Reduced blastocyst formation in reduced culture volume

Purpose

The aim of this prospective sibling oocyte study was to evaluate whether reduced culture volume improves blastocyst formation.

Methods

Twenty-three patients with extended embryo culture until day 5 were selected for the study. After injection, 345 sibling oocytes were individually cultured in either 25 or 7 μl droplets of Origio cleavage medium under oil. On day 3 of development, embryos were transferred to droplets with the corresponding volume of Origio blastocyst culture medium. Fertilization and embryo quality on day 3 and day 5/6 were evaluated.

Results

No statistically significant difference (p = 0.326) in fertilization rate was observed (81.3 versus 83.0 %). There was no significant difference in terms of the number of excellent and good-quality embryos obtained on day 3 between both groups (p = 0.655). Embryo culture in 25 μl droplets led to more embryos with a higher cell number when compared to 7 μl culture (p = 0.024). On day 3, 132 and 131 embryos were considered for further culture until day 5/6. Blastulation rates were significantly higher in the 25 μl group (75.0 versus 61.6 %; p = 0.017) and significantly more day 5 embryos with excellent and good quality were found in this group (54.5 versus 40.5 %; p = 0.026). Finally, the utilization rates expressed per mature oocyte (41.4 versus 29.8 %; p = 0.043), per fertilized oocyte (50.7 versus 36.6 %; p = 0.023), and per day 3 embryo undergoing extended culture to day 5/6 (54.5 versus 39.7 %; p = 0.019) were all significantly higher in the 25 μl group.

Conclusion

Reduced culture volume (7 μl) negatively impacts early development by reducing the cell number on day 3 and both blastocyst formation and quality.     

Effect of culture medium volume and embryo density on early mouse embryonic development: Tracking the development of the individual embryo

Purpose

To determine the optimal volume or density of embryos for the well-of-the-well (WOW) system in order to track the development of individual embryos and to determine whether the WOW system can reverse the negative impact of culturing embryos singly.

Methods

(1) Mouse embryos (groups of nine at the 2-cell stage) were cultured in 6.25 μl, 12.50 μl, 25.00 μl and 50.00 μl of droplets of culture medium under paraffin oil; (2) Groups of three, six, nine and twelve embryos at the 2-cell stage were cultured in 50 μl of droplet of culture medium under paraffin oil; (3) Groups of nine embryos at the 2-cell stage were cultured in 50 μl of droplet under paraffin oil with or without nine micro-wells made on the bottom of the Petri dish into each of which were placed one of the nine embryos (WOW system). Also single 2-cell stage embryos was cultured individually in 5.5 μl of droplet of culture medium under paraffin oil with or without a single micro-well made on the bottom of the Petri dish (WOW system for single culture). At the end of culture, the percentages of blastocyst development, hatching and hatched blastocysts were compared in each group. The blastocysts were fixed for differential staining.

Results

The blastocyst development was significantly higher (P < 0.05) when nine embryos were cultured in 50 μl of droplet of culture medium compared with other volumes. The blastocyst development was significantly reduced (P < 0.05) in single embryo culture compared to group embryo culture with or without the WOW system. The blastocyst development was not improved when single embryo cultured individually in a micro-well was compared to single embryo cultured individually without micro-well. The total cell numbers of blastocysts were significantly higher in group embryo culture than single embryo culture regardless of whether the WOW system was used. In addition, the total cell numbers of blastocysts were significantly higher (P < 0.05) in single embryo culture with the WOW system than without.

Conclusions

Group embryo culture is superior to single embryo culture for blastocyst development. The WOW system with 50 μl of droplet of culture medium can be used to track the individual development of embryo cultured in groups while preserving good embryonic development. The reduced embryonic development with single embryo culture cannot be ameliorated by the WOW system.     

The impact of the protein stabilizer octanoic acid on embryonic development and fetal growth in a murine model

Purpose

The purpose of this study was to determine the effect of the protein stabilizer octanoic acid on blastocyst development, implantation, and fetal growth in a murine model.

Methods

One-cell mouse embryos were collected and individually cultured in medium supplemented with recombinant human serum albumin for 96 h at 5 % oxygen in an EmbryoScope. Embryos were randomly allocated to four octanoic acid groups (0, 400, 800, or 1200 μM). Blastocyst development and cell cycle timings were calculated at 96 h of culture, and experiments were repeated in triplicate. Blastocysts were stained and fixed at 96 h for differential cell counts. Following 96 h of culture, blastocysts were transferred to recipients to determine implantation rates and fetal and placental weights.

