Embryo Fragmentation Research Articles

Preimplantation genetic diagnosis (PGD) for maternal dominant disorders: is polar body (PB) better than blastomere biopsy?

OBJECTIVE: Preimplantation diagnosis (PGD) for maternally inherited dominant or X-linked disorders may be performed by direct genetic analysis of the blastomere(s) or by sequential analysis of the first and second polar bodies. If blastomere biopsy is performed, it is recommended to analyze 2 blastomeres for preventing misdiagnosis due to allele drop-out (ADO). With PB analysis, the genotype of the embryo is predicted by deduction. The objective of this study was to compare polar body biopsy with blastomere biopsy, in PGD for maternally inherited disorders, regarding accuracy of the analysis as well as the effect of the biopsy method on embryo development.DESIGN: A prospective study, comparing 31 PGD cycles for maternal dominant disorders analyzed by PB biopsy, to 51 PGD cycles for dominant disorders, analyzed by 2 blastomeres biopsy.MATERIALS AND METHODS: A total of 241 embryos, analyzed by PB biopsy, were evaluated according to the following parameters: biopsy failure rate, amplification failure rate, rate of non-informative first PB, ADO and contamination rates, and the proportion of embryos in which a blastomere biopsy was performed also. Cleavage rate, degree of fragmentation and development parameters of the biopsed embryos on day 3 were assessed too.RESULTS: Failure of biopsy of 1st and 2nd PBs was encountered in 7.5% and 15.7%, respectively. Blastomere Biopsy after attempted PB biopsy was performed in 117 (48.5%) of the embryos due to homozygosity of the 1st PB and a lack of informative markers that could excluded ADO (n=110) and contamination (n=7). Cleavage rate, degree of fragmentation and development parameters of embryos on day 3 were similar in both groups. Ongoing pregnancy rates per embryo transfer were 22.5% for PB biopsy and 17.6% for 2-blastomere biopsy.CONCLUSIONS: We conclude that PB biopsy is more difficult to perform and genetic analysis of PB is hampered by inability to detect maternal contamination in the 1st PB, and inability to distinguish between homozygousity of 1st PB and ADO. On the other hand, PB biopsy may be associated with less disruption of embryonic development. Therefore, we are recommending on PB biopsy only in analyses of maternal disorders that are based on more than 3 informative maternal polymorphic markers. This reduces the need for an additional blastomere biopsy when 1st PB is homozygote. OBJECTIVE: Preimplantation diagnosis (PGD) for maternally inherited dominant or X-linked disorders may be performed by direct genetic analysis of the blastomere(s) or by sequential analysis of the first and second polar bodies. If blastomere biopsy is performed, it is recommended to analyze 2 blastomeres for preventing misdiagnosis due to allele drop-out (ADO). With PB analysis, the genotype of the embryo is predicted by deduction. The objective of this study was to compare polar body biopsy with blastomere biopsy, in PGD for maternally inherited disorders, regarding accuracy of the analysis as well as the effect of the biopsy method on embryo development. DESIGN: A prospective study, comparing 31 PGD cycles for maternal dominant disorders analyzed by PB biopsy, to 51 PGD cycles for dominant disorders, analyzed by 2 blastomeres biopsy. MATERIALS AND METHODS: A total of 241 embryos, analyzed by PB biopsy, were evaluated according to the following parameters: biopsy failure rate, amplification failure rate, rate of non-informative first PB, ADO and contamination rates, and the proportion of embryos in which a blastomere biopsy was performed also. Cleavage rate, degree of fragmentation and development parameters of the biopsed embryos on day 3 were assessed too. RESULTS: Failure of biopsy of 1st and 2nd PBs was encountered in 7.5% and 15.7%, respectively. Blastomere Biopsy after attempted PB biopsy was performed in 117 (48.5%) of the embryos due to homozygosity of the 1st PB and a lack of informative markers that could excluded ADO (n=110) and contamination (n=7). Cleavage rate, degree of fragmentation and development parameters of embryos on day 3 were similar in both groups. Ongoing pregnancy rates per embryo transfer were 22.5% for PB biopsy and 17.6% for 2-blastomere biopsy. CONCLUSIONS: We conclude that PB biopsy is more difficult to perform and genetic analysis of PB is hampered by inability to detect maternal contamination in the 1st PB, and inability to distinguish between homozygousity of 1st PB and ADO. On the other hand, PB biopsy may be associated with less disruption of embryonic development. Therefore, we are recommending on PB biopsy only in analyses of maternal disorders that are based on more than 3 informative maternal polymorphic markers. This reduces the need for an additional blastomere biopsy when 1st PB is homozygote.

