Søren Ziebe

A randomized clinical trial to evaluate the effect of granulocyte-macrophage colony-stimulating factor (GM-CSF) in embryo culture medium for in vitro fertilization

OBJECTIVE To evaluate the effect of granulocyte-macrophage colony-stimulating factor (GM-CSF) in embryo culture medium on ongoing implantation rate (OIR). DESIGN Multicenter, randomized, placebo-controlled, double-blinded prospective design. SETTING Fourteen Scandinavian fertility clinics. PATIENT(S) A total of 1,332 women with indication for in vitro fertilization or intracytoplasmic sperm injection; 1,149 received embryo transfer (GM-CSF: n = 564; control: n = 585). INTERVENTION(S) Oocytes were fertilized, and embryos cultured and transferred in control medium or test medium containing 2 ng/mL GM-CSF. MAIN OUTCOME MEASURE(S) OIR at gestational week 7, with follow-up at week 12 and birth. RESULT(S) At week 7, OIRs were 23.5% (GM-CSF), and 20.0% (control) (odds ratio [OR] 1.26, 95% confidence interval [CI] 0.91-1.75). At week 12, OIRs were 23.0% (GM-CSF) and 18.7% (control) (OR 1.35, 95% CI 1.06-1.72), and live birth rates were 28.9% and 24.1%, respectively (OR 1.35, 95% CI 1.03-1.78). The effect of GM-CSF was influenced by the human serum albumin concentration in the medium. Birth weight and abnormality incidence were similar in both groups. Exploratory analyses showed that GM-CSF increased OIR in women with previous miscarriage, especially in women with more than one miscarriage. CONCLUSION(S) Addition of GM-CSF to embryo culture medium elicits a significant increase in survival of transferred embryos to week 12 and live birth. Our results are consistent with a protective effect of GM-CSF on culture-induced embryo stress. GM-CSF may be particularly efficacious in women with previous miscarriage. CLINICAL TRIAL REGISTRATION NUMBER NCT00565747.

Association between blastocyst morphology and outcome of single-blastocyst transfer

The aim of this study was to assess the ability of three individual blastocyst morphology parameters - expansion and hatching (EH) stage, inner cell mass (ICM) grade and trophectoderm grade - to predict outcome of a cycle with single-blastocyst transfer. The study was a secondary analysis of data prospectively collected in a large multicentre trial. A total of 618 intracytoplasmic sperm injection patients undergoing ovarian stimulation in a gonadotrophin-releasing hormone antagonist cycle with compulsory single-blastocyst transfer on day 5 were included. In the simple logistic regression analysis, all three blastocyst morphology parameters were statistically significantly (P<0.005 for each) associated with positive human chorionic gonadotrophin, clinical and ongoing pregnancy rates and live birth rates, while only the ICM grade was significantly (P=0.033) associated with early pregnancy loss rate. Blastocyst EH stage was the only significant predictor of live birth (P=0.002) in the multiple logistic regression. In conclusion, although all three blastocyst morphology parameters were related to treatment outcome of fresh single-blastocyst cycles, selection of high-quality blastocysts for transfer should consider first the EH stage. Transfer of a blastocyst with ICM grade A may reduce the risk of early pregnancy loss. Choosing the embryo(s) with the best implantation potential is essential for securing each couple the highest chance of achieving pregnancy after assisted reproduction. The selection of embryo(s) for transfer at the blastocyst stage is based on morphology parameters of expansion and hatching stage, inner cell mass grade and trophectoderm grade. The aim of this study was to assess the relative impact of each parameter in predicting the probability of a successful outcome. The study was a secondary analysis of data prospectively collected in a large multicentre trial. A total of 618 patients who underwent single-blastocyst transfer on day 5 were included. Statistical analysis showed that all three blastocyst morphology parameters were significantly associated with positive human chorionic gonadotrophin (βHCG), clinical and ongoing pregnancy rates and live birth rates. Only the inner cell mass grade was significantly associated with early pregnancy loss between the positive βHCG test and confirmation of ongoing pregnancy 10-11weeks after transfer. The expansion and hatching stage was the only significant predictor of live birth in the multiple logistic regression analysis. In conclusion, although all three blastocyst morphology parameters were related to treatment outcome of fresh single-blastocyst cycles, selection of high-quality blastocysts for transfer should consider first the expansion and hatching stage. Transfer of a blastocyst with inner cell mass grade A may reduce the risk of early pregnancy loss.

