Morphokinetics of vitrified and warmed blastocysts predicts implantation potential.

PURPOSE: It was studied whether morphokinetics of blastocoele re-expansion and hatching in vitrified-warmed blastocysts is predictive of implantation, clinical pregnancy, and live birth. METHODS: In 144 patients aiming for single warmed blastocyst transfer, blastocysts were cultured in a new time-lapse system (Miri® TL) immediately after warming. Video sequences with an image interval of 5 min were annotated and the corresponding morphokinetic variables were correlated with pregnancy outcome. In detail, tRE (start of re-expansion), tCRE (completion of re-expansion), tAH (hatching from the manipulated zona pellucida), and presence of collapses were recorded. RESULTS: In the pregnant group, tRE and tCRE were significantly lower (0.69 ± 0.45 h and 2.16 ± 0.94 h) as compared to the non-pregnant group (1.23 ± 1.08 h and 2.70 ± 1.20 h). Both variables and the duration of re-expansion (tCRE-tRE) allowed for distinction between "non-pregnant," "loss of pregnancy," and live birth/ongoing pregnancy. Presence and number of collapses showed no correlation with outcome. CONCLUSIONS: Time-lapse imaging of vitrified-warmed blastocysts offers additional selection criteria allowing for prediction of implantation potential. As a consequence, cumulative pregnancy rate could be increased and time-to-pregnancy reduced.

Treatment with Ca2+ ionophore improves embryo development and outcome in cases with previous developmental problems: a prospective multicenter study.

STUDY QUESTION: Does calcium ionophore treatment (A23187, calcimycin) improve embryo development and outcome in patients with a history of developmental problems/arrest? SUMMARY ANSWER: Application of A23187 leads to increased rates of cleavage to 2-cell stage, blastocyst formation and clinical pregnancy/live birth. WHAT IS KNOWN ALREADY: Studies on lower animals indicate that changes in intracellular free calcium trigger and regulate the events of cell division. In humans, calcium fluctuations were detected with a peak shortly before cell division. Interestingly, these calcium oscillations disappeared in arrested embryos. Mitotic division blocked with a Ca(2+) chelator could be restored by means of ionophores in an animal model. STUDY DESIGN, SIZE, DURATION: This prospective, multicenter (five Austrian centers), uncontrolled intervention study (duration 1 year) includes 57 patients who provided informed consent. PARTICIPANTS/MATERIALS, SETTING, METHODS: Inclusion criteria were complete embryo developmental arrest in a previous cycle (no transfer), complete developmental delay (no morula/blastocyst on Day 5), or reduced blastocyst formation on Day 5 (≤15%). Severe male factor patients and patients with <30% fertilization rate after ICSI were excluded because these would be routine indications for ionophore usage. The total of the 57 immediately preceding cycles in the same patients constituted the control cycles/control group. In the treatment cycles, all metaphase II-oocytes were exposed to a commercially available ready-to-use ionophore for 15 min immediately after ICSI. After a three-step washing procedure, in vitro culture was performed as in the control cycles, up to blastocyst stage when achievable. MAIN RESULTS AND THE ROLE OF CHANCE: Fertilization rate did not differ (75.4 versus 73.2%); however, further cleavage to 2-cell stage was significantly higher (P < 0.001) in the ionophore group (98.5%) when compared with the control cycles (91.9%). In addition, significantly more (P < 0.05) blastocysts formed on Day 5 in the study compared with the control group (47.6 versus 5.5%, respectively) and this was associated with a significant increase (P < 0.01) in the rates of implantation (44.4 versus 12.5%), clinical pregnancy (45.1 versus 12.8%) and live birth (45.1 versus 12.8%). All babies born at the time of writing (22/28) were healthy. LIMITATIONS, REASONS FOR CAUTION: The frequency of patients showing embryo developmental problems was expected to be low; therefore, a multicenter approach was chosen in order to increase sample size. In one-third of the cycles, the clinician or patient requested a change of stimulation protocol; however, this did not influence the developmental rate of embryos. WIDER IMPLICATIONS OF THE FINDINGS: This is the first evidence that developmental incompetence of embryos is an additional indication for ionophore treatment. The present approach is exclusively for overcoming cleavage arrest. STUDY FUNDING/COMPETING INTERESTS: No funding received. T.E. reports fees from Gynemed, outside the submitted work. All co-authors have no interest to declare.