In vitro ageing of mouse eggs and cytoplasts results in amplification of microtubule organizing centers and leads to fragmentation following activation

OBJECTIVE: Mature mouse eggs have a propensity to fragment following ageing in vitro. This has been attributed to apoptosis. Here we examined the cytoskeletal changes in activated and non-activated in-vitro aged intact and enucleated eggs. DESIGN: Eggs were collected from mice following superovulation, sorted and allocated to different experimental groups. MATERIALS AND METHODS: Intact or enucleated eggs were cultured until 40 hours post hCG, at which point some were ethanol activated; they were examined by light and confocal microscopy (LSM). RESULTS: A proportion of the aged intact eggs (4/15) showed an increase in the activity of microtubule organizing centers (MTOCs) while aged cytoplasts (17/19) showed both a more substantial increase in MTOC activity and an increase in MTOC number. Only about a quarter (47/203) of the aged intact eggs were activated, but almost all of those underwent fragmentation within 2 hours. The predominant pattern of fragmentation was one of several large fragments- some containing chromosomal material. LSM examination of these eggs invariably revealed abnormal progression of meiosis, with irregular spindle forms and abnormal chromosome segregation. About forty percent (79/194) of the aged cytoplasts also fragmented upon exposure to ethanol, but with a different fragmentation pattern. Initially, multiple peripheral small sized fragments formed surrounding one large central fragment which itself underwent further fragmentation later. These patterns of fragmentation fairly closely correlate with the MTOC status within the cytoplasm after aging. CONCLUSIONS: These experiments show that in vitro ageing results in MTOC amplification, which includes extensive polymerization and acetylation of cytoplasmic tubulin, and leads to fragmentation of aged eggs and cytoplasts following activation. Therefore, MTOC amplification is probably a major contributing factor to the failure of development following activation of aged eggs. The possibility that MTOC amplification is a result of one or several of the same critical events that constitute apoptosis cannot be excluded, but the immediate consequence of this aberration per se is abnormal cell division, not cell death. These changes are reminiscent of a condition referred to as centrosome amplification in malignant cells which is believed to be a potential cause of aneuploidy and genetic instability in many cancers. The relevance of these findings to fragmentation and mosaicism in human embryos should be investigated.

The effect of media pH on embryo development and cycle outcome

OBJECTIVE: There has been debate on the optimal embryo culture conditions during in vitro fertilization cycles. This study was undertaken to determine the effect of media pH on embryo quality, the development of early cleaving embryos, and cycle outcome. DESIGN: Prospective, non-randomized study. MATERIALS AND METHODS: Fifteen patients with a minimum of 15 oocytes at retrieval were included in the study. Study patients had their oocytes split between a test incubator with an increased CO2 concentration and a control incubator set at a standard CO2 level. This study design allowed for the patient to act as their own control group. The oocytes from both incubators were observed for fertilization, the presence of early cleaving embryos, and Day 3 development at set hours. The embryos were graded using a standard morphology assessment protocol in order to choose the best quality embryos for Day 3 transfer regardless of which incubator was used for culture. Embryos that were of good quality and not chosen for transfer were subsequently frozen on Day 3. Patients were monitored post transfer and a positive pregnancy defined as the presence of an intrauterine sac. Data were analyzed using the unpaired t-test and the Fisher's exact test, with significance set at P < 0.05. RESULTS: The average pH values of the test and control incubators were significantly different (7.30 vs. 7.38, p < 0.0001). The lowered pH did result in a significantly higher number of blastomeres on Day 3 (7.11 vs. 6.25, p = 0.025). In addition, a higher percentage of embryos transferred were derived from the test incubator (22.0% vs. 10.7%, p = 0.045). While there was a trend towards an increased percentage of early cleaving embryos from the test incubator (22.0% vs. 17.2%) the difference was not significant (p = 0.08). There was no difference in the fertilization rate, embryo fragmentation rate, the development of embryos to at least the five cell stage on Day 3, or the percentage of frozen and discarded embryos. Nine cycles were examined where all of the transferred embryos came from exclusively one incubator. The clinical pregnancy rate was determined to be identical in these two groups (33.3%). CONCLUSIONS: This study demonstrated the role that media pH has on embryo quality as assessed by standard morphological criteria. Ongoing studies are being performed to further establish the effect of decreased media pH on clinical outcomes.