Results

Blastocyst development, hatching rates, developmental kinetics, and total number of cells were negatively affected by octanoic acid at concentrations commonly used in human IVF. Implantation was not affected by octanoic acid but fetal and placental weights at 800 μM octanoic acid were increased relative to control.

Conclusions

Octanoic acid, a standard additive to human protein supplements used in IVF, can have long-term negative effects on embryonic and fetal development. The use of octanoic acid for human embryo culture should be monitored and reduced.     

Confirmation rates of array-CGH in day-3 embryo and blastocyst biopsies for preimplantation genetic screening

Purpose

The purpose of this study was to compare the confirmation rate of day-3 embryo biopsy (blastomere) and trophectoderm biopsy using array-comparative genomic hybridization (array-CGH) technology.

Methods

A blinded study was conducted to re-analyse 109 embryos previously diagnosed as chromosomally abnormal by array-CGH. Preimplantation genetic screening (PGS) was performed using array-CGH on day 3 (n = 50) or day 5 (n = 59). Partial chromosome gains or losses were excluded (n=6), and only whole chromosome aneuploidies were considered. Re-analysis of whole blastocysts was carried out following the same array-CGH protocol used for PGS.

Results

The PGS result was confirmed in the whole blastocyst in (a) 49/50 (98 %) abnormal embryos after day-3 biopsy and (b) 57/59 (96.6 %) abnormal embryos after trophectoderm biopsy. One embryo (1/50; 2 %) was diagnosed as abnormal, with monosomy 18, on day 3, and software analysis of the whole blastocyst gave a euploid result; however, a mosaic pattern was observed for monosomy 18 in the whole blastocyst. Two trophectoderm biopsy cases (3.4 %) did not have the abnormalities (trisomy 7, and trisomy 1 and 4, respectively) verified in the whole embryo. Concordance rates for both biopsy strategies and for individual chromosomes were evaluated by Fisher’s exact test and showed no significant differences.

Conclusions

Both types of biopsies showed similar high concordance rates with whole blastocyst results. Therefore, regarding the confirmation rates shown in this work, day-3 embryo biopsies can be representative of the whole embryo and both types of biopsy can be used for clinical analysis in PGS following the described array-CGH protocol.     

Ongoing and cumulative pregnancy rate after cleavage-stage versus blastocyst-stage embryo transfer using vitrification for cryopreservation: Impact of age on the results

Purpose

To determine if blastocyst transfer increases the ongoing and cumulative pregnancy rates, compared with day 3 embryo transfer, in women of all ages when at least 4 zygotes are obtained.

Methods

Prospective study including patients undergoing a first IVF/ICSI treatment and assigned to cleavage stage (n = 46) or blastocyst (n = 58) embryo transfer. Supernumerary embryos were vitrified and patients failing to achieve an ongoing pregnancy after fresh embryo transfer would go through cryopreserved cycles. The main outcome measure was the ongoing pregnancy rate after the fresh IVF/ICSI transfer and the cumulative ongoing pregnancy rate. Results were also analyzed according to age (under 35 and 35 or older).

Results

A majority of patients (96.6 %) had a blastocyst transfer when at least 4 zygotes were obtained. The ongoing pregnancy rate was significantly higher in the day-5 group compared with the day-3 group (43.1 % vs. 24 %, p = 0.041). The cumulative ongoing pregnancy rate was higher (but not significantly) with blastocyst than with cleavage stage embryos (56.8 % vs. 43.4 %, p = 0.174). When analysed by age, patients 35 or older showed significantly higher ongoing pregnancy rate (48.4 % vs. 19.3 %, p = 0.016) and cumulative ongoing pregnancy rate (58 % vs. 25.8 %, p = 0.01) in the day-5 group compared to the day-3 group, while no such differences were observed in women under 35.

Conclusions

Blastocyst transfer can be suggested whenever there are at least 4 zygotes. While there are no differences in women under 35, the benefit of this option over cleavage stage transfer could be significant in women 35 or older.     

Effects of group culture on the development of discarded human embryos and the construction of human embryonic stem cell lines

Purpose

To explore the effect of group culture on the developmental potential of discarded embryos in in vitro fertilization/intracytoplasmic sperm injection (IVF/ICSI) cycles and establish the human embryonic stem cell lines for future research.

