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This proceedings report presents the outcomes from an international workshop supported by the European Society of Human Reproduction and Embryology (ESHRE) and Alpha Scientists in Reproductive Medicine, designed to establish consensus on definitions and recommended values for Indicators for the assisted reproductive technology (ART) laboratory. Minimum performance-level values ('competency') and aspirational ('benchmark') values were recommended for a total of 19 Indicators, including 12 Key Performance Indicators (KPIs), five Performance Indicators (PIs), and two Reference Indicators (RIs).

Oocyte donation is an efficient alternative to using own oocytes in IVF treatment for different indications. Unfortunately, 'traditional' (fresh) egg donations are challenged with inefficiency, difficulties of synchronization, very long waiting periods and lack of quarantine measures. Given the recent improvements in the efficiency of oocyte cryopreservation, it is reasonable to examine if egg donation through oocyte cryopreservation has merits. The objective of the current manuscript is to review existing literature on this topic and to report on the most recent outcomes from two established donor cryobank centres. Reports on egg donation using slow freezing are scarce and though results are encouraging, outcomes are not yet comparable to a fresh egg donation treatment. Vitrification on the other hand appears to provide high survival rates (90%) of donor oocytes and comparable fertilization, embryo development, implantation and pregnancy rates to traditional (fresh) egg donation. Besides the excellent outcomes, the ease of use for both donors and recipients, higher efficiency, lower cost and avoiding the problem of synchronization are all features associated with the benefit of a donor egg cryobank and makes it likely that this approach becomes the future standard of care. Oocyte donation is one of the last resorts in IVF treatment for couples challenged with infertility problems. However, traditional (fresh) egg donation, as it is performed today, is not very efficient, as typically all eggs from one donor are given to only one recipient, it is arduous as it requires an excellent synchronization between the donor and recipient and there are months or years of waiting time. Because of the development of an efficient oocyte cryopreservation technique, it is now possible to cryo-store donor (as well as non-donor) eggs, maintaining their viability and allowing their use whenever there is demand. Therefore, creating a donor oocyte cryobank would carry many advantages. In the present manuscript, the current experience with oocyte donation using cryopreservation technology is reviewed. The outcomes of two recently established donor egg cryobanks at Instituto Valenciano de Infertilidad in Spain and Reproductive Biology Associates in the USA (involving a large number of cases) demonstrate that egg cryo-survival is high and that fertilization, embryo development, implantation and pregnancy rates are similar to those reported after fresh egg donation. It also provides additional advantages of being more efficient, more economical, easier for both donors and recipients and potentially also safer, because eggs can now be quarantined for 6 months (or longer) to retest for infectious diseases in the donors. It is the opinion of the authors, based on several advantages associated with the use of donor egg cryobanking, that in the future there will be fewer traditional egg donations and increasingly more cryo-egg donations.

Until recently, success in oocyte cryopreservation has been very limited mainly due to poor understanding of the complex physiological processes that lead to cell damage during cryopreservation. In the past three decades, however, a wealth of information has been collected using various different animal models, which has led to development of new technologies and optimization of existing ones. The use of these models has provided the opportunity for research that may not have been possible with human material. Today, results of these studies still continue to form the basis of oocyte cryobiology. This review discusses these studies, especially the physiological impacts of cryopreservation on oocyte biology. It will also focus on the role that animal models have played in improvement strategies, validation before translating new techniques into the human model and the advances made in the human in IVF because of these animal models. Finally, existing investigations and their potential impact in other areas of research will be discussed. Until recently, success in oocyte cryopreservation has been very limited mainly due to poor understanding of the complex physiological processes that lead to cell damage during cryopreservation. In the past three decades, however, a wealth of information has been collected using various different animal models, which has led to development of new technologies and optimization of existing ones. The use of these models provided the opportunity for research that may not have been possible with human material. Today, animal models still continuously provide imperative data that facilitate further advancements in oocyte cryobiology. This review will focus on the physiological impacts, current improvement strategies and future applications of oocyte cryopreservation using animal models as they benefit not only human oocyte cryopreservation procedures, but also the human species through their usefulness in agriculture, medicine and conservation.

