Maria Köster

Which morphological scoring system is relevant in human embryo development

OBJECTIVESnIn the past several scoring systems were proposed for early human development aiming to assist in the identification of the best embryos. Scoring criteria are usually assessed at static developmental time points by microscopy. For almost every scoring system controversial results on its benefit can be found in the literature. With the introduction of time-lapse imaging static assessment of developmental parameters needs to be revised. The objective of this study was to critical review the strategy of static assessment by using an embryo monitoring system to study time-dependent variations of scoring criteria.nnnSTUDY DESIGNnHuman oocytes were subjected to intracytoplasmic sperm injection and subsequently incubated in an embryo monitoring device. Images from individual oocytes were captured at given time intervals allowing a time-lapse analysis of early embryo development.nnnMAIN OUTCOME MEASURESnScoring of pronuclear morphology, early cleavage and embryo morphology up to day 3 of development was performed at standard time points and compared to the morphological fate present in time intervals prior and after standard assessment.nnnRESULTSnPronuclear morphology showed a high variability within very short time intervals. First cleavage can be observed at very early time points questioning the criterion early cleavage. Embryo morphology can change within short time intervals and thus may be misleading if assessment is done at a static time point.nnnCONCLUSIONSnScoring of early embryo development has limitations if based on static observation only. Time-lapse imaging will lead to revised scoring systems emphasizing the need for a new look on embryological parameters.

Oocyte zona birefringence intensity is associated with embryonic implantation potential in ICSI cycles

A retrospective study recently showed that oocytes presenting with a high birefringence of the inner zona layer were more often associated with conception cycles. To further investigate these findings, a prospective study was conducted between September 2005 and September 2006 including intracytoplasmic sperm injection (ICSI) cycles presenting with at least two embryos for transfer. Using a polarization imaging system, oocytes were classified prior to ICSI treatment as having either a high zona birefringence (HZB) or a low zona birefringence (LZB) of the zona pellucida. Using zona birefringence as the only selection criterion, two fertilized oocytes, preferably derived from HZB oocytes, were selected for further culture and transfer. The required criteria were met by 135 ICSI cycles (124 patients; 34.9 +/- 4.1 years of age). Embryos for transfer were used in 20 cycles derived from HZB/HZB oocytes, in 50 cycles from HZB/LZB oocytes and in 65 from LZB/LZB oocytes. The corresponding implantation (P < 0.025), pregnancy (P < 0.005) and live birth (P < 0.025) rates were significantly different between HZB/HZB and HZB/LZB versus LZB/LZB group. Embryo development was superior in embryos derived from HZB oocytes. This study concludes that oocyte zona birefringence is a good selection criterion and a good predictive criterion for embryo implantation potential.

The benefit of artificial oocyte activation is dependent on the fertilization rate in a previous treatment cycle

Following intracytoplasmic sperm injection (ICSI), some patients present low or zero fertilization rates. Artificial oocyte activation has been proposed as a suitable means to overcome this problem. This study applied artificial oocyte activation in patient cohorts with a history of no fertilization (0%, group 1), fertilization between 1 and 29% (group 2) or fertilization between 30 and 50% (group 3) in initial ICSI cycles. In the following treatment cycles, oocytes were activated after ICSI using calcium ionophore. Fertilization, pregnancy and take-home baby rates were compared with the previous cycle without activation. In group 1, fertilization rate was 41.6%, embryos for transfer were available in 82.1% of cycles, giving a clinical pregnancy rate of 18.8% and take-home baby rate of 12.8%. In group 2, despite a lower transfer rate (87.9% versus 100%, P<0.05), there were higher fertilization and clinical pregnancy rates (44.4% versus 19.3% and 31.4% versus 12.8%, respectively, P<0.05) and take-home baby rate was 24.1% versus 12.8%. In group 3, fertilization rates differed (56.1% versus 36.8%; P<0.001) but all other parameters were similar. Artificial oocyte activation has great potential especially in patients showing compromised fertilization rates below 30% after standard ICSI. Following intracytoplasmic sperm injection (ICSI), some patients present very low or even zero fertilization rates after ICSI. Artificial oocyte activation has been proposed as a suitable means to overcome this problem. We applied artificial oocyte activation in patients which presented a history either no fertilization, fertilization between 0 and 30% or fertilization between 30 and 50% in initial ICSI cycles. In the following treatment cycles, oocytes were activated after ICSI using a calcium ionophore. Fertilization, pregnancy and take-home baby rates were compared to the previous cycle without activation. For the groups with previously 0% or 1-29% fertilization, we noted higher fertilization rates and clinical pregnancy rates per embryo transfer. For the group with moderate fertilization, only fertilization rates differed but all other parameters were not significantly different. From these data we conclude that artificial oocyte activation has a great potential especially in patients which show a compromised fertilization rate below 30% in a standard ICSI cycle.