Methords

Fresh discarded embryos were collected from the IVF/ICSI-ET program in the reproductive medical center of the first affiliated hospital of Zhengzhou university in this study. All zygotes were individually cultured from Day 1 to Day 3. On Day 3, discard embryos were then cultured in group of 1–4 embryos per droplet (30 μl/droplets) with a constant culture medium until Day 5 or 6. Mechanical method was used to isolate the inner cell mass (ICM) of blastocyst from the embryo. Then we inoculated the ICM on feeder layer. After identification of those cells, the human embryonic stem cell lines (hESCs) were established.

Results

In this study, we collected 1,223 fresh discarded embryos and they were sequential cultured to the blastocysts (18.07 %, 221/1,223), in which good quality blastocysts were 61(4.98 %, 61/1,223). There was no significant difference in the patients. The embryos from 1PN, 2PN, 3PN were sequential cultured to the blastocyst s(39.31 %,92/234;12.87 %,64/497;13.21 %,65/492),in which good quality blastocysts was 13.6 %(32/92),2.61 %(13/64), 3.04 %(15/65).1PN embryo’s blastulation rate and quality embryo formation rate was significantly higher than the 2PN and 3PN embryos’ (P <0.05). Three embryos group cultivation has the highest blastulation rate and quality embryo formation rate (P <0.05). In total, we successfully established 4 hESCs lines.

Conclusion

The group culture of human discard embryos can improve the blastulation rate and blastocyst quality to some extent. Three embryos group cultivate is the better culture number. Human discard embryos are good source for establishment of hESCs.     

Effects of granulocyte-macrophage colony-stimulating factor supplementation in culture medium on embryo quality and pregnancy outcome of women aged over 35 years

Purpose

The purpose of this study is to explore whether a low concentration of granulocyte-macrophage colony-stimulating factor (GM-CSF) supplementation in culture medium is beneficial to infertile women aged over 35 years.

Methods

A retrospective cohort study was performed to analyze the embryo quality and pregnancy outcome of 212 controlled ovarian stimulation (COH) cycles with or without GM-CSF addition (n = 117 [GM-CSF, 0.2 ng/mL] vs n = 95 [control]).

Results

No significant difference was observed in cleavage rate (96.2 vs 96.5 %), blastocyst formation rate (53.2 vs 54.0 %), good blastocyst rate (26.8 vs 26.8 %), or available embryo rate (54.2 vs 49.7 %) between the GM-CSF group and the control group. However, the average age of the GM-CSF group (38.41 ± 3.13 years) was significantly 1 year older than that of the corresponding control group (37.45 ± 2.74 years) (P < 0.05). GM-CSF addition greatly decreased the occurrence of biochemical pregnancy (55.6 % [control] vs 20.8 % [GM-CSF], P < 0.05). No case of neonatal malformation was observed in the present study.

Conclusion

Although no benefit of GM-CSF on embryo quality was observed, the addition of this factor significantly decreased the occurrence of chemical pregnancy of women aged over 35 years, indicating the role of GM-CSF in improving implantation competence of embryos derived from elderly infertile women.     

Markers of cellular senescence are elevated in murine blastocysts cultured in vitro: molecular consequences of culture in atmospheric oxygen

Purpose

We aimed to determine whether embryo culture induces markers of cellular senescence and whether these effects were dependent on culture conditions.

Methods

Murine blastocysts were derived in vitro and in vivo and assessed for 2 primary markers of senescence: senescence-associated β-galactosidase (SA-β-gal) and phosphorylated H2A.X (γ-H2A.X), the latter being a mark of DNA oxidative damage. Expression of senescence-associated genes p21, p16, and interleukin 6 (IL6) were also assessed.

Results

Compared with in vivo–derived blastocysts, in vitro embryos had high levels of SA-β-gal, nuclear γ-H2A.X, and p21 mRNA expression, indicating that a senescence-like phenotype is induced by in vitro culture. To determine the role of culture conditions, we studied the effect of oxygen (5 % vs 20 %) and protein supplementation on senescence markers. Blastocysts in reduced oxygen (5 %) had low levels of both SA-β-gal and γ-H2A.X compared with blastocysts cultured in ambient oxygen. Senescence markers also were reduced in the presence of protein, suggesting that antioxidant properties of protein reduce oxidative DNA damage in vitro.