Alpha-tocopherol is a predictor of human embryo fragmentation but not independent of high density lipoprotein (HDL)

OBJECTIVE: We recently reported a negative association between the follicular fluid (FF) HDL-Apolipoprotein A1 (ApoA1) complex with it's known antioxidant activities and human embryo fragmentation [HDL-cholesterol rsp = -0.59 (95% CI -0.78, -0.29) and ApoA1 rsp = -0.54 (95% CI -0.75, -0.21)]. Lipophilic micronutrients, such as α-tocopherol, are also antioxidants previously identified in human FF. It has been reported in mammalian studies that lipophilic micronutrients in FF may play protective roles in follicular/oocyte heath.

Early microsurgical removal of fragments can support the subsequent development of the fragmented human embryos by inhibiting the generation of new fragments

OBJECTIVE: Cytoplasm fragmentation leads to apoptosis or limits the subsequent development of the fragmented embryos due to a reduction of cytoplasm available for cell division and a generation of ROS. The present study was carried out to evaluate the beneficial effect of the microsurgical removal of fragments on the subsequent development of the fragment-removed human embryos.

High pregnancy loss rate following implantation of IVF human embryos with extensive fragmentation

OBJECTIVE: It is often argued that in the absence of absolute certainty about the viability of human embryos based on their morphology, transfer of embryos with multiple cleavage abnormalities is justified - particularly when those are the only embryos available to the patient. However, at least one study has linked extensive fragmentation to increased rates of congenital anomalies. Here, we have re-examined this relationship. DESIGN: Retrospective cohort study. MATERIALS AND METHODS: The embryology database was searched for cases in which embryos with extensive fragmentation (>35%) were transferred exclusively - that is, all transferred embryos had >35% fragmentation. Forty three such cases were identified. Cases (n=1874) in which only embryos with minimal or no fragmentation (0-5%) were replaced were selected as control. Cycle outcomes were evaluated and compared. RESULTS: Pregnancy was confirmed in 13/43 (30%) patients with exclusive transfer of extensively fragmented embryos. Of these, seven pregnancies were biochemical, three were lost between 7 and 9 weeks gestation, and three resulted in birth, giving a live birth rate of 7% per cycle (3/43). All three infants had normal weights and were in apparently good health at birth. Pregnancy rate in the control group was 61.6% (1154/1874) per cycle. The live birth rate in this group was 44.8% (839/1874). Pregnancy loss rate following three consecutive rises in the beta hCG level was therefore 77% (10/13) in the study population and 27% (315/1154) in the control group. The average age in the two groups was not statistically different and the age range was similar. CONCLUSIONS: Implantation failure and early pregnancy loss are the most common outcomes after exclusive transfer of extensively fragmented embryos. This is in sharp contrast to the outcome of transfer when the embryos have minimal fragmentation: a large majority of the latter lead to live birth. Considering the risk of physical and psychological trauma associated with an aborted pregnancy, even in the absence of birth defects, patients for whom only embryos with extensive fragmentation are available should be counseled and warned of the high likelihood of a negative outcome following transfer.

Correlation between preimplantation genetic diagnosis for chromosomal aneuploidies and the efficiency of establishing human ES cell lines

There are several sources from which human embryonic stem cell (hESC) lines can be generated: surplus embryos after in vitro fertilization procedures, one- and three-pronuclear zygotes, early arrested or highly fragmented embryos that have reached the blastocyst stage, or otherwise chromosomally or genetically abnormal embryos after preimplantation genetic diagnosis (PGD). We report on the efficiency of establishing hESC lines from blastocysts with proven meiotic or mitotic errors after sequential testing of both polar bodies and blastomere analysis on day 3. The success rate of establishing hESC lines originating from blastocysts carrying a meiotic error was as low as 2.4% and differed significantly from the success rate of establishing hESC lines originating from blastocysts with balanced meiotic errors (21.6%) or mitotic errors (after sequential testing (9.1%) and after blastomere testing alone (12.2%)). This suggests that it may be reasonable to apply sequential PGD prior to the initiation of hESC culture. Information about the karyotype may in the future help refine the methods and possibly improve the efficiency by which hESC lines are derived from embryos with prezygotic abnormalities. Additionally, it may in general prove very difficult to obtain abnormal hESC lines for scientific study from aneuploid PGD embryos, which will limit our ability to study the biological consequences of chromosomal abnormalities. Furthermore, the success rates for generating aneuploid cell lines originating from fertilized oocytes carrying a prezygotic nondisjunction error seem to mirror the miscarriage rates during pregnancy of embryos carrying such errors.