During vitrification, the glass-like solidification is the phase-transition process from liquid to solid. Phase transition is one of the major factors suspected to affect the physiology of the oocyte, such as the structure of the meiotic spindle. Therefore, it is very important to investigate the systematic and morphological alterations of the metaphase-II spindle and chromosome arrangement during complete course of a vitrification and warming process. B6D2F1 (C57BL/6 X DBA/2) mouse oocytes were cryopreserved by minimum volume cooling (MVC) method of vitrification in a solution with 15% ethylene glycol, 15% dimethylsulphoxide and 0.5 mol/l sucrose. To examine the spindle, oocytes were fixed before, during and after vitrification and were analysed by immunocytochemistry and confocal microscopy. It was shown that spindles in all oocytes could be maintained through the vitrification and warming process, even though they were exposed to extreme temperature and two rounds of phase transition. According to the sequential observations, chromosome alignment was maintained throughout the complete course of vitrification, warming and post-warming stage. The impact of phase transition was barely detectable when the oocyte was exposed to the vitrification and warming process. The oocyte spindle was able to recover immediately after warming.

The aim of this study was to determine whether the denuding procedure causes the polar body to move within the perivitelline space. Only those patients undergoing IVF who had unused in-vitro matured (IVM) oocytes were included in this study. IVM oocytes were initially viewed under a non-invasive, polarized light microscope. A laser was used to mark the location of the polar body on the zona. Oocytes were subjected to the denuding procedure with a 150 microm, 135 microm and 125 mum diameter pipette. After each pipetting, the oocytes were viewed again to determine whether the polar body had moved. After denuding, the oocyte was left to culture overnight and viewed 24 h later. After denuding with the 150 microm, 135 microm and 125 microm pipettes and after 24 h in culture, the average angle between the spindle and polar body was 15.4 +/- 10.4 degrees , 16.1 +/- 10.1 degrees , 20.9 +/- 11.7 degrees , and 26.7 +/- 18.2 degrees , respectively (P = 0.0021). Slight changes in angles were noted between denuding with the different diameter pipettes. The largest changes in angles were seen after 24 h in culture.

This study aimed to evaluate oocyte vitrification efficiency using in-vivo matured (IVO) versus rescued in-vitro matured (IVM) oocytes. The results show that oocyte survival (85% versus 81%), fertilization (86% versus 76%) and cleavage rate (98% versus 89%) was not significantly different in IVO oocytes compared with rescued IVM sibling oocytes. The fertilized oocytes from IVO and IVM groups were cultured to blastocyst stage; however, embryo development was significantly reduced in the rescued IVM group (72% versus 15%). Embryo transfer was only performed with the embryos derived from IVO oocytes on day 5; 42 blastocysts were transferred to 18 recipients; 16 of 18 recipients had positive beta-human chorionic gonadotrophin and a total of 26 fetal cardiac activities were detected in 15 recipients (implantation: 26/42, 61.9%). Ten of the 15 recipients have delivered 19 healthy babies, and the other five pregnancies are still ongoing. These data indicate that the combination of oocyte vitrification and rescued IVM not only yield a new strategy to extend the pool of total fertilizable oocytes, but also demonstrate that the efficiency of vitrified/warmed oocytes can be comparable to fresh oocytes with regard to clinical outcomes.

Increasing the efficiency of the IVF procedure by improving pregnancy/implantation rates and at the same time lowering (or avoiding) the risks of multiple gestations are the primary goals of the current assisted reproductive technology. These aims require a much improved gamete/embryo testing and selection procedure, which, using the current approach of microscopy-based morphology evaluation is unlikely to be achieved. Therefore, alternative or additional, non-invasive techniques have been proposed which may be able to detect alterations of the culture environment surrounding gametes/embryos reflective of the (patho-)physiological processes. One of the most recently applied approaches is to measure metabolomic changes in the culture medium of embryos and oocytes (‘exometabolomics'). Initial studies have demonstrated that different types of spectrophotometric tests, including Raman and near-infrared (NIR) techniques, are similarly well capable of detecting specific changes of the ‘secretome' (exometabolome). These studies have also demonstrated that metabolomic measurements correlate well with embryo development and morphology assessment. Furthermore, viability index on oocytes/embryos established by metabolomic tests may be a stronger predictor for implantation potential than traditional morphological assessment. Although the results of these initial investigations are promising, further prospective studies are required to define clearly the potential benefits and most relevant applications of this novel non-invasive technology in the field of assisted reproduction.