Progesterone supplementation during early gestation after in vitro fertilization has no effect on the delivery rate

OBJECTIVE To compare the delivery rate with IVF or ICSI in women who did and did not receive progesterone supplementation in the first 3 weeks after a positive hCG test result. DESIGN Retrospective study. SETTING Fertility Clinic, Rigshospitalet University Hospital, Copenhagen, Denmark. PATIENT(S) 200 pregnant women who did not receive progesterone (intervention group) and 200 pregnant women who received progesterone for 3 weeks after a positive hCG result. INTERVENTION(S) In the study group, vaginal progesterone therapy was withdrawn on the day of positive hCG result. In the control group, treatment with progesterone, 600 mg/d, was continued for 3 weeks after a positive hCG result. Both groups received 600 mg of progesterone starting on the day of embryo replacement until testing positive for pregnancy 14 days after embryo transfer. MAIN OUTCOME MEASURES Delivery rate. RESULT(S) The number of deliveries was 126 in the study group and 128 in the control group. CONCLUSION(S) The delivery rate was the same in pregnant women who received and those who did not receive progesterone for 3 weeks after a positive hCG result. Progesterone supplementation for more than 2 weeks after embryo transfer may therefore yield no benefit in terms of pregnancy.

Impact of recombinant FSH dose adjustments on ovarian response in the second treatment cycle with IVF or ICSI in “standard” patients treated with 150 IU/day during the first cycle

Background.  A dose of 150 IU/day of recombinant follicle stimulating hormone (rFSH) is commonly used as a “standard” dose for “standard” patients in the first in vitro fertilization (IVF) treatment cycle. In the second cycle, the starting dose is adjusted in those patients who had an inappropriate response during the first cycle. The purpose of the study was to assess the impact of dose adjustments on ovarian response.

Total Cytoplasmic Volume as Biomarker of Fragmentation in Human Embryos

AbstractPurpose: To use computer-controlled, multilevel embryo morphology analysis to quantify the reduction in cytoplasmic volume from the zygote stage to the combined volume of the individual blastomeres as an expression for the degree of fragmentation. Methods: Zygotes and their corresponding embryos from patients referred to ICSI treatment were analyzed. Sequences of digital images were taken focussing at 5-μm intervals through the zygotes and embryos. Assessment of cytoplasmic volumes and fragmentation were based on these sequences. Results: The mean cytoplasmic reduction of the embryos were significant linear increasing with increasing degree of fragmentation assessed by traditional evaluation (P<0.001). Embryos scored to be highly fragmented had on average a cytoplasmic reduction of 63% larger than embryos assessed to have no fragmentation. In total, 68% of the embryos had a cytoplasmic reduction lying within their allocated fragmentation group. Conclusion: Assessment of embryonic fragmentation based on computer-controlled, multilevel embryo analysis of the blastomere volumes expressed in relation to the volume of the preceding zygote may allow a more precise and standardized evaluation of fragmentation than the traditional fragmentation assessment.

Syncytin-1 and its receptor is present in human gametes

Main purpose and research questionTo determine whether the true fusogen Syncytin-1 and its receptor (ASCT-2) is present in human gametes using qRT-PCR, immunoblotting and immunofluorescence.MethodsDonated oocytes and spermatozoa, originating from a fertility center in tertiary referral university hospital, underwent qRT-PCR, immunoblotting and immunofluorescence analyzes.ResultsQuantitative RT-PCR of sperm samples from sperm donors showed that syncytin-1 is present in all samples, however, protein levels varied between donors. Syncytin-1 immunoreactivity predominates in the sperm head and around the equatorial segment. The receptor ASCT-2 is expressed in the acrosomal region and in the sperm tail. Moreover, ASCT-2, but not syncytin-1, is expressed in oocytes and the mRNA level increases with increasing maturity of the oocytes.ConclusionsSyncytin and its receptor are present in human gametes and localization and temporal appearance is consistent with a possible role in fusion between oocyte and sperm.