Polar Body Biopsy

Polar body biopsy combined with array comparative genomic hybridization allows detection of maternal chromosomal aberrations. Although it has limitations, it can be seen as an alternative to blastomere and trophectoderm biopsy.

Gamete competence assessment by polarizing optics in assisted reproduction

BACKGROUNDnThe purpose of this study was first to give an overview of the historical development of polarization microscopy, second to describe the various applications of this technique in assisted reproduction techniques (ART) and third to discuss the potential benefit of polarization microscopy as a predictor for IVF success.nnnMETHODSnThe history of polarization microscopy was undertaken by performing a backward search in the scientific literature using Google and internet sites of several Societies for Microscopy and Cell Biology. Studies of polarization microscopy in ART were identified by using a systematic literature search in PubMed and Scopus.nnnRESULTSnA total of 62 articles were identified by the direct search and further relevant articles were found by screening the cited literature in these articles. The topics relevant for assisted reproduction were spindle and zona imaging in combination with IVF success, meiotic cell cycle progression, pharmaceutical studies and cryopreservation. A separate topic was the use of sperm birefringence in ART.nnnCONCLUSIONSnThe majority of studies are observational studies and were not performed in a randomized manner and there is no direct comparison of techniques using other gamete selection markers. Despite this, most studies show that polarization microscopy may help us to further increase our knowledge on gametes and meiosis. Whether certain applications such as spindle or zona imaging may lead to an increase in IVF success is unclear at present. Publications on the use of polarization microscopy on sperm are still very limited.

Application of a ready-to-use calcium ionophore increases rates of fertilization and pregnancy in severe male factor infertility

OBJECTIVEnTo analyze whether a ready-to-use calcium ionophore improves outcomes, from fertilization to live birth, in patients with severe male factor infertility.nnnDESIGNnArtificial oocyte activation offered to applicable patients over a 20-month period.nnnSETTINGnSpecialized inxa0vitro fertilization (IVF) centers in Austria and Germany.nnnPATIENT(S)nTwenty-nine azoospermic and 37 cryptozoospermic men.nnnINTERVENTION(S)nMature oocytes treated with a ready-to-use Ca(2+)-ionophore (GM508 Cult-Active) immediately after intracytoplasmic sperm injection (ICSI).nnnMAIN OUTCOME MEASURE(S)nRates of fertilization, implantation, clinical pregnancy, and live birth.nnnRESULT(S)nPatients had had 88 previous cycles without artificial activation that resulted in a fertilization rate of 34.7%, 79 transfers (89.8%), and 5 pregnancies, which all spontaneously aborted except one. After artificial oocyte activation, the fertilization rate was 56.9%. In terms of fertilization rate, both azoospermic (64.4%) and cryptozoospermic (48.4%) men statistically significantly benefited from use of the ionophore. In 73 transfer cycles, positive β-human chorionic gonadotropin levels were observed in 34 cases (46.6%) and 29 cycles (39.7%) that ended with a clinical pregnancy. The corresponding implantation rate was 33.3%. Four spontaneous abortions occurred (11.8%), and 32 healthy children were born.nnnCONCLUSION(S)nThis is the first prospective multicenter study on artificial oocyte activation in severe male factor infertility. Present data indicate that a ready-to-use calcium ionophore can yield high fertilization and pregnancy rates for this particular subgroup. In addition to fertilization failure after ICSI, severe male factor infertility is an additional area for application of artificial oocyte activation.