Using sibling oocytes, the objective of this study was to compare the intracytoplasmic sperm injection (ICSI) fertilization rates to those achieved with conventional IVF in patients with high rates of oocyte immaturity. This study was observational in nature, and included 91 patients who were treated using split insemination techniques. The fertilization rates for the ICSI group and the IVF group were 41.1 +/- 15.0% and 53.2 +/- 19.8%, respectively (Plt: 0.0001). There was no significant difference in day-3 embryo quality between the two groups. There was a significantly higher number of embryos frozen in the IVF group than in the ICSI group: 357 (84.8%) and 297 (76.7%), respectively (P = 0.037). Furthermore, the number of embryos either transferred or frozen was significantly higher in the IVF group than the ICSI group: 459 of 1173 (39.1%) and 385 of 1268 (30.4%), respectively (P0.0001). These data indicate that conventional IVF results in a higher fertilization rate than ICSI. Furthermore, IVF provided more embryos available for transfer or cryopreservation when compared with ICSI, thereby optimizing the patient's cycle.

The predictable answer to the provocative question of whether programmable freezers are still needed in the embryo laboratory is an even more provocative 'no'. However, such a radical statement needs strong support. Based on the extensive literature of the past 5 years, the authors collected arguments either supporting or contradicting their opinion. After an overview of the causes of cryoinjuries and strategies to eliminate them, the evolution of vitrification methods is discussed. Special attention is paid to the biosafety issues. The authors did not find any circumstance in oocyte or embryo cryopreservation where slow freezing offers considerable advantages compared with vitrification. In contrast, the overwhelming majority of published data prove that the latest vitrification methods are more efficient and reliable than any version of slow freezing. Application of the proper vitrification methods increases the efficiency of long-term storage of stem cells and opens new perspectives in cryopreservation of oocytes, both for IVF and somatic cell nuclear transfer. However, lack of support from regulatory authorities, and conservative approachs regarding novel techniques can slow down the implementation of vitrification. The opinion of the authors is that vitrification is the future of cryopreservation. The public have the final say in whether they want and allow this future to arrive.

During mitosis, a spindle checkpoint detects chromosome misalignment and halts the cell cycle progression. In meiosis of female germ cells, however, it is debatable whether such a checkpoint is present. This research employed a unique model in the mouse, mitotic chromosomes transferred to meiotic cytoplasts to investigate whether a meiotic oocyte's microtubule apparatus can effectively separate mitotic metaphase chromosomes, and whether a spindle checkpoint exists during its division. The intact germinal vesicle (GV) oocytes, enucleated GV cytoplasts, and enucleated GV cytoplasts at 15 hin-vitro maturation were transferred with a metaphase fibroblast cell. When mitotic chromosomes were transferred into enucleated or intact mouse GV oocytes, the first bipolar meiotic spindles were established and the reconstructed oocytes were able to extrude polar bodies. However, none of the reconstructed oocytes showed complete and accurate alignment of chromosomes, except the enucleated GV cytoplasts reconstructed after maturation. The spindle formation and polar body extrusion suggest that the first meiotic spindle was functional, and the chromosome misalignment did not prevent the onset of anaphase. The data indicate that a spindle checkpoint, providing surveillance of misaligned chromosomes, was overridden or compromised by the incompatibility between somatic chromosomes and meiotic spindles during the first meiotic division.

The birth of Louise Brown, the first IVF baby, in 1978 marked a breakthrough in infertility treatment. In recent decades, several important new techniques have been introduced. One limiting factor has been the requirement to use reproductive cells (gametes) for fertilization and for embryonic development. Somatic cell nuclear transfer (cloning) has been successful in mammals, opening a potential new approach for the treatment of human infertility. In addition, nuclear transfer to achieve embryo development starting from somatic cells instead of gametes, and the creation of artificial oocytes/spermatozoa has been attempted. The present paper reviews the various alternative approaches to haploidization of somatic cells. It has been observed that chromosome segregation (of the donor somatic nucleus) may take place; however, this process is largely random, thus leading to major cytogenetic abnormalities. An alternative approach is related to stem cell technology, to be further explored in the future. Culture conditions may be adjusted so that the totipotent embryonic stem cells will differentiate to specific gametes, sperm cells or egg cells. Injecting spermatozoa produced in this manner into recipient oocytes has led to pronuclear formation and early cleavage stages in some embryos. Finally, the birth of parthenogenetic mice indicates that some of these epigenetic problems can be overcome, and that some of the embryos may survive to birth.