Conclusion

Elevated SA-β-gal, γ-H2A.X, and p21 suggest that in vitro stress can induce a senescence-like phenotype. Reduced oxygen during embryo culture minimizes these effects, providing further evidence for potential adverse effects of culturing embryos at ambient oxygen concentrations.     

A quantitative approach to blastocyst quality evaluation: morphometric analysis and related IVF outcomes

Purpose

To quantify blastocyst morphologic parameters with a feasible and standardized tool, investigating their predictive value on implantation outcome.

Method

The study retrospectively analyzes 124 blastocysts from 75 patients. Quantitative measurements of blastocyst expansion, inner cell mass and trophoectoderm were taken using digital image analysis software.

Result(s)

Blastocysts areas were found to be ranging from 11626.2 up to 35076.4 μm². The area of an early blastocyst is A ≤ 18500 μm² with a mean diameter d = 140 ± 9 μm, and the area of an expanded blastocyst is A ≥ 24000 with d = 190 ± 9 μm. While blastocyst mean area was not related to implantation rate, more expanded blastocysts displayed a significantly higher implantation rate. Trophoectoderm cell number is a predictor of positive outcome: since a higher of cells (25.6 ± 11.3 vs 16.3 ± 12.8) `forming a tightly knit epithelium is prognostic of implantation potential. Conversely, inner cell mass size is significantly related to implantation only in expanded blastocysts (3122.7 ± 739.0 vs. 2978.1 ± 366.0 μm²).

Conclusion(s)

Evaluation of blastocyst morphology with a digital image system could be a valuable tool to standardize blastocyst grading based on quantitative parameters. Therefore, digital analysis may be helpful in identifying the best blastocyst to transfer.     

Effect of micro-vibration culture system on embryo development

Purpose

Micro-vibration culture system was examined to determine the effects on mouse and human embryo development and possible improvement of clinical outcomes in poor responders.

Materials and methods

The embryonic development rates and cell numbers of blastocysts were compared between a static culture group (n = 178) and a micro-vibration culture group (n = 181) in mice. The embryonic development rates and clinical results were compared between a static culture group (n = 159 cycles) and a micro-vibration culture group (n = 166 cycles) in poor responders. A micro-vibrator was set at a frequency of 42 Hz, 5 s/60 min duration for mouse and human embryo development.

Results

The embryonic development rate was significantly improved in the micro-vibration culture group in mice (p < 0.05). The cell numbers of mouse blastocysts were significantly higher in the micro-vibration group than in the static culture group (p < 0.05). In the poor responders, the rate of high grade embryos was not significantly improved in the micro-vibration culture group on day 3. However, the optimal embryonic development rate on day 5 was improved in the micro-vibration group, and the total pregnancy rate and implantation rate were significantly higher in the micro-vibration group than in the static culture group (p < 0.05).

Conclusions

Micro-vibration culture methods have a beneficial effect on embryonic development in mouse embryos. In poor responders, the embryo development rate was improved to a limited extent under the micro-vibration culture conditions, but the clinical results were significantly improved.     

Increased body mass index negatively impacts blastocyst formation rate in normal responders undergoing in vitro fertilization

Purpose

The aim of this study is to investigate the effect of female BMI and metabolic dysfunction on blastocyst formation rate.

Methods

This was a retrospective cohort study that was performed in an academic center for reproductive medicine. Patients who were normal weight, overweight with metabolic dysfunction, or obese who had ≥6 oocytes retrieved in a fresh IVF cycle were included in the study. The blastocyst formation rate was calculated from the number of ≥5 cell embryos on day 3 observed in culture until day 5 or day 6. Only good quality blastocysts were included in the calculation as defined by a morphologic grade of 3BB or better.

Results

The blastocyst formation rate was significantly better in the normal-weight controls versus overweight/obese patients (57.2 versus 43.6 %, p < 0.007). There was no difference in blastocyst formation between the patients with a BMI 25–29.9 kg/m² with metabolic dysfunction and those with a BMI ≥30 kg/m².

Conclusion

The maternal metabolic environment has a significant impact on embryo quality as measured by blastocyst formation. A decreased blastocyst formation rate is likely a significant contributor to poorer reproductive outcomes in overweight and obese women with infertility.     

