Markus Montag

Spindle imaging in human oocytes: the impact of the meiotic cell cycle

Recent studies using polarized light microscopy revealed a correlation between the presence of a spindle in human metaphase II meiotic oocytes and the fertilization rate following intracytoplasmic sperm injection (ICSI). Using a new spindle imaging system, it was possible to visualize the spindle image and the conventional light microscopic view of the oocyte simultaneously. Using this system, time-lapse studies of the meiotic cycle of human oocytes were performed. The video sequences showed that during the transition from metaphase I to metaphase II, the spindle completely disappears for approximately 40-60 min. These data support the idea that at least in some oocytes, the absence of the spindle is more likely an indicator for physiological progression through an important developmental stage of meiosis rather than a cellular disturbance. In view of the low fertilization rates of oocytes with absence of spindles as reported in the literature, the underlying problem could simply be the incorrect timing of ICSI.

Outcome of laser-assisted polar body biopsy and aneuploidy testing

Polar body biopsy and subsequent fluorescence in-situ hybridization (FISH) analysis allows detection of maternally derived chromosomal aneuploidies in human oocytes during IVF treatment. The development of a diode laser technique for the partial opening of the zona pellucida has stimulated the use of this technique to assist polar body biopsy. Laser-assisted polar body biopsy was performed in 140 IVF cycles from patients of advanced maternal age (> or =35 years). A total of 921 oocytes were treated by a laser for partial zona opening and polar body removal. FISH was performed for chromosomes 13, 16, 18, 21 and 22 and results were available for 903 oocytes (98%). In all, 443 oocytes (49.1%) were euploid and of these, 293 were fertilized. A total of 214 embryos were transferred in 120 embryo transfer cycles (1.78 per embryo transfer) resulting in 27 clinical pregnancies (22.5% per embryo transfer) with an implantation rate of 15.4%. Subsequently, five women aborted (18.5%) and 24 healthy children were born from the remaining 22 pregnancies, which gives a take home baby rate of 18.3% per transfer cycle. It is concluded that polar body biopsy using a diode laser system is as efficient as standard polar body biopsy using zona drilling.

New approaches to embryo selection

Embryo selection has been an important topic since the introduction of assisted reproduction, with embryo morphology being the most obvious criterion. Although morphology serves as indicator for overall IVF laboratory quality, its statistical assessment limits the possibility to identify the most implantation-competent embryos. In order to reach a direct picture of the developing embryo, invasive procedures such as preimplantation genetic screening or transcriptome and proteome analysis of biopsied embryonic tissue were initially prioritized and are still under investigation. More recently, focus has shifted towards noninvasive techniques that maintain the integrity of the embryo. Metabolomic profiling of culture medium from growing embryos attracted much research. Although successful in a pilot study, that approach failed in a randomized controlled trial. Other metabolomics studies are on their way but not yet available for routine clinical use. The most promising strategy at present is the combined evaluation of morphology and developmental kinetics using time-lapse imaging. This has brought new insights into certain characteristics that enable deselection of embryos at an early stage of development and to identify others with high potential for successful implantation. However, there is still considerable room for improvement. Further strategies will most likely involve the combination of several different approaches.

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.

Retransplantation of Cryopreserved Ovarian Tissue: the First Live Birth in Germany