Cytokines and chemokines in follicular fluids and potential of the corresponding embryo: the role of granulocyte colony-stimulating factor

The cytokine/chemokine levels of individual follicular fluids (FFs) were measured to determine whether a biomarker could be linked to the developmental potential of the derived embryo. Fluid was collected from 132 individual FFs that were the source of oocytes subsequently fertilized and transferred. In each, a bead-based multiplex sandwich immunoassay (Luminex) was used to measure 28 cytokines and chemokines simultaneously. Significantly higher levels of interleukin (IL-2) and interferon (IFN-gamma) were detected in FF for embryos that underwent early cleavage. IL-12 was significantly higher in FF corresponding to highly fragmented embryos and the chemokine CCL5 was significantly higher in FF related to the best quality (Top) embryos. The level of granulocyte colony-stimulating factor (G-CSF) in individual FF samples was correlated with the implantation potential of the corresponding embryo. The area under the receiver operating characteristics curve, which distinguished the embryos that definitely led to delivery from those that did not, was 0.84 (0.75-0.90) (P = 0.0001) for FF G-CSF. FF G-CSF was significantly lower in patients older than 36 years compared with those <30-year old. When the FF G-CSF was 20 pg/ml or higher, the ratio between Top and non-Top embryos was significantly higher than for the group with FF G-CSF below 20 pg/ml (45 versus 20.45%, P = 0.007). Individual FF composition is related to the development of the corresponding in vitro generated embryo and its potential of implantation. Individual FF G-CSF may provide a non-invasive biomarker of implantation that needs to be evaluated together with in vitro observation to select the oocyte, and hence the embryo, to transfer.

Distributions of high-density lipoprotein particle components in human follicular fluid and sera and their associations with embryo morphology parameters during IVF

High-density lipoprotein (HDL) is the sole lipoprotein present in follicular fluid (FF). The objectives of this study were to examine HDL lipid composition and associated enzyme activities in FF and serum and to relate these levels to embryo morphology parameters in women undergoing in vitro fertilization (IVF). Serum and FF were prospectively obtained from 60 women undergoing IVF. HDL lipids, apolipoprotein AI (ApoAI), paraoxonase 1 (PON1) and paraoxonase 3 (PON3) activities were determined. Bivariate analysis and ordinal logistic regression models were employed to examine the associations between biochemical measures and embryo morphology parameters [embryo cell number (ECN) and embryo fragmentation score (EFS)] as surrogate markers of oocyte health. All biochemical parameters were significantly (P < 0.05) lower in FF than serum except PON3 levels which were significantly higher. FF-HDL cholesterol (OR 0.66, 95%CI 0.46-0.96) and ApoAI (OR 0.13, 95%CI 0.03-0.97) levels were negative predictors for EFS; however, their effects were not independent and the level of one moderated the effect of the other. Limited to Day 3 embryo transfers, FF-PON1-arylesterase activity was a significant positive predictor for ECN (OR 1.09, 95%CI 1.01-1.17). In this pilot study, our data suggests that HDL and its component proteins within FF may play protective roles in the health of the human oocyte and subsequent early embryo development. We describe for the first time the activities of PON1 and PON3 in FF. We suspect that PON3 activity may be locally generated due to higher activities in FF compared with serum.

Human embryonic stem cell derivation from poor-quality embryos

During in vitro fertilization, embryos deemed clinically useless based on poor morphology are typically discarded. Here we demonstrate a statistical correlation between the developmental stage of such poor-quality embryos and the yield of human embryonic stem (hES) cell lines. Early-arrested or highly fragmented embryos only rarely yield cell lines, whereas those that have achieved blastocyst stage are a robust source of normal hES cells.