Dexamethasone attenuates the embryotoxic effect of endometriotic peritoneal fluid in a murine model

Purpose

The in vitro fertilization (IVF) pregnancy rate of women with advanced stage endometriosis is nearly half that of the general population, suggesting incomplete targeting of the pathophysiology underlying endometriosis-associated infertility. Compelling evidence highlights inflammation as the etiologic link between endometriosis and infertility and a potential target for adjunctive treatment. The objective of this study was to examine the effect of dexamethasone on murine embryos exposed to human endometriotic peritoneal fluid (PF) using the established murine embryo assay model.

Methods

PF was obtained from women with and without severe endometriosis. Murine embryos were harvested and randomly allocated to five groups of culture media conditions: (1) human tubal fluid (HTF), (2) HTF and 10 % PF from women without endometriosis, (3) HTF and 10 % PF from women with endometriosis (PF-E), (4) HTF with PF-E and 0.01 mcg/mL dexamethasone, and (5) HTF with PF-E and 0.1 mcg/mL dexamethasone. Embryos were cultured in standard conditions and evaluated for blastocyst development.

Results

A total of 266 mouse embryos were cultured. Baseline blastulation rates were 63.6 %. The addition of peritoneal fluid from women with endometriosis decreased the blastocyst development rate to 38.9 % (P = 0.008). The addition of 0.1 mcg/mL of dexamethasone to the culture media restored the blastulation rate to near baseline levels (61.2 %; P = 0.019).

Conclusions

The results of our in vitro study demonstrate the capacity of dexamethasone to mitigate the deleterious impact of endometriotic PF on embryo development. If confirmed in vivo, dexamethasone may prove a useful adjunct for the treatment of endometriosis-associated infertility.     

Effects of oxygen tension and IGF-I on HIF-1α protein expression in mouse blastocysts

Purpose

Hypoxia inducible factors (HIFs) are key regulators of oxygen homeostasis in response to reduced oxygenation in somatic cells. In addition, HIF-1α protein can be also induced by insulin-like growth factor I (IGF-I) treatment in various cell lines under normoxic condition. However, the expression and function of HIF-1α in embryogenesis are still unclear. Therefore, the objectives of this study were to examine the expression of HIF-1α in mouse blastocysts cultured under hypoxic and normoxic conditions, and to determine whether oxygen tension and IGF-I influence embryonic development through stimulation of HIF-1α expression.

Methods

Mouse embryos were cultured from the 1-cell to blastocyst stage under 5 % or 20 % O₂ in both the absence and presence of IGF-I.

Results

The embryonic development rates to the blastocyst stage were not affected by oxygen tension or IGF-I treatment. HIF-1α protein was localized to the cytoplasm of blastocysts, and its levels were independent of oxygen concentration or IGF-I treatment. Blastocysts cultured under 5 % O₂ exhibited significantly higher total cell numbers (83.4 ± 18.1) and lower apoptotic index (3.7 ± 1.5) than those cultured under 20 % O₂ (67.4 ± 15.6) (6.9 ± 3.5) (P < 0.05). IGF-I reduced the apoptotic index in both oxygen conditions, but a significant decrease was detected in the 20 % O₂ group.

Conclusions

HIF-1α may not be a major mediator that responds to change in oxygen tension within blastocysts, inconsistent with that of somatic cells. Supplementation of culture media with IGF-I has been shown to promote embryo development by an anti-apoptotic effect, instead of increasing HIF-1α protein expression.     

Vitrification can modify embryo cleavage stage after warming. Should we change endometrial preparation?

Purpose

Studies have shown that embryo metabolism and cell cleavage after warming vitrified embryos is faster than after thawing frozen embryos. We study vitrified embryo transfer (VET) results depending on the developmental stage of warmed embryos and the duration of progesterone treatment before embryo transfer.

Methods

We designed a prospective study, patients were randomized in two groups, starting progesterone three (D + 3) or four days (D + 4) before embryo transfer. We recruited 88 patients with embryos vitrified on day 3.

Results

We didn’t find statitistical differences in pregnancy rate when we transferred embryos in D + 3 vs D + 4 (38.2 % vs 40.5 % p ≥ 0.05). The day after warming, 54.6 % of embryos had developed to morula or early blastocyst, 32.4 % to cleavage stage and 13 % didn’t cleave. Transfers were with morula/blastocysts stage embryos (52.1 %; n:37), cleavage stage embryos (18.3 %; n:13) or mixed (29.6 %; n:21). Implantation rate was significantly higher in morula/blastocyst stage than in cleavage stage or mixed transfers (44 %, 22 % and 16.3 %; p = 0.011). Pregnancy and implantation rates were significantly higher in morula/blastocyst transfers on D + 4 than on D + 3 (68.7 % and 64.7 % vs 33.3 %, and 33.3 %, p = 0.033 and p = 0.034).