BACKGROUND Cryopreserved ovarian tissue can be retransplanted to restore fertility after radiation or chemotherapy. To date, 15 live births after retransplantation have been reported worldwide. We report the first pregnancy and the first live birth after retransplantation in Germany. CASE REPORT A 25-year-old female patient received initial chemotherapy and radiation of the mediastinum for Hodgkins lymphoma in 2003 and suffered a relapse two years later. Ovarian tissue was laparoscopically removed and cryopreserved, and she was then treated with high-dose chemotherapy and stem cell transplantation. She remained in remission for 5 years and she could not conceive during this time. The cryopreserved ovarian tissue was thawed and laparoscopically retransplanted into a peritoneal pouch in the ovarian fossa of the right pelvic wall. Three months later, her menopausal symptoms resolved, and she had her first spontaneous menstruation. Six months after retransplantation, after two normal menstrual cycles, low-dose follicle stimulating hormone (FSH) treatment induced the appearance of a dominant follicle in the tissue graft. Ovulation was then induced with human chorionic gonadotropin (HCG), whereupon the patient conceived naturally. After an uncomplicated pregnancy, she bore a healthy child by Caesarean section on 10 October 2011. Histological examination of biopsy specimens revealed that the ovarian tissue of the graft contained follicles in various stages of development, while the original ovaries contained only structures without any reproductive potential. CONCLUSION This was the first live birth after retransplantation of cryopreserved ovarian tissue in Germany and also the first case with histological confirmation that the oocyte from which the patient conceived could only have come from the retransplanted tissue. In general, young women who will be undergoing chemotherapy and/or radiotherapy for cancer must be informed and counseled about the available options for fertility preservation.

Gamete competence assessment by polarizing optics in assisted reproduction

BACKGROUND The 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. METHODS The 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. RESULTS A 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. CONCLUSIONS The 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.

Time-lapse culture with morphokinetic embryo selection improves pregnancy and live birth chances and reduces early pregnancy loss: a meta-analysis

Embryo evaluation and selection is fundamental in clinical IVF. Time-lapse follow-up of embryo development comprises undisturbed culture and the application of the visual information to support embryo evaluation. A meta-analysis of randomized controlled trials was carried out to study whether time-lapse monitoring with the prospective use of a morphokinetic algorithm for selection of embryos improves overall clinical outcome (pregnancy, early pregnancy loss, stillbirth and live birth rate) compared with embryo selection based on single time-point morphology in IVF cycles. The meta-analysis of five randomized controlled trials (n = 1637) showed that the application of time-lapse monitoring was associated with a significantly higher ongoing clinical pregnancy rate (51.0% versus 39.9%), with a pooled odds ratio of 1.542 (P < 0.001), significantly lower early pregnancy loss (15.3% versus 21.3%; OR: 0.662; P = 0.019) and a significantly increased live birth rate (44.2% versus 31.3%; OR 1.668; P = 0.009). Difference in stillbirth was not significant between groups (4.7% versus 2.4%). Quality of the evidence was moderate to low owing to inconsistencies across the studies. Selective application and variability were also limitations. Although time-lapse is shown to significantly improve overall clinical outcome, further high-quality evidence is needed before universal conclusions can be drawn.

Morphokinetics and embryo aneuploidy: has time come or not yet?

Morphokinetic analysis of early human embryos in combination with trophectoderm biopsy and chromosomal enumeration by array technology has been shown to identify embryos with single and/or multiple aneuploidies. In a time-lapse imaging study, aneuploid embryos showed a delayed initiation of blastocyst formation and also reached the full blastocyst stage later compared with euploid embryos. Based on these findings, a model for a risk factor classification for aneuploidy of human embryos has been established. However, time values that allow differentiation between different risk classes have to be considered carefully as timings that are prognostic for one clinic may not apply directly to another clinic. Thus, risk models may need to be verified in specific IVF settings. Further studies are needed to verify that time-lapse imaging does allow subselection of embryos with a high aneuploidy risk and thus does hold the chance to restrict invasive biopsy procedures to those embryos where there is uncertainty regarding the chromosomal status.

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

OBJECTIVE To investigate the effects of a dynamic fluidic culture system on early in vitro folliculogenesis in standardized ovarian cortex biopsies. DESIGN Cortical small strips were cultured for 6 days in a conventional static or in a dynamic fluidic culture system. SETTING University-affiliated laboratory with an associated cryobank facility. PATIENT(S) Ovarian 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. INTERVENTION(S) None. MAIN OUTCOME MEASURE(S) The 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. RESULT(S) The 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. CONCLUSION(S) This 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.