Marcos Meseguer

The use of morphokinetics as a predictor of embryo implantation

BACKGROUND Time-lapse observation presents an opportunity for optimizing embryo selection based on morphological grading as well as providing novel kinetic parameters, which may further improve accurate selection of viable embryos. The objective of this retrospective study was to identify the morphokinetic parameters specific to embryos that were capable of implanting. In order to compare a large number of embryos, with minimal variation in culture conditions, we have used an automatic embryo monitoring system. METHODS Using a tri-gas IVF incubator with a built-in camera designed to automatically acquire images at defined time points, we have simultaneously monitored up to 72 individual embryos without removing the embryos from the controlled environment. Images were acquired every 15 min in five different focal planes for at least 64 h for each embryo. We have monitored the development of transferred embryos from 285 couples undergoing their first ICSI cycle. The total number of transferred embryos was 522, of which 247 either failed to implant or fully implanted, with full implantation meaning that all transferred embryos in a treatment implanted. RESULTS A detailed retrospective analysis of cleavage times, blastomere size and multinucleation was made for the 247 transferred embryos with either failed or full implantation. We found that several parameters were significantly correlated with subsequent implantation (e.g. time of first and subsequent cleavages as well as the time between cleavages). The most predictive parameters were: (i) time of division to 5 cells, t5 (48.8-56.6 h after ICSI); (ii) time between division to 3 cells and subsequent division to 4 cells, s2 (≤ 0.76 h) and (iii) duration of cell cycle two, i.e. time between division to 2 cells and division to 3 cells, cc2 (≤ 11.9 h). We also observed aberrant behavior such as multinucleation at the 4 cell stage, uneven blastomere size at the 2 cell stage and abrupt cell division to three or more cells, which appeared to largely preclude implantation. CONCLUSIONS The image acquisition and time-lapse analysis system makes it possible to determine exact timing of embryo cleavages in a clinical setting. We propose a multivariable model based on our findings to classify embryos according to their probability of implantation. The efficacy of this classification will be evaluated in a prospective randomized study that ultimately will determine if implantation rates can be improved by time-lapse analysis.

Use of cryo-banked oocytes in an ovum donation programme: a prospective, randomized, controlled, clinical trial

BACKGROUND An efficient oocyte cryopreservation method is mandatory to establish a successful egg-banking programme. Although there are increasing reports showing good clinical outcomes after oocyte cryopreservation, there is still a lack of large controlled studies evaluating the effectiveness of oocyte cryo-banking. In this study, we aimed to compare the outcome of vitrified-banked oocytes with the gold standard procedure of employing fresh oocytes. METHODS A randomized, prospective, triple-blind, single-centre, parallel-group controlled-clinical trial (NCT00785993), including 600 recipients (alpha = 0.05 and power of 80% for sample-size calculation) selected among 1032 eligible patients from November 2008 to September 2009, was designed to compare the outcome of vitrified-banked oocytes with the gold standard procedure of employing fresh oocytes. The study was designed to establish the superiority of the ongoing pregnancy rate (OPR) of fresh oocytes over that of vitrified oocytes, by performing a likelihood ratio test in a logistic regression analysis expressed as odds ratio (OR) with 95% confidence interval (CI). A limit of 0.66 for OR of vitrified versus fresh groups was defined to set up a possible conversion from superiority to non-inferiority. Randomization was performed 1:1 based on a computer randomization list in vitrification (n = 300) or fresh groups (n = 300). The primary end-point was the OPR per randomized patient i.e. intention-to-treat population (ITT). Secondary end-points were clinical pregnancy (CPR), implantation (IR) and fertilization rates, respectively. Additionally, embryo developmental characteristics were recorded. RESULTS There were no differences in donor ovarian stimulation parameters, demographic baseline characteristics for donors and recipients, ovum donation indications or male factor distribution between groups (NS). The OPR per ITT was 43.7 and 41.7% in the vitrification and fresh groups, respectively. The OR of OPR was 0.921 in favour of the vitrification group. Nevertheless, the 95% CI was 0.667-1.274, thus the superiority of fresh group with respect to OPR was not proven (P = 0.744). Non-inferiority of the vitrified group compared with the fresh group was shown with a margin of 0.667, which was above the pre-established non-inferiority limit of 0.66. CPR per cycle (50.2 versus 49.8%; P = 0.933) or per embryo-transfer (55.4 versus 55.6% ; P = 0.974), and IR (39.9 versus 40.9%; P = 0.745) were similar for patients receiving either vitrified or fresh oocytes. The proportion of top-quality embryos obtained either by inseminated oocyte (30.8 versus 30.8% for Day-2; and 36.1 versus 37.7% for Day-3, respectively) or by cleaved embryos (43.6 versus 43.8% for Day-2 and 58.4 versus 60.7% for Day-3, respectively) was similar between groups (NS). CONCLUSIONS This controlled-randomized, clinical trial confirmed the effectiveness of oocyte cryo-storage in an ovum donation programme, failing to demonstrate the superiority of using fresh oocytes with respect to the use of vitrified egg-banked ones in terms of OPR. Instead, the non-inferiority of vitrified oocytes was confirmed. These findings involve highly relevant issues that may open a new range of possibilities in ART.