MUC1 in human testis and ejaculated spermatozoa and its relationship to male fertility status

MUC1 is a cell surface glycoprotein with a previously described antiadhesive role involved in different aspects of reproductive function. We found MUC1 expressed in male germ cell line and within the ejaculated sperm, but its presence in mature sperm does not seem to be related to male fertility. This was confirmed after analysis of results from assisted reproduction techniques with oocyte donation related with MUC1, although higher MUC1 expression is related to sperm recovery after preparation.

L-carnitine as an antiapoptotic supplement in mouse embryo culture media

ObjectiveHuman embryos generated from in vitro fertilization (IVF) show varying degrees of cytoplasmic fragmentation. Reports suggest that cytoplasmic fragmentation in human embryos arises from apoptosis. Mitochondrial membrane potential and energy production in preimplantation embryos have recently been the focus of many studies. L-Carnitine (LC) has no major side effects on embryonic cells, interactions with other drugs or teratogenicity. It acts by stabilizing the mitochondrial membrane and protecting the cell from apoptosis. The aim of our study was to investigate the antiapoptotic effect of LC supplementation in mouse embryo culture media.DesignProspective in vitro study.Materials and methodsA total of 240 mouse embryos (8-cell) were randomly assigned into 4 groups: group 1: control (HTF media only); groups 2 (Actinomycin D 5 ng/mL; dose based on previous literature) group 3–4: (Actinomycin D 5 ng/mL + two concentrations of LC (0.3 and 0.6 mg/mL). LC concentrations were based on pilot study by our group to optimize the dose of LC in mouse embryo culture media. Embryos were incubated at 37°C in 5% CO2. Assessment of embryos development was done after 48 hours by examining the percentage blastocyst development rate (%BDR). After TUNEL staining, the blastocysts were examined by confocal microscopy. Apoptosis was measured by examining the percentage of TUNEL positive blastomeres.ResultsTableaP<0.05 considered significant using Fisher's exact test for %BDR differences; bP<0.05 was significant for differences in apoptosis using Kruskal-Wallis or Wilcoxon test. cP differences between Actinomycin group and Actinomycin + LC groups. Open table in a new tab ConclusionsL-Carnitine improves the blastocyst development rate and reduces apoptosis. Supplementing in vitro culture media with L-Carnitine may improve the fertilization outcome. ObjectiveHuman embryos generated from in vitro fertilization (IVF) show varying degrees of cytoplasmic fragmentation. Reports suggest that cytoplasmic fragmentation in human embryos arises from apoptosis. Mitochondrial membrane potential and energy production in preimplantation embryos have recently been the focus of many studies. L-Carnitine (LC) has no major side effects on embryonic cells, interactions with other drugs or teratogenicity. It acts by stabilizing the mitochondrial membrane and protecting the cell from apoptosis. The aim of our study was to investigate the antiapoptotic effect of LC supplementation in mouse embryo culture media. Human embryos generated from in vitro fertilization (IVF) show varying degrees of cytoplasmic fragmentation. Reports suggest that cytoplasmic fragmentation in human embryos arises from apoptosis. Mitochondrial membrane potential and energy production in preimplantation embryos have recently been the focus of many studies. L-Carnitine (LC) has no major side effects on embryonic cells, interactions with other drugs or teratogenicity. It acts by stabilizing the mitochondrial membrane and protecting the cell from apoptosis. The aim of our study was to investigate the antiapoptotic effect of LC supplementation in mouse embryo culture media. DesignProspective in vitro study. Prospective in vitro study. Materials and methodsA total of 240 mouse embryos (8-cell) were randomly assigned into 4 groups: group 1: control (HTF media only); groups 2 (Actinomycin D 5 ng/mL; dose based on previous literature) group 3–4: (Actinomycin D 5 ng/mL + two concentrations of LC (0.3 and 0.6 mg/mL). LC concentrations were based on pilot study by our group to optimize the dose of LC in mouse embryo culture media. Embryos were incubated at 37°C in 5% CO2. Assessment of embryos development was done after 48 hours by examining the percentage blastocyst development rate (%BDR). After TUNEL staining, the blastocysts were examined by confocal microscopy. Apoptosis was measured by examining the percentage of TUNEL positive blastomeres. A total of 240 mouse embryos (8-cell) were randomly assigned into 4 groups: group 1: control (HTF media only); groups 2 (Actinomycin D 5 ng/mL; dose based on previous literature) group 3–4: (Actinomycin D 5 ng/mL + two concentrations of LC (0.3 and 0.6 mg/mL). LC concentrations were based on pilot study by our group to optimize the dose of LC in mouse embryo culture media. Embryos were incubated at 37°C in 5% CO2. Assessment of embryos development was done after 48 hours by examining the percentage blastocyst development rate (%BDR). After TUNEL staining, the blastocysts were examined by confocal microscopy. Apoptosis was measured by examining the percentage of TUNEL positive blastomeres. ResultsTableaP<0.05 considered significant using Fisher's exact test for %BDR differences; bP<0.05 was significant for differences in apoptosis using Kruskal-Wallis or Wilcoxon test. cP differences between Actinomycin group and Actinomycin + LC groups. Open table in a new tab aP<0.05 considered significant using Fisher's exact test for %BDR differences; bP<0.05 was significant for differences in apoptosis using Kruskal-Wallis or Wilcoxon test. cP differences between Actinomycin group and Actinomycin + LC groups. ConclusionsL-Carnitine improves the blastocyst development rate and reduces apoptosis. Supplementing in vitro culture media with L-Carnitine may improve the fertilization outcome. L-Carnitine improves the blastocyst development rate and reduces apoptosis. Supplementing in vitro culture media with L-Carnitine may improve the fertilization outcome.