Conclusions

Our findings suggest that a majority of embryos will develop to morula/blastocyst stage after warming. VET results with morula/blastocysts, and after four days of progesterone supplementation, are better than with cleavage stage embryos.     

Uterine flushing with supernatant embryo culture medium in vitrified warmed blastocyst transfer cycles: a randomized controlled trial

Purpose

Does transfer of supernatant embryo culture fluid (stimulation of endometrial embryo transfer - SEET) prior to vitrified warmed blastocyst transfer result in better clinical pregnancy and live birth rates than direct vitrified warmed blastocyst transfer?

Methods

This randomized controlled trial compared SEET group and direct transfer group (control) in 60 women undergoing vitrified warmed blastocyst transfers. The duration of the study was 3 years. The patients were undergoing vitrified warmed blastocyst transfer at university level infertility centre. Sixty women were randomized to SEET (n = 30) or control (n = 30).

Results

Data was available for analysis from all the 30 women in the SEET group and 30 women in the control group. There were no drop outs in the trial. The implantation rate was significantly lower in the SEET group compared to the control group (27 vs. 44 %, P = 0.018). The clinical pregnancy rates were similar in both the groups (47 vs. 53 %) but the live birth rate was also significantly lower in SEET group (23 vs. 50 %, P = 0.03).

Limitations

The sample size based on clinical pregnancy rates was small and hence not adequately powered to detect differences in live birth rates. Lack of blinding leading to possible bias cannot be ruled out.

Conclusion

There was no evidence of an improvement in clinical pregnancy rate following SEET in vitrified warmed blastocyst transfer compared to direct transfer.     

Effect of cumulus cell removal 4 h post-insemination on fertilization and embryo quality: a prospective randomized sibling-oocyte study

Purpose

The study was designed to evaluate whether cumulus cell removal 4 h post-insemination could influence fertilization and embryo quality.

Methods

The study included 61couples undergoing standard long down regulation protocol from July 2011 to May 2012. Sibling oocytes of each patient were randomly assigned to either the 4 h group or the 20 group. For the 4 h group, cumulus cells were removed 4 h after gamete coincubation; for the 20 group, cumulus cells removal was performed 20 h after insemination. Fertilization rate, embryo quality, pregnancy rate and implantation rate were assessed.

Results

A total of 801 sibling cumulus-oocyte complexes (COCs) were randomized to the 4 h group (421 COCs) or 20 h group (380 COCs). There was no difference in the two pronuclei, one pronucleus and grade 1–2 embryo rate. Three pronuclei rate was significantly higher in the 4 h group compared to the 20 h group (12.6 % vs. 8.2 %, P = 0.041). Comparison of embryo transfer cycles in which either embryos from the 4 h group or 20 h group were transferred did not reveal any statistically significant differences in pregnancy or implantation rates.

Conclusion

The results of the present study indicate that cumulus cell removal 4 h post-insemination may increase the percentage of tripronuclear zygotes. However, normal fertilization rate, embryo development, clinical pregnancy rate and implantation rates are not influenced by the timing of cumulus cell removal.     

Effects of transfer of embryos independently cultured in essential and sequential culture media on pregnancy rates in assisted reproduction cycles

Purpose

Several culture media are available to be used in ART. However it is uncertain whether embryos would preferably benefit from one type of medium or the association of different media.

Methods

We performed this study to evaluate the impact of simultaneous transfer of embryos independently cultured in two distinct culture media, on pregnancy outcome. A total of 722 couples who underwent infertility treatment were sequentially allocated into three groups: those who had half of the embryos individually cultured in MEM and the other half cultured in sequential media (MEM + Seq Group) (n = 243); those who had all embryos cultured only in sequential medium (Seq Group) (n = 239); and those who had all embryos cultured only in MEM (MEM Group) (n = 240).

Results

The pregnancy rate was higher in the MEM + Seq group (51.8 %) than the Seq group (36.7 %) (p < 0.001). However the pregnancy rate observed in the MEM group was similar to the others (44.2 %). When a logistic regression test was applied it demonstrated that the number of transferred embryos did not interfere in the pregnancy rates.