© 2008 Published by Reproductive Healthcare Ltd, Duck End Farm, Dry Drayton, Cambridge CB3 8DB, UK Zsolt Peter Nagy obtained his MD (1986) and his specialism in Obstetrics and Gynaecology (1996) at the Semmelweis Medical University in Budapest. His PhD was granted at the Free University of Brussels in 1997, where he worked from 1991–1997 in the Centre for Reproductive Medicine as part of the team that developed the procedure for intracytoplasmic sperm injection. He developed a new technique for fusion between karyoplast and cytoplast for nuclear transfer (2000). He is author or co-author of more than 100 publications. At present, he is the scientific and laboratory director of Reproductive Biology Associates in Atlanta, USA.

For a long time, the cryopreservation of gametes and embryos remained a major hurdle for the clinicians and scientists in terms of success. However, recent technical advancement in the field of cryobiology has opened up various options for freezing gametes and embryos at different developmental stages. The tendency of the IVF world to switch over to natural-cycle IVF and to elective single-embryo transfer has put cryotechnology in the forefront of research. Still, the intricacies of the cold-induced changes in human gametes and embryos that could affect the intracellular and developmental processes need to be known. The transcriptomics, proteomics and metabolomic platforms hold promise for elucidating these complex processes during cryopreservation processes.

The aim of the current study was to evaluate whether near-infrared (NIR) spectroscopy-generated metabolomic data obtained from oocyte culture samples would correlate with nuclear maturity status and derived embryo development. A total of 412 oocyte culture samples were collected from 43 patient cycles. Metabolomic profiles of metaphase I and II oocytes were obtained by NIR spectroscopy and were significantly different from each other and from profiles of prophase I (germinal vesicle) oocytes (P +/- 0.001 at the 95% confidence interval). Additionally, NIR spectroscopic analysis of culture medium of oocytes that developed to grade A embryos on day 3 demonstrated significantly higher viability indices (0.62 +/- 0.23) than those that developed to grades C/D (0.42 +/- 0.26; P0.006); and on day 5 grade A (0.37 +/- 0.20) was also higher than grades C/D (0.14 +/- 0.21; P0.02). Metabolomic profiles of oocytes that resulted in pregnancy had higher viability indices (0.87 +/- 0.27) than those that did not (0.44 +/- 0.17; P0.0001). The results of the current study demonstrate that metabolomic profiling from spent culture medium of the oocyte is related to nuclear maturity, is able to predict embryo development at day 3 and day 5 stages, and relates to embryo viability.

Italian legislation regarding reproductive medicine prohibits embryo storage while allowing cryopreservation of supernumerary oocytes. This study evaluated the effect of fresh oocytes obtained from natural unstimulated cycles on the clinical success rates derived from the use of frozen-thawed (FR-TH) oocytes obtained following ovarian stimulation. For 36 women, intracytoplasmic sperm injection was performed on FR-TH oocytes supplemented by a fresh oocyte, if available, derived from a natural cycle in which gonadotrophin-releasing hormone-antagonist was used for premature LH surge control. The retrieval rate of fresh oocytes was 61.1% and survival rate of FR-TH oocytes was 43.6%. The fertilization rate of fresh and FR-TH oocytes was 70% and 52.5%, respectively. Fifty embryos were transferred, 14 of them developed from fresh oocytes and 36 from FR-TH oocytes. Six pregnancies occurred in 10 cycles in which the embryos developed from fresh and FR-TH oocytes (pregnancy rate 60.0%) and two in 12 patients in whom the embryos were obtained from only FR-TH oocytes (pregnancy rate 16.7%) (P < 0.05). In summary, the data demonstrate that the transfer of embryos derived from oocytes cryopreserved following a previous ovarian stimulation and an embryo developed from a fresh one retrieved in natural cycle ensures an excellent clinical outcome.

Increasing the efficiency of the IVF procedure by improving pregnancy/implantation rates and at the same time lowering (or avoiding) the risks of multiple gestations are the primary goals of the current assisted reproductive technology. These aims require a much improved gamete/embryo testing and selection procedure, which, using the current approach of microscopy-based morphology evaluation is unlikely to be achieved. Therefore, alternative or additional, non-invasive techniques have been proposed which may be able to detect alterations of the culture environment surrounding gametes/embryos reflective of the (patho-)physiological processes. One of the most recently applied approaches is to measure metabolomic changes in the culture medium of embryos and oocytes ('exometabolomics'). Initial studies have demonstrated that different types of spectrophotometric tests, including Raman and near-infrared (NIR) techniques, are similarly well capable of detecting specific changes of the 'secretome' (exometabolome). These studies have also demonstrated that metabolomic measurements correlate well with embryo development and morphology assessment. Furthermore, viability index on oocytes/embryos established by metabolomic tests may be a stronger predictor for implantation potential than traditional morphological assessment. Although the results of these initial investigations are promising, further prospective studies are required to define clearly the potential benefits and most relevant applications of this novel non-invasive technology in the field of assisted reproduction.