In vitro development of polyspermic porcine oocytes: Relationship between early fragmentation and excessive number of penetrating spermatozoa

Embryo development during in vitro culture of polyspermic porcine oocytes was investigated in the present study. After in vitro fertilization (IVF) of in vitro matured oocytes, putative zygotes were centrifuged to visualize pronuclei. Two pronuclear (2PN) and poly-pronuclear (PPN) zygotes were selected and cultured in vitro. Their development to the blastocyst stage and total cell numbers, dead cell rates and ploidy at the blastocyst stage and morphology of resultant embryos after first cleavage were compared. A cleavage rate of PPN embryos was lower than that of 2PN (61.3% and 82.2%, respectively), however, the ability of cleaved embryos to develop to the blastocyst stage did not differ between the PPN and the 2PN groups (22.4% and 32.9%, respectively). Also there was no difference in total cell numbers and rates of dead cells between PPN and 2PN blastocysts. The majority of blastocysts in 2PN group were found to be diploid. In contrast, blastocysts in PPN group showed heterogeneous status in their ploidy including polyploidy and mixoploidy, whereas a remarkable proportion (31.3%) of them was found to be diploid. After the first cleavage (at 36 h after IVF), there was no difference in the number of nuclei/embryo between the two groups, nevertheless embryos in PPN group had significantly higher numbers of blastomeres than that of embryos in 2PN group, mainly due to an increased frequency of anuclear blastomeres. The present results indicate that correction of embryo ploidy in polyspermic embryos can occur during IVC. Nevertheless the frequency of partial fragmentation in polyspermic embryos is increased.

142 RELATIONSHIP BETWEEN APOPTOSIS IN BOAR SEMEN AND DNA FRAGMENTATION IN PORCINE EMBRYOS

Although apoptosis in somatic cells and in spermatocytes and spermatids in vivo is well established, the presence and significance of apoptosis in ejaculated animal sperm and its correlation with developmental competence of preimplantation embryos is still unresolved. The aim of this experiment was to study the relationship between apoptosis in boar semen and DNA fragmentation in porcine embryos. Two ejaculates from the same boar were used in the experiment. Both fresh ejaculates were analyzed using Vybrant Apoptosis Assay Kit #4 (Molecular Probes, Inc., Eugene, OR, USA). Then one of them was diluted in Biosolwens plus extender, stored for 5 days at 15°C and analyzed using YO-PRO-1/PI assay, which detect changes in the membrane of boar spermatozoa, based on the slight increase of membrane permeability. Both fresh and stored semen were used for insemination, of superovulated gilts (8 per group). After 5.5 days of insemination embryos were flushed out of the uterus and DNA integrity of obtained embryos were analyzed. DNA fragmentation and caspase-3 activity were detected in embryos using kits, Roche Diagnostics (Mannheim, Germany) and PhiPhiLux G2D2 (Calbiochem, San Diego, CA, USA), respectively. For both the fresh and stored semen, 3 groups of sperm were observed under a fluorescence microscope. In the fresh semen, 3 and 2% of apoptotic sperm, 13 and 9% of necrotic sperm, 84 and 89% of live sperm in first and second ejaculated, respectively, were observed. In stored semen, 14% of apoptotic sperm, 27% of necrotic, and 59% of live sperm were noted. In total, 141 expanded blastocysts for DNA fragmentation were analyzed. The results are summarized in Table 1. In conclusion, apoptosis in fresh boar semen was lower than in stored semen and was correlated with the TUNEL nucleus index in blastocysts. The expression of caspase-3 was positively correlated with cells positive for TUNEL. Table 1. Relationship between apoptosis in boar semen and DNA fragmentation in porcine embryos This study was supported by Polish Research Committee, grant no. 2 P06D 024 30.