Conclusions

Our results suggests that offering different culture conditions for sibling embryos with subsequent transfer of embryos that were kept in distinct culture media, might increase pregnancy rates in assisted reproduction cycles.     

The importance of the cleavage stage morphology evaluation for blastocyst transfer in patients with good prognosis

Purpose

To evaluate: (i) the influence of morphology at cleavage stage on blastocyst formation and implantation, and (ii) whether the transfer of low-quality embryos on day-three would be a better approach than the transfer at blastocyst stage.

Methods

This study included 8,444 embryos obtained from 1,125 patients undergoing ICSI cycles between January/2011 and September/2013. The influence of the quality of the embryo on days-two and -three on blastocyst formation and implantation success was evaluated. Moreover, the implantation potential of low-quality embryos, at cleavage stage, transferred on day-three was compared with the implantation potential of low-quality embryos, at cleavage stage, transferred on day-five.

Results

Low-quality embryos on day-two had an approximate 20 % decreased chance of achieving the blastocyst stage, and blastocysts derived from low-quality embryos on day-two had a nearly 40 % decrease in the implantation chance. Low-quality embryos on day-three had a 30 % decreased chance of achieving the blastocyst stage, and blastocysts derived from low-quality embryos on day-three had an almost 40 % decreased implantation chance. The implantation rate didn’t differ when low-quality embryos on the cleavage stage were transferred on day-three or left in culture and transferred on day-five.

Conclusions

The transfer of low-quality embryos on day-three is a better approach than transfer at the blastocyst stage. In addition, the embryo morphology evaluation at the cleavage stage is still needed for the selection of the embryo with the best implantation potential in extended embryo culture programmes.     

The effect of two distinct levels of oxygen concentration on embryo development in a sibling oocyte study

Purpose

This prospective randomized study used sibling oocytes of 258 women with ≥8 oocytes to compare the effect of 5 % O₂ versus 20 % O₂ concentrations on embryo development and clinical outcome.

Methods

Oocytes of each case were divided between incubators with either 5 % or 20 % O₂ concentration. Outcome measures were fertilization, cleavage, embryo quality, blastocyst formation, and implantation, pregnancy and live birth rates.

Results

Fertilization and cleavage rates were similar in both groups. The 5 % O₂ group had significantly more blastomeres (P < 0.05) and more top-quality embryos on day 3 (P < 0.02), as well as significantly more available embryos for transfer (31.6 % vs. 23.1 % for the 20 % O₂ group; P < 0.0001). There were significantly more cycles with good embryos in the 5 % group (76/258) than in the 20 % group (38/258) (P < 0.0001). Implantation and pregnancy rates were significantly higher for 5 % O₂ embryos (P < 0.03 and P < 0.05, respectively). Live birth rates per embryo transfer were 34.2 % and 15.8 %, respectively, P < 0.05.

Conclusions

Implantation, pregnancy and live birth rates are higher, and more good quality embryos are available for transfer and freezing with reduced rather than with atmospheric oxygen concentrations during embryo incubation.     

Comparison of fertilization and embryonic development in sibling in vivo matured oocytes retrieved from different sizes follicles from in vitro maturation cycles

Purpose

To examine the fertilization and developmental potential of sibling mature oocytes collected from different follicle sizes on day of retrieval in in vitro maturation (IVM) cycles.

Methods

Two hundred thirty eight hCG-primed IVM cycles were performed in 213 patients with polycystic ovaries. If sibling mature oocytes were retrieved on day of collection, they were divided into two groups, Group 1 (n = 78): M-II oocytes obtained from follicles size 10–14 mm; Group 2 (n = 192): M-II oocytes obtained from follicles size <10 mm.

Results

Of the 238 cycles, 63 cycles had more than one M-II oocytes retrieved (total M-II oocytes = 270) both from Groups 1 and 2. There were no significant differences between the two groups for oocyte diameter (117.2 mm vs. 116.9 mm), fertilization (79.5% vs. 72.4%) and good quality embryo on day 3 (66.1% vs. 56.8%).

Conclusions

The M-II oocytes retrieved from the cohort of follicles (<10 mm diameter) can produce the same quality of embryos as that from large follicles, likely contributing to improve the clinical outcome.