Transfer of human embryos at the blastocyst stage may offer considerable benefits including an increased implantation rate and a decreased risk of multiple pregnancies; however, blastocyst culture requires an efficient and reliable in-vitro embryo culture system. In this study, the effect of the Well-of-the-Well (WOW) system consisting of microwells formed on the bottom of the culture dish was tested in three mammalian species, including humans. The WOW system resulted in significant improvement when comparing the drops for culture of in-vitro-matured and parthenogenetically activated porcine oocytes, and in-vivo-derived mouse zygotes. In human embryos, using a sibling oocyte design, embryos cultured in WOW developed to the blastocyst stage in a significantly higher proportion than did embryos cultured traditionally (55% in WOW and 37% in conventional culture; P < 0.05). In a separate study, also in human, a total of 48 patients with a cumulative 214 unsuccessful previous IVF cycles were selected for the trials. In subsequent intracytoplasmic sperm injection cycles, oocytes/embryos were cultured individually in the WOW system or in microdrops. Transferable quality blastocyst development (48.9% of cultured zygotes) was observed in the WOW system. Ninety-four blastocysts transferred to 45 patients resulted in clinical pregnancy rates of 48.9%, including nine twin pregnancies, seven single pregnancies, five miscarriages and one ectopic pregnancy. The results indicate that the WOW system provides a promising alternative for microdrop culture of mammalian embryos, including human embryos.

Virtually all normal cells can reproduce themselves. The germ cells, however, can initiate reproduction of the entire organism. A special sequence of events, meiosis, is responsible for generating mature germ cells bearing a haploid genome. The basic features of meiosis, i.e. two cell divisions with no intervening DNA replication, resulting in a halving of the chromosome complement, are evident throughout evolution. The idea of generating an artificial gamete was recently put forward to provide an ultimate solution for the treatment of infertility. Currently, there are different strategies to create artificial gametes in vitro, such as converting somatic cells from mitotic division to meiotic division directly (somatic cell haploidization), or de-differentiating somatic cells into embryonic stem (ES) cells and re-differentiating ES cells into gametes, or extracting adult stem cells and re-differentiating them into gametes.

Recent clinical reports not only show that cryopreserved embryos can be successfully used for human fertility treatment, but also that cryopreserved oocytes may be used successfully as an adjunct to human assisted reproductive technologies. Vitrification is known to establish a glass-like solid state during the cooling process. The high concentration of cryoprotectants and an extremely rapid rate of cooling are responsible for the formation of the solid state, and also prevent formation of intracellular ice crystals. Hence, in theory, vitrification should minimize cryo-injuries, and therefore has great promise for oocyte and embryo cryopreservation. This article describes two pregnancies from vitrified–warmed blastocysts obtained after intracytoplasmic sperm injection fertilization of vitrified–warmed oocytes. Vitrification was employed to cryopreserve the oocytes and the subsequent blastocysts. The results present the intriguing implication that vitrification may serve as an efficient method for clinical oocyte cryopreservation and embryo re-cryopreservation.

Intracytoplasmic sperm injection (ICSI) provides an excellent outcome in a consistent manner, and is therefore used worldwide as a routine procedure. Since its introduction, few modifications have been made to its methodology. Recently, a combination of ICSI with micro-hole drilling by laser (LA-ICSI) of the zona pellucida appeared to decrease oocyte degeneration rates and to improve embryo quality and implantation. Cytoplasmic transfer is a more recently introduced procedure where the objective is to improve the quality of patients' oocytes by transferring cytoplasm from a good quality donor oocyte, in cases where it is assumed that cytoplasm is compromised. Nuclear transfer, involving exchange of nuclei between donor and receptor oocytes, is still an experimental procedure, the objective being similar to cytoplasmic transfer in improving oocyte/embryo quality. A nuclear transfer procedure involving somatic cells for reproductive purposes should not be used in humans, for ethical and technical considerations. On the other hand, nuclear transfer for therapeutic purposes to obtain stem cells may be considered in respect of its unique potential in medicine. Finally, the most recently emerged new concept under investigation is the haploidization of somatic cells for the purpose of creating artificial gametes.

