O. Shebl

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.

Automatic user-independent zona pellucida imaging at the oocyte stage allows for the prediction of preimplantation development

OBJECTIVEnTo analyze whether a change in three-dimensional structure of the zona pellucida could indicate suboptimal gamete quality.nnnDESIGNnProspective study.nnnSETTINGnWomens general hospital.nnnPATIENT(S)nA total of 72 patients who gave informed consent.nnnINTERVENTION(S)nThe birefringence of all oocytes was prospectively analyzed with an automatic user-independent polarization microscopy imaging system.nnnMAIN OUTCOME MEASURE(S)nBirefringence of the inner zona layer, preimplantation development, implantation, and pregnancy.nnnRESULT(S)nIn approximately one third of all gametes (244/712), the systems automatic detection of the inner zona layer did not succeed. This phenomenon was a negative predictor of compaction (P<0.01), blastulation (P<0.001), and pregnancy (P<0.001). In cases of successful zona imaging, the score based on the birefringence of the inner zona layer was a strong predictor of blastocyst formation but not of embryo quality or pregnancy (P>0.05). Interestingly, antagonist protocol resulted in lower zona scores as compared with the long protocol (P<0.05).nnnCONCLUSION(S)nCombining the information from both undetected and detected oocytes, zona imaging was a helpful tool in oocyte selection. This knowledge might further help to reduce both the time in culture and the number of concepti considered for transfer.

Endometriosis: a premenopausal disease? Age pattern in 42,079 patients with endometriosis

PurposeThe objectives of this study were to examine the age distribution among women suffering from endometriosis and to establish that endometriosis is not a disease that occurs only in premenopausal women. The null hypothesis was that there are also postmenopausal women with endometriosis.MethodsIn a retrospective epidemiological study, a descriptive analysis of data from the Federal Statistical Office in Germany for 2005 and 2006 was carried out. A total of 42,079 women in Germany were admitted for surgical treatment due to histologically confirmed endometriosis during this period. The patients’ age distribution was examined and they were assigned to 5-year age groups and then to premenopausal, perimenopausal, and postmenopausal subgroups.ResultsA total of 20,835 women in 2005 and 21,244 in 2006 were admitted to hospital for the treatment of endometriosis. In the premenopausal group (age 0–45xa0years), there were 33,814 patients (80.36xa0%); 23 patients (0.05xa0%) in this premenopausal group were younger than 15. There were 7,191 patients (17.09xa0%) in the perimenopausal group (45–55xa0years), and the postmenopausal group (55–95xa0years) included 1,074 patients (2.55xa0%).ConclusionsThe assumption that endometriosis is a disease of the premenopausal period and in women of reproductive age needs to be called into question, as well as the influence of estrogen in fully developed endometriosis. Due to the relatively high prevalence of the condition in patients aged over 40, physicians should consider endometriosis in cases of unclear pelvic pain in this age group.

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.

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.

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 5.0–42.1%). 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.

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.

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.

Developmental capacity and pregnancy rate of tetrahedral- versus non-tetrahedral-shaped 4-cell stage human embryos

PurposeThe arrangement of the blastomeres within the 4-cell stage embryo reflects the orientation of the cleavage planes during the second division. To examine their relevance, the developmental capacity and the pregnancy rate were compared between tetrahedral-shaped and non-tetrahedral-shaped 4-cell stage human embryos.MethodsThe study included 3,546 4-cell stage embryos. The arrangement of the blastomeres at the 4-cell stage was annotated as being tetrahedral or non-tetrahedral on day 2 of preimplantation development. Embryo quality was compared on day 3 and day 5. Pregnancy rates were calculated per single embryo transfer on day 3 or day 5.ResultsIn total, 2,803 4-cell stage embryos (79xa0%) displayed a tetrahedral arrangement and 743 (21xa0%) displayed a non-tetrahedral arrangement. Tetrahedral-shaped embryos developed more into high-quality embryos on day 3 (pu2009<u20090.001) and day 5 (pu2009=u20090.036) and had a higher blastulation rate (pu2009=u20090.009). Though, the number of high-quality embryos selected for transfer did not differ between both groups on day 3 (pu2009=u20090.167) and day 5 (pu2009~u20091). Three hundred thirty single embryo transfers were analysed. No significant difference in clinical pregnancy was found between both groups after transfer on day 3 (pu2009=u20090.209) and day 5 (pu2009=u20090.653).ConclusionsThe arrangement of the blastomeres according to their previous cleavage planes was correlated to the developmental potential of the 4-cell stage embryo up to the blastocyst stage. If embryo transfers are performed on day 3 and day 5 of development using embryos of adequate quality, the blastomere arrangement at the 4-cell stage had no predictable value regarding pregnancy success.