Live birth after artificial oocyte activation using a ready-to-use ionophore: a prospective multicentre study.

Artificial oocyte activation has been proposed as a suitable means to overcome the problem of failed or impaired fertilization after intracytoplasmic sperm injection (ICSI). In a multicentre setting artificial oocyte activation was applied to 101 patients who were diagnosed with fertilization abnormalities (e.g. less than 50% fertilized oocytes) in a previous conventional ICSI cycle. Female gametes were activated for 15 min immediately after ICSI using a ready-to-use Ca(2+)-ionophore solution (A23187). Fertilization, pregnancy and live birth rates were compared with the preceding cycle without activation. The fertilization rate of 48% in the study cycles was significantly higher compared with the 25% in the control cycles (P < 0.001). Further splitting of the historical control group into failed (0%), low (1-30%) and moderate fertilization rate (31-50%) showed that all groups significantly benefitted (P < 0.001) in the ionophore cycle. Fewer patients had their embryo transfer cancelled compared with their previous treatments (1/101 versus 15/101). In total, 99% of the patients had an improved outcome with A23187 application resulting in a 28% live birth rate (35 babies). These data suggest that artificial oocyte activation using a ready-to-use compound is an efficient method.

Influence of intracervical and intravaginal seminal plasma on the endometrium in assisted reproduction: a double-blind, placebo-controlled, randomized study.

OBJECTIVE: To investigate the effect of intracervical and intravaginal application of seminal plasma on the endometrium, as assessed by endometrial/subendometrial vascularization and endometrial volume between the day of oocyte retrieval and the day of embryo transfer in an in-vitro fertilization/intracytoplasmic sperm injection (IVF/ICSI) cycle. METHODS: This was a double-blind, placebo-controlled, randomized study including patients undergoing a first or second IVF/ICSI cycle. Homologous seminal plasma or placebo (sodium chloride) was injected into the cervix and posterior vaginal fornix just after follicle aspiration. Three-dimensional power Doppler examination was performed 30 min before oocyte retrieval and 30 min before embryo transfer. Main outcome measures were changes in vascularization flow index (VFI), flow index (FI) and vascularization index (VI) of the endometrium/subendometrium using VOCAL™ (Virtual Organ Computer-aided AnaLysis) and endometrial volume. RESULTS: One hundred patients agreed to participate in the study. Twenty-three patients were excluded, mainly as a result of canceled embryo transfer. Data were analyzed from 40 patients receiving seminal plasma and 37 receiving placebo. No significant differences between the two groups were seen in VFI, FI or VI of the endometrium or subendometrium or in endometrial volume on the day of oocyte pick-up and on the day of embryo transfer. CONCLUSION: Neither endometrial/subendometrial vascularization parameters nor endometrial volume seem to be affected by the application of seminal plasma in patients undergoing their first or second IVF/ICSI cycle.

Healthy twin live-birth after ionophore treatment in a case of theophylline-resistant Kartagener syndrome.

PURPOSE: To evaluate whether it is a feasible option to target the oocyte (with Ca(2+)-ionophore) in case that sperm motility cannot be restored in Kartagener syndrome. METHODS: A case of a male Kartagener syndrome with exclusively immotile spermatozoa that did not react to the dimethylxanthine theophylline. Thus, half of the associated oocytes were treated for 15 min with the ready-to-use- ionophore CultActive immediately after ICSI whereas the other 50 % were injected with routine ICSI without artificial oocyte activation. Rates of fertilization, blastulation, pregnancy and live birth were evaluated. RESULTS: Fertilization check revealed that none of the conventionally injected but 4/6 (66.7 %) of the artificially activated oocytes showed two pronuclei. Three embryos were of good and one of fair quality. Corresponding blastocyst formation rate was 3 out of 4 (75 %). A double embryo transfer led to a healthy twin birth in the 34th week of gestation (two boys with a birth weight of 1724 g and 2199 g). CONCLUSIONS: This case indicates that Ca(2+)-ionophore treatment in cycles from theophylline-resistant Kartagener syndrome patients is a feasible option. The future will show if routine application of A23187 in Kartagener or primary cilia dyskinesis patients will be of benefit.

