Kersti Lundin

Preimplantation genetic screening in women of advanced maternal age caused a decrease in clinical pregnancy rate: a randomized controlled trial

BACKGROUND Advanced maternal age (AMA) is an important parameter that negatively influences the clinical pregnancy rate in IVF, in particular owing to the increased embryo aneuploidy rate. It has thus been suggested that only transferring euploid embryos in this patient group would improve the pregnancy rate. The purpose of this study was to test whether employing preimplantation genetic screening (PGS) in AMA patients would increase the clinical pregnancy rate. METHODS We conducted a two-center, randomized controlled trial (RCT) to analyze the outcome of embryo transfers in AMA patients (>or=38 years of age) after PGS using FISH analysis for chromosomes X, Y, 13, 16, 18, 21 and 22. The PGS group was compared with a control group. The primary outcome measure was clinical pregnancy rate after 6-7 weeks of gestation per randomized patient. RESULTS The study was terminated early as an interim analysis showed a very low conditional power of superiority for the primary outcome. Of the 320 patients calculated to be included in the study, 56 and 53 patients were randomized into the PGS and control groups, respectively. The clinical pregnancy rate in the PGS group was 8.9% (95% CI, 2.9-19.6%) compared with 24.5% (95% CI, 13.8-38.3%) in the control group, giving a difference of 15.6% (95% CI, 1.8-29.4%, P = 0.039). CONCLUSIONS Although the study was terminated early, this RCT study provides evidence against the use of PGS for AMA patients when performing IVF. TRIAL REGISTRATION NUMBER ISRCTN38014610.

Derivation of a Xeno‐Free Human Embryonic Stem Cell Line

Elimination of all animal material during both the derivation and long‐term culture of human embryonic stem cells (hESCs) is necessary prior to future application of hESCs in clinical cell therapy. The potential consequences of transplanting xeno‐contaminated hESCs into patients, such as an increased risk of graft rejection [Stem Cells 2006;24:221–229] and the potential transfer of nonhuman pathogens, make existing hESC lines unsuitable for clinical applications. To avoid xeno‐contamination during derivation and culture of hESCs, we first developed a xeno‐free medium supplemented with human serum, which supports long‐term (>50 passages) culture of hESCs in an undifferentiated state. To enable derivation of new xeno‐free hESCs, we also established xeno‐free human foreskin fibroblast feeders and replaced immunosurgery, which involves the use of guinea pig complement, with a modified animal‐product‐free derivation procedure. Here, we report the establishment and characterization (>20 passages) of a xeno‐free pluripotent diploid normal hESC line, SA611.

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.

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.

Fertilization and pregnancy after intracytoplasmic microinjection of acrosomeless spermatozoa

Spermatozoa lacking acrosomes were injected into the cytoplasm of mature human oocytes. In two subsequent cycles, 12 of 28 (43%) oocytes were fertilized, and the ET of the second cycle resulted in a twin pregnancy. This report describes, to the best of our knowledge, the first case of successful fertilization and delivery after using acrosomeless spermatozoa. Our hope is that with increasing experience with microinjection, in the near future, this type of infertility will not remain a serious problem.

Chromosomal integrity maintained in five human embryonic stem cell lines after prolonged in vitro culture

There have been recent reports of human embryonic stem cell (hESC) lines developing chromosomal aberrations after long-term culture, indicating an unstable genomic status due to the in vitro milieu. This raises concern, since it would limit their use in therapeutics. In this study the chromosomal status of five well-characterized hESC lines, SA002, SA002.5, AS034.1.1, SA121 and SA461, was monitored during long-term in vitro culture. The criteria of defined hESCs were met by all of the five hESC lines (four diploid and one trisomic for chromosome 13). The genomes were screened for chromosomal aberrations and rearrangements using comparative genomic hybridization (CGH), interphase fluorescence in situ hybridization (FISH) and traditional karyotyping on several occasions while in culture. The genomic integrity was shown to be maintained after repeated freeze-thaw procedures and continuous culture in vitro for up to 22 months (148 passages). We discuss the most common de novo chromosomal aberrations reported in hESCs, as well as their possible origin.

There Is a Cutoff Limit in Diameter Between a Blastomere and a Small Anucleate Fragment

AbstractPurpose: To document the DNA content of blastomeres/fragments from early human preembryos and to determine if there is a “cutoff” diameter at which a cell should be consi- dered an anucleate fragment rather than a blastomere. Methods: Surplus embryos from in vitro fertilization were used. Individual cells were measured, fixated, and stained for DNA. Results: In day 2 preembryos, only 2% of cells with a diameter <45 μm contained DNA, compared with 67% of those ≥45 μm. In day 3 preembryos, 3% of cells <40 μm contained DNA, compared with 66% of those ≥40 μm. Conclusions: It is suggested that cells <45 μm in day 2 preembryos, and <40 μm in day 3 preembryos should be classified as fragments, and cells larger than this, as blastomeres. This may influence the embryo scoring system for in vitro fertilization. We therefore recommend that cells within this critical range should be measured when scoring preembryos for embryo transfer.