Human Endometrial Mucin MUC1 Is Up-Regulated by Progesterone and Down-Regulated In Vitro by the Human Blastocyst

Abstract Expression of MUC1 in endometrial epithelium has been suggested to create a barrier to embryo attachment that must be lifted at the time of implantation. In this study, we investigated the hormonal regulation of human endometrial MUC1 in hormone replacement therapy cycles and in the human blastocyst. We also analyzed the embryonic regulation of MUC1 in human endometrial epithelial cells (EECs) during the apposition and adhesion phases of human implantation using two different in vitro models. Our results indicate that endometrial MUC1 mRNA and immunoreactive protein increase in receptive endometrium compared to nonreceptive endometrium. Human blastocysts express MUC1, as demonstrated by reverse transcription-polymerase chain reaction and immunocytochemistry, localized at the trophectoderm. In vitro, MUC1 was present at the surface of primary cultures of human EEC, and presence of a human blastocyst (i.e., apposition phase) increases EEC MUC1 protein and mRNA compared to control EEC lacking embryos. Interestingly, when human blastocysts were allowed to attach to the EEC monolayer (i.e., adhesion phase), MUC1 was locally removed in a paracrine fashion on EEC at the implantation site. These results demonstrate a coordinated hormonal and embryonic regulation of EEC MUC1. Progesterone combined with estradiol priming induces an up-regulation of MUC1 at the receptive endometrium. During the apposition phase, presence of a human embryo increases EEC MUC1. However, at the adhesion phase, the embryo induces a paracrine cleavage of EEC MUC1 at the implantation site. These findings strongly suggest that MUC1 may act as an endometrial antiadhesive molecule that must be locally removed by the human blastocyst during the adhesion phase.

Female obesity impairs in vitro fertilization outcome without affecting embryo quality

OBJECTIVE To compare embryo quality and reproductive outcome in our IVF program according to the womens body mass index (BMI). DESIGN Retrospective study. SETTING University-affiliated infertility clinic, between January 2001 and April 2007. PATIENT(S) Women undergoing a total of 6,500 IVF-intracytoplasmic sperm injection (ICSI) cycles. INTERVENTION(S) Six thousand five hundred IVF-ICSI cycles were included and divided into four groups: lean (<20 kg/m(2); n = 1,070; 16.5%); normal (20-24.9 kg/m(2); n = 3,930; 60.5%); overweight (25-29.9 kg/m(2); n = 1,081; 16.6%); and obese (> or =30 kg/m(2); n = 419; 6.4%). MAIN OUTCOME MEASURE(S) Comparison of embryo quality and reproductive outcome (implantation, pregnancy, miscarriage, and live birth rates) among BMI groups. RESULT(S) No difference in insemination procedure, fertilization rate, day of ET, mean number of transferred and cryopreserved embryos, percentage of blastocyst transfers, or embryo quality on day 2 and 3 was found among groups. However, implantation, pregnancy, and live birth rates were poorer in obese women. In fact, pregnancy and live birth rates were reduced progressively with each unit of BMI (kilograms per square meter) with a significant odds ratio of 0.984 (95% confidence interval 0.972-0.997) and 0.981 (95% confidence interval 0.967-0.995), respectively. In addition, the cumulative pregnancy rate after four IVF cycles was reduced as BMI increased. CONCLUSION(S) Female obesity impairs IVF outcome, but embryo quality is not affected, pointing to an alteration in the uterine environment.

Testicular sperm extraction (TESE) and ICSI in patients with permanent azoospermia after chemotherapy

BACKGROUND Patients persistently azoospermic after chemotherapy have been considered traditionally as sterile unless sperm was frozen before therapy. Recent advances during the last decade combining testicular sperm extraction (TESE) and ICSI in patients with non-obstructive azoospermia allow these males to father their own genetic offspring. METHODS A retrospective study was conducted of 12 patients with non-obstructive azoospermia after chemotherapy undergoing TESE between 1995 and 2002. Cancer type and anti-neoplastic treatments were recorded, together with maximum testicular volume, serum FSH levels and testicular histopathology. When TESE was successful, spermatozoa were cryopreserved for performing ICSI later. RESULTS In five patients (41.6%) motile spermatozoa for cryopreservation and ICSI were retrieved. Four of them had received chemotherapy for testicular cancer, and one had been treated by chemotherapy/radiotherapy for Hodgkins disease. Clinical and histological parameters were unable to predict with certainty TESE outcome in an individual patient. Eight ICSI cycles were performed on five couples and one pregnancy was obtained which resulted in the delivery of a healthy girl. CONCLUSION Some patients with permanent azoospermia after chemotherapy can be successfully treated by TESE-ICSI. This procedure, however, may have potential genetic risks. Therefore, freezing semen before starting gonadotoxic therapy is the strategy of choice, and patients should be counselled accordingly.