Efficacy of a sperm-selection chamber in terms of morphology, aneuploidy and DNA packaging.

Since most current techniques analysing spermatozoa will inevitably exclude these gametes from further use, attempts have been made to enrich semen samples with physiological spermatozoa with good prognosis using special sperm-processing methods. A particular sperm-selection chamber, called the Zech-selector, was found to be effective in completely eliminating spermatozoa with DNA strand breaks. The aim of this study was to further analyse the subgroup of spermatozoa accumulated using the Zech-selector. In detail, the potential of the chamber to select for proper sperm morphology, DNA status and chromatin condensation was tested. Two samples, native and processed semen, of 53 patients were analysed for sperm morphology (×1000, ×6300), DNA packaging (fragmentation, chromatin condensation) and chromosomal status (X, Y, 18). Migration time (the time needed for proper sperm accumulation) was significantly correlated to fast progressive motility (P=0.002). The present sperm-processing method was highly successful with respect to all parameters analysed (P<0.001). In particular, spermatozoa showing numeric (17.4% of patients without aneuploidy) or structural chromosomal abnormalities (90% of patients without strand-breaks) were separated most effectively. To summarize, further evidence is provided that separating spermatozoa without exposure to centrifugation stress results in a population of highly physiological spermatozoa.

Planar embryos have poor prognosis in terms of blastocyst formation and implantation.

Normally, day-2 embryos show a crosswise arrangement of four cells with three blastomeres lying side by side. Cleavage anomalies include embryos that are characterized by a particular planar constellation of four blastomeres with presumed incomplete cleavage. Since little is known on the developmental fate of such conceptuses, within a 10-month period all consecutive patients were screened for day-2 planar embryos. A total of 64/2070 embryos with suboptimal blastomere configuration were detected (3.1%). In conventional IVF, planar embryos were significantly less frequent (0.7%) as compared with intracytoplasmic sperm injection (2.8%; P<0.05) and cases of testicular sperm extraction (5.4%; P<0.01). Interestingly, embryos with a cleavage anomaly showed better morphology both on day 2 (P<0.005) and day 3 (P<0.001). In contrast, blastocyst formation (P<0.001) and blastocyst quality (P=NS) was higher in tetrahedral embryos. There was a significant increase in implantation rate if tetrahedral embryos could be transferred compared with when planar embryos had to be transferred (P<0.01). It may be postulated that, in planar embryos, the mitotic spindle might have been affected, e.g. sperm centrosome composition or function, which in turn might have led to the observed cleavage anomaly. Normally, day-2 embryos show a crosswise arrangement of four cells with three blastomeres lying side by side. Cleavage anomalies include more planar embryos that are characterized by a particular flat constellation of four blastomeres with presumed premature cleavage (like a tetrafoliate clover). Since little is known on the developmental fate of such embryos within a 10-month study period, all consecutive patients were screened for the presence of day-2 planar embryos (study group). A total of 64 (out of 2070) embryos with abnormal blastomere configuration were detected (3.1%). Interestingly, in conventional IVF (0.7%), the presence of planar embryos was significantly less frequent as compared with intracytoplasmic sperm injection (2.8%; P<0.05) and cases of testicular biopsy (5.4%; P<0.01). Embryos from the study group showed better morphology both on day 2 (P<0.005) and day 3 (P<0.001). In contrast, blastocyst formation (survival to day 5 of preimplantation development) was higher in the normally cleaved control group (P<0.001) and so was blastocyst quality; however, the latter parameter did not reach level of significance. This was also reflected in a significantly higher implantation rate in the control group (P<0.01). Based on present data, it may be postulated that, in planar embryos, the mitotic spindle (which involves the sperm centrosome) might have been affected, which in turn might have led to an incomplete cleavage.

Easy sperm processing technique allowing exclusive accumulation and later usage of DNA-strandbreak-free spermatozoa.