Association between blastocyst morphology and outcome of single-blastocyst transfer

The aim of this study was to assess the ability of three individual blastocyst morphology parameters - expansion and hatching (EH) stage, inner cell mass (ICM) grade and trophectoderm grade - to predict outcome of a cycle with single-blastocyst transfer. The study was a secondary analysis of data prospectively collected in a large multicentre trial. A total of 618 intracytoplasmic sperm injection patients undergoing ovarian stimulation in a gonadotrophin-releasing hormone antagonist cycle with compulsory single-blastocyst transfer on day 5 were included. In the simple logistic regression analysis, all three blastocyst morphology parameters were statistically significantly (P<0.005 for each) associated with positive human chorionic gonadotrophin, clinical and ongoing pregnancy rates and live birth rates, while only the ICM grade was significantly (P=0.033) associated with early pregnancy loss rate. Blastocyst EH stage was the only significant predictor of live birth (P=0.002) in the multiple logistic regression. In conclusion, although all three blastocyst morphology parameters were related to treatment outcome of fresh single-blastocyst cycles, selection of high-quality blastocysts for transfer should consider first the EH stage. Transfer of a blastocyst with ICM grade A may reduce the risk of early pregnancy loss. Choosing the embryo(s) with the best implantation potential is essential for securing each couple the highest chance of achieving pregnancy after assisted reproduction. The selection of embryo(s) for transfer at the blastocyst stage is based on morphology parameters of expansion and hatching stage, inner cell mass grade and trophectoderm grade. The aim of this study was to assess the relative impact of each parameter in predicting the probability of a successful outcome. The study was a secondary analysis of data prospectively collected in a large multicentre trial. A total of 618 patients who underwent single-blastocyst transfer on day 5 were included. Statistical analysis showed that all three blastocyst morphology parameters were significantly associated with positive human chorionic gonadotrophin (βHCG), clinical and ongoing pregnancy rates and live birth rates. Only the inner cell mass grade was significantly associated with early pregnancy loss between the positive βHCG test and confirmation of ongoing pregnancy 10-11weeks after transfer. The expansion and hatching stage was the only significant predictor of live birth in the multiple logistic regression analysis. In conclusion, although all three blastocyst morphology parameters were related to treatment outcome of fresh single-blastocyst cycles, selection of high-quality blastocysts for transfer should consider first the expansion and hatching stage. Transfer of a blastocyst with inner cell mass grade A may reduce the risk of early pregnancy loss.

Cumulative impact of adding frozen-thawed cycles to single versus double fresh embryo transfers

Randomized control trials have shown that single embryo transfer (SET) results in lower live birth rates than double embryo transfer (DET), while observational, retrospective studies find no decrease in overall live birth rate when using a SET policy. The cumulative (fresh transfer followed by frozen - thawed transfers of embryos from the same stimulated cycle) live birth rate after the first and the second stimulated cycle of SET and DET respectively has been analysed. All couples who received their first fresh embryo transfer at Sahlgrenska University Hospital during 2003 and 2004 were included (n = 689). The live birth rates after DET versus SET in the first and second fresh cycles were 29.7 (47/158) versus 23.9% (127/531) and 30.8 (41/133) versus 22.0% (45/205). The cumulative live birth rate per patient after the addition of frozen-thawed embryo transfers were similar: 33.5 (53/158) and 34.8% (185/531) for DET and SET respectively after the first cycle and 32.3 (43/133) versus 32.2% (66/205) after the second cycle. A logistic regression analysis showed no significant correlation for SET or DET with cumulative live birth. Thus, cumulative live birth rates are similar after SET and DET in a routine IVF programme with a majority of SET transfers, although a higher number of frozen-thawed cycles were needed in the SET group.

Reinsemination of one-day-old oocytes by use of intracytoplasmic sperm injection *

OBJECTIVE To evaluate the possible advantages of reinseminating oocytes by use of intracytoplasmic sperm injection (ICSI). DESIGN Clinical study. SETTING In vitro fertilization unit with research facilities. PATIENTS Fifty-seven couples who were part of our regular IVF program. INTERVENTIONS Nonfertilized oocytes from IVF cycles with no or very low fertilization were microinjected with spermatozoa approximately 25 hours after oocyte pick-up. MAIN OUTCOME MEASURES Fertilization and pregnancy rates. RESULTS A mean fertilization rate of 46.5% was obtained when reinseminating the oocytes on day 2 using the ICSI procedure. Of 57 cycles with completely or almost completely failed fertilization, 29 patients received ET after reinsemination by ICSI. Two of these transfers resulted in pregnancies (6.9% per ET) and two healthy babies were born. CONCLUSION Despite this relative success, considering both the extra work involved and the potential genetic risk, it is doubtful whether ICSI on day 2 should be recommended as a routine procedure. For training and research purposes, however, this approach can be of value.