O-204: Angiotensin II (Ang II) regulates apoptosis in human granulosa-lutein cells (GL)

The human ovary has a complete and functional gonadotrophin-sensitive renin-angiotensin system and both types of AngII receptors, AT1 and AT2, are present in ovarian cells. We have shown that AngII is present in high concentrations in granulosa cells and follicular fluid of human preovulatory follicles and in luteal cells. AngII regulates ovulation and modulates steroidogenesis in rat luteal cells and human GL cells. Other investigators have shown that AngII regulates luteal cell apoptosis. Recently, we have shown that apoptosis of human GL cells correlates with several indicators of IVF outcome, such as embryo fragmentation and pregnancy rate, and is regulated by intraovarian growth factors. The aim of the present study was to test whether AngII could induce in vitro apoptosis of gonadotrophin-exposed human IVF GL cells. Following AngII exposure, apoptosis was assessed by caspace expression. Controls included the non-specific angiotensin receptor blocker Saralasin and the specific AT2 receptor (AT2-R) blocker CGP-42112A. GL cells from 87 IVF-intra-cytoplasmic sperm injection (ICSI) cycles were analyzed. Ovarian stimulation was performed using recombinant FSH and LH or HMG. The pregnancy rate for these cycles was 56.32%. Following oocyte recovery GL cells were collected from pooled follicular fluid (FF), washed and purified using a 50% percoll gradient and anti-CD45-coated magnetic beads. Cells were cultured without serum for 48 hours in the presence or absence of 10-11, 10-9, 10-7M AngII, with and without Saralasin10-5 or CGP-42112A 10-5M. The percentage of apoptotic cells was determined under a fluorescence microscope by staining with 10μm caspace FITV-VAD-FMK (a fluorescent marker for activated caspaces) and 0.5μg/ml propidium iodide. Statistical analysis was carried out using the student-t test. AngII in concentrations present in human follicle fluid caused increased apoptosis of GL cells compared to untreated control cells (10-11M p=0.312; 10-9M p<0,05; 10-7M p<0,05, respectively). This effect was partially inhibited by both Saralasin and CGP-42112A (18.15% and 33.01%, respectively, p<0,05). In these preliminary studies on human GL cells Ang II at concentrations that are present in FF from human IVF cycles (J Soc Gynecol Investig. 1994 1:118-27) is proapoptotic. The partial blockade by angiotensin receptor antagonists is consistent with a receptor-mediated action. Ovarian AII has been linked to fertility, steroidogenesis and polycystic ovarian syndrome. Further studies are underway to determine the relevance of these results to the outcome of IVF cycles and other clinical scenarios.

P-289: The effect of fragmentation index on embryo survival upon freeze thawing

Evaluation of the effect of embryo morphology on pregnancy outcome following single embryo transfers found that increasing blastomere number predicted improved pregnancy outcome better than fragmentation index. The objective of the present study was to determine if less fragmented embryos have a better chance of surviving freeze/thawing.