Sperm DNA fragmentation is increased in poor-quality semen samples and correlates with failed fertilization, impaired preimplantation development and reduced pregnancy outcome. Common sperm preparation techniques may reduce the percentage of strandbreak-positive spermatozoa, but, to date, there is no reliable approach to exclusively accumulate strandbreak-free spermatozoa. To analyse the efficiency of special sperm selection chambers (Zech-selectors made of glass or polyethylene) in terms of strandbreak reduction, 39 subfertile men were recruited and three probes (native, density gradient and Zech-selector) were used to check for strand breaks using the sperm chromatin dispersion test. The mean percentage of affected spermatozoa in the ejaculate was 15.8 ± 7.8% (range 5.0­42.1%). Density gradient did not significantly improve the quality of spermatozoa selected(14.2 ± 7.0%). However, glass chambers completely removed 90% spermatozoa showing strand breaks and polyethylene chambers removed 76%. Both types of Zech-selectors were equivalent in their efficiency, significantly reduced DNA damage (P < 0.001) and,with respect to this, performed better than density gradient centrifugation (P < 0.001). As far as is known, this is the first report ona sperm preparation technique concentrating spermatozoa unaffected in terms of DNA damage. The special chambers most probably select for sperm motility and/or maturity.

Group culture of human zygotes is superior to individual culture in terms of blastulation, implantation and life birth.

This prospective study tested a new type of culture dish for the effects of individual culture and autotrophic factors. Within a 6-month period, 72 patients with nine or more fertilized eggs were enrolled in this prospective evaluation. Their 936 zygotes were split into three subgroups (individual culture, individual culture with contact to neighbours, group culture). All concepti were cultured in 30 µl drops (medium change on day 3) until blastocyst stage. On day 5, a single-blastocyst transfer was performed and the remaining blastocysts of good quality were vitrified. Fertilization rates were 69% for IVF and 81% for intracytoplasmic sperm injection. Blastulation was 48%. Single-blastocyst transfer resulted in a clinical pregnancy rate of 54%. Group culture was superior in terms of compaction (P<0.01) and blastulation (P<0.001) as compared with individual culture. A better blastocyst quality was observed in group culture (P<0.05). As a trend, more life births were achieved with blastocysts derived from group culture. As far as is known, this is the first evidence that grouping embryos improves preimplantation development in human and it is recommended that culture volume should be reduced or embryo density increased.

Morphological analysis at compacting stage is a valuable prognostic tool for ICSI patients.

In terms of treatment outcome, little prognostic power is attributed to day-4 morphology. A day-4 score was applied to 56 patients separating non-compacting embryos from compacting (some areas of compaction) and fully compacted embryos. The latter were further subdivided according to the morphology of compaction. Grade C1 embryos represented optimal quality, while grades C2 (exclusion of fragments) and C3 (exclusion of blastomeres) were characterized by a loss of cytoplasm. Grade 4 embryos (C4) showed incomplete compaction with several blastomeres not yet incorporated into cell mass. Pooled embryos without compaction showed a reduced (P < 0.001) blastulation (28.8%) as compared with compacting embryos with the same cell number (68.8%), which, in turn, revealed lower (P < 0.05) rates of blastulation as compared with concepti that completed compaction process (84.6%). Among fully compacted embryos grade C1 had a better (P < 0.01) blastocyst formation rate (94.4%) as compared with grade C3 (68.2%). Grade C1 embryos showed significantly higher rates of top-quality blastocysts as compared with grade C2 (P < 0.05) and C3 (P < 0.01). Blastocysts deriving from grade C1/C4 embryos led to a higher pregnancy rate as compared with the C2/C3 counterparts (P < 0.05). This modified score allows for adequate prediction of both blastocyst formation/quality and pregnancy.

Morphology of vitrified/warmed day-5 embryos predicts rates of implantation, pregnancy and live birth.

Although some post-thaw morphological predictors of pregnancy have been investigated in slow freezing of blastocysts, no such data have been published for vitrified and warmed blastocysts. Therefore, a prospective four-part score was applied to vitrified/warmed day-5 embryos to evaluate whether certain morphological parameters could serve as predictors of implantation, pregnancy and live birth. All morulae/blastocysts that were considered to be viable after warming were scored according to a previously unpublished grading system based on re-expansion, hatching (out of an artificial gap in the zona pellucida), extensive cytoplasmic granulation and presence of necrotic foci. Overall, 74% (202/273) of the vitrified concepti were found to be viable after warming. Early blastocysts showed better survival versus extended/hatching blastocysts (P < 0.01). Of the morphological parameters analysed, immediate re-expansion (P < 0.05) and hatching (P < 0.001) were positive predictors of the rates of implantation, pregnancy and live birth. The opposite holds for extensive cytoplasmic granulation (P < 0.05), which was negatively related. Accurate scoring of warmed blastocysts (within the first 2 h) allows for prediction of pregnancy outcome, and thus will help to further reduce the number of transferred embryos.