This retrospective review study, carried out in a private IVF clinic, compared pregnancy and implantation rates with day 3 versus day 5 embryo transfers in a selected group of patients. Participants were patients who failed to achieve pregnancy in at least one previous attempt with embryo transfer on days 2 or 3, and had more than five oocytes fertilized. A total of 296 patients who had undergone day 3 (group A) transfers were compared with 154 who had undergone day 5 transfers (group B). Interventions were intracytoplasmic sperm injection (ICSI), day 3 and day 5 embryo transfer. Outcome measures were pregnancy, implantation, multiple gestation and blastocyst formation rates. Overall, 86.4% of embryos were at the six- to eight-cell stage at 72 h and 30% developed to blastocyst by day 5. The mean number of embryos transferred was 4.0 on day 3 and 3.0 on day 5. Pregnancy and implantation rates were 34.8 and 11.5% in group A, versus 45.3 and 18.5% in group B. Multiple gestation rate was 47.1% in group A and 28.5% in group B. Prolonging embryo culture in vitro to day 5 improved embryo selection and implantation rates. A significant decrease in high order gestations was achieved by reducing the number of embryos transferred, without compromising the pregnancy rates.

An inadvertent consequence of intracytoplasmic sperm injection (ICSI) is the degeneration of some of the microinjected oocytes. Most patients may not suffer any disadvantage through losing oocyte(s) during micromanipulation; however, in some circumstances, this can result in a reduction of the chances for pregnancy. This study reports a clinical pregnancy obtained by a novel approach using laser-assisted micro-opening of the zona pellucida prior to ICSI to secure a non-traumatic microinjection that avoids degeneration of oocytes. A total of 12 oocytes were obtained from the 36 year old patient in her third IVF treatment cycle, following two previously failed attempts where very high degeneration rates of oocytes after ICSI were recorded, together with suboptimal embryo quality. Five of the 11 matured (MII) oocytes were submitted to conventional ICSI and the other six MII oocytes first underwent laser-assisted opening of the zona pellucida (5-7 microm hole size was created with a 1.48 microm diode laser) before microinjection (LA-ICSI). Three of the five conventionally microinjected oocytes degenerated while one oocyte fertilized normally and developed to a good quality embryo. After the LA-ICSI procedure, one of the six oocytes degenerated and four oocytes fertilized normally; of these, two developed to excellent quality embryos, one to a good quality embryo and one to a poor quality embryo. The three best embryos (LA-ICSI group) were transferred to the patient on day 3. Rising serum human chorionic gonadotrophin concentrations were measured 12 days after transfer and on week 7 two implantation sites were detected, together with regular heart activity. The results of the present report suggest that laser-assisted ICSI may provide a safer approach to non-traumatic microinjection of oocytes than conventional ICSI, thereby minimizing the risk of degeneration and possibly also improving embryo quality. Therefore, it is suggested that laser-assisted ICSI might be applied in all cases associated with difficult zona pellucida penetration or/and fragile oolemma, or where patients have very few oocytes available, to improve the chances for pregnancy.

This report presents a case of triplet heterotopic gestation after intracytoplasmic sperm injection (ICSI)-IVF treatment, with a left ruptured ectopic tubal implantation with a living embryo and successful outcome of the concurrent intrauterine twin gestation. A couple whose infertility was caused by oligoasthenozoospermia was referred for ICSI treatment. Three good quality embryos were transferred at the request of the patient. Early gestational control was performed by ultrasound at weeks 5 and 7 of gestation. The patient reported to the centre during week 7 with severe abdominal pain and with signs of peritoneal irritation. Transvaginal ultrasound revealed an extra-uterine ruptured implantantion. During the concomitantly performed laparoscopic procedure, a living embryo was observed after opening the extra-uterine embryonic sac. Heartbeat activity was present and lasted for 5 min after surgical resection of the tubal implantation. The patient was discharged from hospital without complications. The intrauterine twin gestation was not affected and two healthy infants were born at week 38 of gestation. Heterotopic pregnancy should be ruled out in patients submitted to IVF-embryo transfer, although no predisposing factors are present in some cases. Precise diagnosis may be delayed due to some important characteristics of the IVF-embryo transfer treatment. Nevertheless, this condition should be diagnosed by ultrasound before tubal rupture to avoid obvious complications. Laparoscopy remains the gold standard for diagnosis and treatment in these cases. The presentation of the heterotopic pregnancy was recorded on video and may be viewed on the internet at www.rbmonline.com/Article/710.