Embryo quality, blastocyst and ongoing pregnancy rates in oocyte donation patients whose embryos were monitored by time-lapse imaging

PurposeIn the current study, our aim was to demonstrate that EmbryoScope incubation conditions is comparable to standard laboratory incubation circumstances by comparing embryo quality, development and ongoing pregnancy rates between the EmbryoScope (ES) and a standard incubator (SI). We analyzed 478 embryos from 60 couples undergoing oocyte donation were included in the study.MethodsAll embryos retrieved from a patient were randomly distributed in the ES or SI. We calculated blastocyst development rate, blastocyst viability and ongoing pregnancy rate for embryo transfers from ES, SI and mixed (one embryo from the ES and one from the SI). Statistical analysis was conducted by Chi square tests, considering p < 0.05 significant.ResultsNo significant differences were found between the ES and SI from all the parameters evaluated.ConclusionsThus we concluded that time-lapse monitoring in the EmbryoScope does not impair embryo quality while allowing for morphological, spatial and temporal analysis of embryo development.

Timing of cell division in human cleavage-stage embryos is linked with blastocyst formation and quality.

Noninvasive markers of embryo quality are being sought to improve IVF success. The present study aimed to discover possible associations between embryo division kinetics in the cleavage stage, the subsequent ability of human embryos to reach the blastocyst stage and the resulting blastocyst morphology. A retrospective cohort study analysed 834 embryos from 165 oocyte donation couples using a time-lapse monitoring system that allowed the recording of the exact timings for key events related to embryo development. Timing parameters were categorized into four quartiles. The probability of an embryo developing to a blastocyst was linked to a strict chronology of development. To further evaluate the relationships between these morphokinetic parameters and subsequent blastocyst formation, the ensuing blastocyst morphology was compared with a viability score based on a hierarchical classification of the cleavage-stage morphokinetic parameters. It is concluded that the kinetics of early embryo development and the potential for human embryos to develop to the blastocyst stage on day 5 are closely related and that time-lapse-based evaluation of the exact timing of early events in embryo development is a promising tool for the prediction of blastocyst formation and quality according to the proposed model.

Microarray analysis in sperm from fertile and infertile men without basic sperm analysis abnormalities reveals a significantly different transcriptome

Sperm analysis following World Health Organization guidelines is unable to explain the molecular causes of male infertility when basic sperm parameters are within a normal range and women do not present gynecologic pathology. Consequently, there is a need for accurate diagnostic tools in this area, and microarray technology emerges as promising. We present, for the first time, preliminary results of a comparison of sperm mRNA expression profiles between fertile and infertile men with normal semen parameters, discovering profound discrepancies between groups, with potential diagnostic and therapeutic possibilities.

Effect of sperm DNA fragmentation on pregnancy outcome depends on oocyte quality

OBJECTIVE To quantify the effect of sperm DNA fragmentation (SDF) on reproductive outcome by evaluating the most statistically significant bias factors using logistic regression. DESIGN Prospective blind observational cohort study. SETTING University affiliated private IVF unit. PATIENT(S) Two hundred ten male partners of couples undergoing in vitro fertilization (IVF) or first intracytoplasmic sperm injection (ICSI) cycles with fresh or thawed sperm with the womens own or donated oocytes. INTERVENTION(S) None. MAIN OUTCOME MEASURE(S) SDF determined before and after swim-up (n=420), odds ratio calculated of the effect of an increase of one unit of SDF on pregnancy, and stratified regression analysis performed to evaluate the confusion effect of oocyte quality, sperm origin, and the fertilization procedure. RESULT(S) The effect of SDF on pregnancy was not affected by sperm origin (fresh or thawed) or fertilization procedure when measured both before and after swim-up. When oocytes from infertile patients were employed, SDF had a statistically significant negative impact on chance of pregnancy. For every 10% increase in SDF, the probability of not achieving pregnancy increased by 1.31. When donated oocytes were employed, SDF did not have a statistically significant effect. CONCLUSION(S) The effect of SDF on the probability of pregnancy can be calculated independent of the fertilization procedure or sperm origin. Oocyte quality conditions the extent of the negative impact of SDF on pregnancy; this can be overcome when good quality oocytes are employed.

Selection of high potential embryos using time-lapse imaging: the era of morphokinetics

Incorporation of time-lapse imaging in the field of IVF has provided much information about embryo development. The combination of the embryo appearance (morphology) and the importance of when and how the cellular processes that lead to this appearance occur (kinetics) are now integrated into the unique concept of morphokinetics. At present, efforts are focused on using this information to improve embryo selection and existing success rates without losing sight of the ever-present objective of implementing a single ET strategy to avoid multiple gestations. Several investigative groups have identified predictive morphokinetic variables for embryo viability and implantation potential. Promising supplementary models of embryo selection based on time-dependent markers have been proposed and are currently being verified in prospective trials. Pending further results of these studies that confirm the clinical validity of time-lapse imaging, the present article tries to summarize the studies conducted to date to take stock of what has been done and the prospects that are emerging from this technology.