The role of mode of conception in the outcome of twin pregnancies.

Artificial reproductive techniques (ART) have become a routine and successful treatment of infertility. Twin pregnancies are more frequent after ART compared to spontaneous conception. Varying findings have been reported for the obstetric and perinatal outcome of ART twin pregnancies as compared to spontaneously conceived twin pregnancies. To evaluate the specific risks of twin pregnancies achieved through ART, literature has been reviewed for obstetric and perinatal risks and differences between artificially and spontaneously conceived twin pregnancies. Eleven studies have been involved in this review. Three of the studies were matched and eight were non-matched studies. Results of analysed studies differed widely. Twin pregnancies resulting from ART showed an increased rate of Caesarean section and tend toward higher risk for preterm birth and low birthweight compared to spontaneously conceived twin pregnancies. Various conditions can influence twin pregnancies outcome after ART. In in-vitro fertilization programs twin pregnancies should be avoided and physicians have to inform patients of the specific risks when transferring more than one embryo.

Developmental fate of ovoid oocytes.

BACKGROUND: Irregularities in composition, thickness and/or color of the zona pellucida may impair optimal function and result in reduced outcome. Anomalies of oocyte shape have not been investigated in detail in this respect. METHODS: Therefore, all patients attending our clinic within a period of 1 year were screened for the presence of ovoid gametes and the corresponding developmental potential was evaluated. For all elongated gametes, a roundness index (RI; length divided by width) was calculated in order to quantify shape. RESULTS: RI did not affect fertilizability (P > 0.05). The degree of dysmorphism was found to be related to cleavage pattern. The more ovoid a gamete was, the higher was the risk of the corresponding zygote not cleaving like a tetrahedron (P < 0.01). Abnormal cleavage (a rather flat array of blastomeres) was associated with delayed compaction (P < 0.01) and blastocyst formation (P < 0.001). The quality of blastocysts was not affected at any stage in ovoid concepti (P > 0.05). CONCLUSIONS: Ovoid oocytes with abnormal cleavage pattern show delayed preimplantation development, probably due to a reduced number of cell-to-cell contacts.

Blood clots in the cumulus-oocyte complex predict poor oocyte quality and post-fertilization development.

Assessment of oocyte maturity and quality (morphological appearance) at the time of retrieval is difficult as the egg is obscured by a large cumulus mass that hinders adequate scoring. Since no data are available on the possible relationship between the cumulus-oocyte complex (COC) and oocyte morphology, this prospective intracytoplasmic sperm injection study was set up in 87 consecutive patients. COC were grouped according to expansion of both corona radiata and cumulus matrix. Special emphasis was placed on recording morphological anomalies of COC (inclusion of blood clots and amorphous clumps). For all mature ovae, quality was assessed and preimplantation development followed up to blastocyst stage if fertilized. The risk of not harvesting an oocyte was higher in COC with blood clots compared with normal cumulus matrices (P = 0.004). COC expansion did not allow for prediction of either nuclear status or quality of the egg. The presence of blood clots within the cumulus matrix was associated with reduced oocyte quality (dense central granulation), fertilization rate and blastocyst formation, compared with unaffected COC (P < 0.05). It may be postulated that COC showing blood inclusions derive from poor quality follicles, which has a detrimental effect on oocyte quality and further cleavage to blastocyst stage. Consequently, mechanical removal of blood clots cannot rescue the corresponding embryo.

Effect of hyaluron derivate gel in prevention of postsurgical peritoneal adhesions--an experimental study in pigs.

