments involving assisted reproductive technology (ART) initiated in Europe during 2009: are there any changes in the trends compared with previous years? SUMMARY ANSWER Despite some fluctuations in the number of countries reporting data, the overall number of ART cycles has continued to increase year by year and, while pregnancy rates in 2009 remained similar to those reported in 2008, the number of transfers with multiple embryos (3+) and the multiple delivery rates declined. WHAT IS KNOWN ALREADY Since 1997, ART data in Europe have been collected and reported in 12 manuscripts, published in Human Reproduction. STUDY DESIGN, SIZE, DURATION Retrospective data collection of European ART data by the EIM Consortium for the European Society of Human Reproduction and Embryology (ESHRE); cycles started between 1st January and 31st December are collected on a yearly basis; the data are collected by the National Registers, when existing, or on a voluntary basis. PARTICIPANTS/MATERIALS SETTING, METHODS From 34 countries (-2 compared with 2008), 1005 clinics reported 537 463 treatment cycles including: IVF (135 621), intracytoplasmic sperm injection (ICSI, 266 084), frozen embryo replacement (FER, 104 153), egg donation (ED, 21 604), in vitro maturation (IVM, 1334), preimplantation genetic diagnosis/screening (PGD/PGS, 4389) and frozen oocyte replacements (FOR, 4278). European data on intrauterine insemination using husband/partners semen (IUI-H) and donor (IUI-D) semen were reported from 21 and 18 countries, respectively. A total of 162 843 IUI-H (+12.7%) and 29 235 IUI-D (+17.3%) cycles were included. Data available from each country are presented in the tables; total values (as numbers and percentages) refer to those countries where all data have been reported. MAIN RESULTS AND THE ROLE OF CHANCE In 21 countries where all clinics reported to the ART register, a total of 399 020 ART cycles were performed in a population of 373.8 million, corresponding to 1067 cycles per million inhabitants. For IVF, the clinical pregnancy rates per aspiration and per transfer were 28.9 and 32.9%, respectively and for ICSI, the corresponding rates were 28.7 and 32.0%. In FER cycles, the pregnancy rate per thawing was 20.9%; in ED cycles, the pregnancy rate per transfer was 42.3%. The delivery rate after IUI-H was 8.3 and 13.4% after IUI-D. In IVF and ICSI cycles, 1, 2, 3 and 4+ embryos were transferred in 24.2, 57.7, 16.9 and 1.2%, respectively. The proportions of singleton, twin and triplet deliveries after IVF and ICSI (combined) were 79.8, 19.4 and 0.8%, respectively, resulting in a total multiple delivery rate of 20.2%, compared with 21.7% in 2008, 22.3% in 2007, 20.8% in 2006 and 21.8% in 2005. In FER cycles, the multiple delivery rate was 13.0% (12.7% twins and 0.3% triplets). Twin and triplet delivery rates associated with IUI cycles were 10.4/0.7% and 10.3/0.5%, following treatment with husband and donor semen, respectively. LIMITATIONS, REASONS FOR CAUTION The method of reporting varies among countries, and registers from a number of countries have been unable to provide some of the relevant data such as initiated cycles and deliveries. As long as data are incomplete and generated through different methods of collection, results should be interpreted with caution. WIDER IMPLICATIONS OF THE FINDINGS The 13th ESHRE report on ART shows a continuing expansion of the number of treatment cycles in Europe, with more than half a million of cycles reported in 2009. The use of ICSI has reached a plateau. Pregnancy and delivery rates after IVF and ICSI remained relatively stable compared with 2008 and 2007. The number of multiple embryo transfers (3+ embryos) and the multiple delivery rate have shown a clear decline.

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.

Incidence of Chromosomal Abnormalities from a Morphologically Normal Cohort of Embryos in Poor-Prognosis Patients

Purpose:Preimplantation genetic diagnosis of aneuploidy was performed on the embryos yielded by 70 poor-prognosis patients, with the aim of transferring those with a normal chromosomal complement, thus possibly increasing the chances of pregnancy.Methods:Multicolor fluorescence in situ hybridization (FISH) was applied for the simultaneous detection of chromosomes X, Y, 13, 16, 18, and 21. Inclusion criteria were (1) a maternal age of 36 years or older (n = 33), (2) three or more previous in vitro fertilization cycles (n = 20), and (3) an altered karyotype (n = 17).Results:A total of 412 embryos underwent FISH, resulting in 234 (57%) that were chromosomally abnormal. Euploid embryos were available for transfer in 59 patients, generating 19 pregnancies (32%), with an implantation rate of 19.9%.Conclusions:High rates of chromosomally abnormal embryos in poor-prognosis patients can determine repeated in vitro fertilization failures when embryo selection is performed on the basis of morphological criteria alone. Hence, the FISH analysis could represent the prevailing approach for the identification of embryos possessing full potential for developing to term.

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.