The impact of culture conditions on early follicle recruitment and growth from human ovarian cortex biopsies in vitro

OBJECTIVEnTo investigate the effects of a dynamic fluidic culture system on early in vitro folliculogenesis in standardized ovarian cortex biopsies.nnnDESIGNnCortical small strips were cultured for 6 days in a conventional static or in a dynamic fluidic culture system.nnnSETTINGnUniversity-affiliated laboratory with an associated cryobank facility.nnnPATIENT(S)nOvarian cortex from postpuberal female cancer patients (26.1 ± 1.3 y) who opted for cryopreservation of their tissue for fertility protection before gonadotoxic cancer therapy. With informed consent of the Institutional Ethics Committee, part of the tissue was available for patient-related research studies.nnnINTERVENTION(S)nNone.nnnMAIN OUTCOME MEASURE(S)nThe viability and proliferative capacity of the cortex biopsies were evaluated by chemiluminescent microparticle immunoassay for detection of in vitro produced E2 and P in the supernate, by viable follicle counting via calcein staining, by histologic analyses, and by total RNA preparation and reverse transcription for real-time polymerase chain reaction of selected early folliculogenesis genes.nnnRESULT(S)nThe data support the notion that early follicle development can be better achieved in vitro in a dynamic fluidic culture system. The findings are based on the presence of more viable follicles, higher expression levels of early folliculogenesis genes KIT-L, INHB, and GDF9, and the absence of premature luteinization of follicles.nnnCONCLUSION(S)nThis study provides evidence that dynamic fluidic culture is a promising approach for investigating early follicular recruitment and growth in cortical biopsies. It may serve as a first step in a multistep culture system to design a complex in vitro system for complete folliculogenesis.

Temperature-induced sperm nuclear vacuolisation is dependent on sperm preparation.

The influence of temperature during incubation on the degree of sperm nuclear vacuolisation was assessed by two different experiments. In a first experiment, motile spermatozoa from 24 patients were prepared by the swim‐up technique and incubated either at room temperature or at 37u2003°C for up to 4u2003h. The presence of sperm nuclear vacuoles was determined by contrast‐enhanced high magnification microscopy. No statistically significant difference was found in the degree of sperm nuclear vacuoles in both groups (RT: 45.6u2003±u200317.6%; 37u2003°C: 48.4u2003±u200317.0%) following 4u2003h of incubation. In a second experiment, spermatozoa from six patients were either prepared by swim‐up or washed and incubated at 37u2003°C. After 4u2003h of incubation, a significant increase in sperm nuclear vacuolisation was found in washed sperm (from 51.5u2003±u200315.4% to 68.6u2003±u20039.0%; Pu2003<u20030.05) but not in swim‐up sperm (from 51.5u2003±u200315.4% to 48.2u2003±u200317.1%; n.s.). Our data show that the mode of sperm preparation does influence sperm nuclear vacuolisation at 37u2003°C (Experiment II). However, sperm nuclear vacuolisation is unaffected by temperature in motile sperm after preparation and isolation by swim‐up.

Analysis of the Zona Pellucida as an Indicator of Oocyte Developmental Potential

In human-assisted reproductive technologies (ART) the search for parameters to assess the quality and developmental potential of human gametes and embryos is of great importance. There is a clear need to gather as much information as possible in order to allow for identification of those gametes or embryos which may finally implant and give birth to a healthy child. Although experience is a key factor in embryology, the overall trend is to replace subjective judgment and decision making with objective devices which assist in the process of gamete and embryo classification. The technology of such a device should rely on physiological properties, it should be easy to use, and should include an automatic user interface which will facilitate acceptance in the daily routine laboratory work. A technique which is available in all embryology laboratories is microscopy. Different microscopic technologies can be used to visualize subcellular structures within cells. One such technology is polarization microscopy.