BACKGROUND/AIMS: The aim of this study was to assess the efficacy of a hyaluronic acid derivate gel in reducing adhesions in pigs undergoing laparoscopic abdominal cavity surgery. METHODOLOGY: 20 domestic female pigs underwent laparoscopy in general anesthesia. 4 defined serosal defects have been made (hepatoduodenal ligament, parietal, anterior stomach wall and jejunal mesentery). The defects have been covered with 20 mL Hyalobarrier Gel in 10 randomly selected pigs. 2 weeks postoperatively the pigs got reoperated for evaluation of potential adhesions. RESULTS: 17 pigs remained for evaluation. 2 pigs died during introduction of anesthesia, one at the end of the operation. None of the pigs died due to a reaction to the gel. We found adhesions in 33% of the gel group (n=3 out of 9) and in 87.5% in the group without gel (7 out of 8): p < 0.05. Most of the adhesions have been found at the parietal defect (7 out of 17 at all). CONCLUSIONS: The hyaluronic acid gel was highly efficacious and reduced the number and extent of adhesions throughout the abdomen following laparoscopic peritoneal surgery significantly.

[Multiple pregnancies after ART: problems and possible solutions]. / Mehrlingsschwangerschaften nach ART: Probleme und Losungsansatze.

Multiple pregnancies following an assisted reproduction technique (ART) should be seen as a complication, and for that reason they should be avoided. In contrast to singleton pregnancies following ART, the multiple pregnancies are associated with a higher prenatal, neonatal and maternal risk; furthermore this results in a financial burden for the health care system. This paper gives an overview of the latest literature and different attempts of European countries, trying to reduce the multiple pregnancy rate. An efficient reduction is only possible by single-embryo transfers. There should be strict and cross-national regulation for the choice of women who should have a transfer of more than one embryo.

Incomplete denudation of oocytes prior to ICSI enhances embryo quality and blastocyst development.

BACKGROUND: Granulosa cells are essential mediators of oocyte maturation and fertilization. Because of the denudation of oocytes in preparation for ICSI, any potential positive effect of surplus cumulus cells (CCs) on further development would be unable to exert further effect. In order to evaluate the actual influence of adhering cumulus cells on further preimplantation development, this prospective study was carried out. METHODS: Sibling cumulus-oocyte complexes for 57 ICSI patients were split into a study group (incomplete denudation, n = 314) and a control group (complete denudation, n = 336). According to the cumulus cell pattern after partial denudation, mature gametes from the study group were further subdivided into type A oocytes, which showed several prominent CC clusters (n = 202), and type B (n = 75), which showed a more homogeneous pattern with CC covering the whole surface of the gamete. RESULTS: In immature oocytes, presence of adhered CCs led to a significant increase in resumption of meiosis (P < 0.01). Fertilization rate (P < 0.05) and ability to cleave (P < 0.01) was impaired in the study group, because of difficulties in ICSI of type B oocytes. By contrast, embryo morphology on days 2 (P < 0.01) and 3 (P < 0.05), as well as blastocyst formation, was better (P < 0.05) in the study group (55 blastocysts out of 88 zygotes) as compared to that in the control group (49/105). CONCLUSION: These data indicate that co-culture of oocytes with attached CCs may enhance preimplantation development.

Basal level of anti-Müllerian hormone is associated with oocyte quality in stimulated cycles.

BACKGROUND: Serum anti-Müllerian hormone (AMH) levels provide a powerful means for predicting ovarian response, which is reflected not only by the size of the primordial follicle pool but also by the quality of the oocytes. Considering a mutual interdependence between AMH-expressing somatic cells and gametes, this prospective morphological study was set up to evaluate whether extreme AMH levels represent diminished oocyte quality and developmental incompetence. METHODS: A total of 141 consecutive ICSI patients were subdivided into three groups using the 25th and 75th percentiles of the serum AMH levels (cycle day 3). In these three groups, morphology of all oocytes and fertilization rate, embryo quality and blastocyst formation were evaluated, and FSH, LH and estradiol (E(2)) levels were also measured. RESULTS: Cycle cancellation rate was correlated with AMH levels (P < 0.05). AMH groups 1 (<1.66 ng/ml) and 3 (>4.52 ng/ml) showed oocytes of lower quality [dark central granulation, aggregation of smooth endoplasmic reticulum (sER)] compared with the median group 2 (1.66-4.52 ng/ml). Basal serum FSH did not allow for adequate prognosis in terms of gamete appearance. Fertilization and further cleavage up to blastocyst stage was not affected by AMH levels. CONCLUSIONS: AMH seems to be superior to FSH in predicting both oocyte number and quality.