P-218: Pregnancy rates according to embryo fragmentation number in women with decreased egg reserve

P-218: Pregnancy rates according to embryo fragmentation number in women with decreased egg reserve

Predictors of embryo fragmentation and outcome after fragment removal in in vitro fertilization

To assess predictors of embryo fragmentation in IVF as well as to compare cycle outcomes between low-grade embryos subjected to defragmentation and high-grade embryos not undergoing defragmentation. A retrospective, case-control trial. A university hospital IVF program. Three hundred twenty-seven nondonor, fresh IVF cycles. Predictors of fragmentation. We evaluated age, basal FSH and E(2) levels, the number of retrieved oocytes, and fertilization rates. Outcome assessments following defragmentation included rates of implantation, clinical pregnancy, spontaneous abortion, and live birth. Increased age and lower number of oocytes and embryos were associated with embryo fragmentation. Lower-grade embryos after defragmentation yielded rates of implantation, clinical pregnancy, live birth, spontaneous abortion, and fetal defects equivalent to high-grade embryos. Fragmented embryos correlate with poorer prognosis cycles; however, fragmented embryos that undergo defragmentation result in equivalent clinical outcomes to high-grade, nondefragmented embryos.

No advantage of laser-assisted over conventional intracytoplasmic sperm injection: a randomized controlled trial [NCT00114725

BackgroundIntracytoplasmic sperm injection (ICSI) is a component of infertility treatment often employed when conventional in vitro fertilization is unlikely to be successful. Despite good clinical results with ICSI, the procedure is typically associated with degeneration of a significant percentage (approximately 10%) of the treated oocytes. The cause of this degeneration remains unclear. Speculation that damage caused by oocyte compression during the injection procedure may be responsible has led to the development of a novel technique known as laser-assisted ICSI. This procedure involves drilling a small hole through the zona pellucida with a laser prior to sperm injection. Preliminary studies have suggested that laser-assisted ICSI may dramatically reduce oocyte degeneration rates. The objective of this study was to examine whether the reported benefits of laser-assisted ICSI could be verified on a larger, less-selected group of patients.MethodsOocytes retrieved from 59 patients scheduled for ICSI were randomly divided into equal treatment and control groups. Oocytes in the treatment group were inseminated by laser-assisted ICSI, while oocytes in the control group were inseminated by conventional ICSI. Outcome variables (oocyte fertilization and degeneration, embryo cell numbers and fragmentation on days 2 and 3, and compaction and blastocyst formation rates) were compared between treatment and control groups by paired-sample t-test. Subgroup analysis was performed according to zona pellucida and oolemma breakage patterns.ResultsNo significant differences between treatment and control groups were observed for any of the measured outcome variables. However, fragile zonae pellucidae were associated with significantly poorer embryo quality, and fragile oolemmas that broke easily upon insertion of the injection needle were associated with a significantly higher oocyte degeneration rate. Nevertheless, there were also no between-treatment differences in clinical outcomes within these patient subpopulations.ConclusionContrary to previous reports based on smaller sample sizes, the results of this study suggest that there is no benefit of laser-assisted ICSI, either for the general population of ICSI patients, or for patients prone to zona pellucida or oolemma fragility.

Use of a medium buffered with N-hydroxyethylpiperazine-N-ethanesulfonate (HEPES) in intracytoplasmic sperm injection procedures is detrimental to the outcome of in vitro fertlization

This study was conducted to determine whether N-hydroxyethylpiperazine-N-ethanesulfonate (HEPES)-buffered medium used for the microinjection of sperm into oocytes may be detrimental for the embryo. Controlled randomized study. Private IVF center. Women (n = 708) undergoing ICSI. The women were randomized into two study groups: 2,204 oocytes from 357 women were treated using a medium buffered with bicarbonate without HEPES during the ICSI procedure, and 2,168 oocytes from 351 women were treated using a medium buffered with HEPES during the ICSI procedure. Fertilization rate, degeneration rate, triploid rate, cleavage rate, embryo quality, pregnancy rate, implantation rate, and abortion rate. Oocytes treated with a HEPES-buffered medium showed a statistically significant higher rate of triploid and degenerated oocytes after fertilization with ICSI compared with oocytes treated with a medium without HEPES. The embryos obtained from oocytes microinjected with a HEPES-buffered medium showed a statistically significant higher rate of highly fragmented embryos compared with the controls. Pregnancy rate and implantation rate were statistically significantly lower in the patient group with oocytes treated with the HEPES-buffered medium. The other parameters evaluated did not show any statistically significant differences. Our study showed that the use of media buffered with HEPES, during the microinjection of sperm into the oocytes, is detrimental for IVF outcome and should be avoided.