Assisted reproductive technology in Europe, 2011: results generated from European registers by ESHRE

STUDY QUESTION The 15th European IVF-monitoring (EIM) report presents the results of treatments involving assisted reproductive technology (ART) initiated in Europe during 2011: are there any changes in the trends compared with previous years? SUMMARY ANSWER Despite some fluctuations in the number of countries reporting data, while the overall number of ART cycles has continued to increase year by year, the pregnancy rates in 2011 decreased slightly to those reported in 2010, and the number of transfers with multiple embryos (3+) and the multiple delivery rates declined. WHAT IS KNOWN ALREADY Since 1997, ART data in Europe have been collected and reported in 14 manuscripts, published in Human Reproduction. STUDY DESIGN, SIZE, DURATION Retrospective data collection of European ART data by the EIM Consortium for the European Society of Human Reproduction and Embryology (ESHRE); cycles started between 1 January and 31 December 2011 are collected on a yearly basis. The data are collected by National Registers, when existing, or on a voluntary basis by personal information. PARTICIPANTS/MATERIALS SETTING, METHODS From 33 countries (+2 compared with 2010), 1064 clinics reported 609 973 treatment cycles including: IVF 138 592, ICSI 298 918, frozen embryo replacement (FER) 129 693, egg donation (ED) 30 198, in vitro maturation 511, preimplantation genetic diagnosis/screening 6824 and frozen oocyte replacements 5237. European data on intrauterine insemination (IUI) using husband/partners semen (IUI-H) and donor semen (IUI-D) were reported from 861 IUI laboratories in 24 countries. A total of 174 390 IUI-H and 41 151 IUI-D cycles were included. MAIN RESULTS AND THE ROLE OF CHANCE In 17 countries where all clinics reported to the ART register, a total of 361 972 ART cycles were performed in a population of 285 million inhabitants, corresponding to 1269 cycles per million inhabitants. For all IVF cycles, the clinical pregnancy rates per aspiration and per transfer were stable with 29.1 and 33.2%, respectively, and for ICSI, the corresponding rates also were stable with 27.9 and 31.8%, respectively. In FER cycles, the pregnancy rate per thawing increased to 21.3% if compared with previous years. In ED cycles, the pregnancy rate per fresh transfer decreased to 45.8% (47.4% in 2010) and increased to 33.6% (33.3% in 2010) per thawed transfer. The delivery rate after IUI-H decreased to 8.3 (8.9 in 2010), and to 12.2% (13.8% in 2010) after IUI-D. In IVF and ICSI cycles, 1, 2, 3 and 4+ embryos were transferred in 27.5, 56.7, 14.5 and 1.3% of cycles, respectively. The proportions of singleton, twin and triplet deliveries after IVF and ICSI (added together) were 80.8, 18.6 and 0.6%, respectively, resulting in a total multiple delivery rate of 19.2% compared with 20.6% in 2010, 20.2% in 2009, 21.7% in 2008, 22.3% in 2007 and 20.8% in 2006. In FER cycles, the multiple delivery rate was 13.2% (12.8% twins and 0.4% triplets). Twin and triplet delivery rates associated with IUI cycles were 9.7/0.6% and 7.3/0.3%, following IUI-H and IUI-D treatment, respectively. LIMITATIONS, REASONS FOR CAUTION The method of reporting varies among countries, and registers from a number of countries have been unable to provide some of the relevant data such as initiated cycles and deliveries. As long as data are incomplete and generated through different methods of collection, results should be interpreted with caution. WIDER IMPLICATIONS OF THE FINDINGS The 15th ESHRE report on ART shows a continuing expansion of the number of treatment cycles in Europe, with more than 600 000 cycles reported in 2011. Since 2006, the proportion of IVF to ICSI cycles has reached a plateau after a small decrease in 2009. Pregnancy and delivery rates after IVF remained relatively stable compared with 2010 and 2009. The pregnancy rate per aspiration in ICSI cycles declined for the first time by 0.9%. The multiple delivery rate is lower than ever before. STUDY FUNDING/COMPETING INTERESTS The study had no external funding; all costs are covered by ESHRE. There are no competing interests.

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.

The in vivo and in vitro efficiency and efficacy of PGD for aneuploidy

Preimplantation genetic diagnosis for aneuploidy was implemented on 1782 morphologically normal embryos generated in vitro by patients with a poor prognosis of pregnancy. Only 592 of them (34%) were diagnosed as chromosomally normal. Embryo transfer was accomplished in 240 cycles resulting in 79 clinical pregnancies (33%) and an implantation rate of 22.6%. The in vitro efficiency of the procedure was established by analysing all the blastomeres obtained from 311 non transferrable embryos and resulted to be 97.1%. The in vivo efficiency of the technique was calculated with the data derived from the prenatal diagnoses by examination of the infants at birth and was 97.8%. In consideration of the reported inaccuracy rate, patients are still recommended to undergo prenatal diagnosis. The transfer of PGD selected embryos in women of advanced reproductive age reduces by half the risk of having a trisomic pregnancy.

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.