Birthweight distribution in ART singletons resulting from embryo culture in two different culture media compared with the national population

STUDY QUESTION Is there a difference in birthweight distribution in ART singletons born after IVF culture in two different culture media? SUMMARY ANSWER There is no effect of culture media on both crude and adjusted birthweight distributions in ART singletons from nulliparous mothers. WHAT IS KNOWN ALREADY Studies on human ART singletons have reported a difference in birthweight in singletons following IVF culture in different culture media. However, other studies comparing different culture media have not shown any significant differences in birthweight. STUDY DESIGN, SIZE, DURATION This study was a retrospective comparison of birthweights in IVF/ICSI singletons conceived after fresh embryo transfer following embryo culture in Cook or Medicult medium and in a national cohort of naturally conceived singletons in nulliparous women. The study compares four independent groups consisting of singletons in nulliparous women from Cook-d2: 2-day culture in Cook medium at Rigshospitalet (n = 974), Medicult-d2: 2-day culture in Medicult EmbryoAssist medium at Rigshospitalet (n = 147), Medicult-d3: 3-day culture in Medicult EmbryoAssist medium with and without added GM-CSF (n = 204), and DK: pregnancies from the Danish birth registry (n = 106842). PARTICIPANTS/MATERIALS, SETTING, METHODS The study compares the birthweights of singletons from nulliparous women in the four independent groups mentioned above; Cook-d2: Medicult-d2: Medicult-d3: and DK. In addition, distributions of large and small for gestational age infants were compared between the groups and a multiple linear regression analysis was used to determine which factors determined birthweight. MAIN RESULTS AND THE ROLE OF CHANCE We found no significant difference in the crude birthweight distributions between singletons born after culture in Cook-d2 or Medicult-groups. Singleton girls from the Cook-d2 group weighed 3302 ± 28 g, versus 3252 ± 76 in the Medicult-d2 group (difference 50 g; P = 0.547). Singleton boys from the Cook-d2 group weighed 3430 ± 27 g, versus 3354 ± 56 in the Medicult-d2 group (difference 76 g; P = 0.279). In the background population, mean birthweights for singleton girls and boys were 3383 ± 2.4 g and 3494 ± 2.5 g, respectively. The mean birthweights of girls, 3315 ± 61 g, and boys, 3383 ± 64 g, in the Medicult-d3 group were not significantly different from that in the Medicult-d2 group. When pooling data from all culture media groups, we found the same slightly lower mean birthweight in IVF/ICSI singletons when compared with the national birth cohort as has been previously reported (Cook-d2 + Medicult-d2 + d3 versus birth cohort; girls: P < 0.001, boys: P < 0.001). We also pooled data on boys and girls and calculated the mean birthweight for the Cook-d2, Medicult-d2 and Medicult-d3 groups and found no significant differences. LIMITATIONS, REASONS FOR CAUTION The retrospective design and the inherent risk of confounding factors is a limitation. Selection bias cannot be excluded as the embryos cultured in Cook-d2 and Medicult-d2 were from single centre studies while data in Medicult-D3 group were derived from a multicentre study. WIDER IMPLICATIONS OF THE FINDINGS This large cohort of singletons born after IVF/ICSI shows no difference in crude mean birthweight after culture in two different culture media, indicating that if such a difference exists, this must be specific for certain culture media. As expected we found a slightly lower mean birthweight in ART compared with naturally conceived singletons. This suggests that parental characteristics or IVF technique related factors other than type of culture medium may influence the birthweight in ART singletons. STUDY FUNDING/COMPETING INTERESTS No external funding was used for this study. No conflicts of interest are declared.

Requirements for Human Chorionic Gonadotropin and Recombinant Human Luteinizing Hormone for Follicular Development and Maturation

AbstractPurpose:Our purpose was to evaluate the requirements for human chorionic gonadotropin (hCG) and recombinant luteinizing hormone (rec-LH) for follicular development and maturation in mice.Methods:We carried out ovarian stimulation of immature mice. Output parameters were the preembryos created in vivo and frequency of blastocyst formation in vitro. Results:hCG at 0 to 1 IU resulted in a dose-dependent recovery of preembryos (0 to 39.7 ± 4.3; mean ± SE) per mouse. hCG at 1 and 10 hCG gave similar results, whereas higher doses significantly reduced the number of preembryos. Potential for blastocyst formation was independent of hCG dose. hCG and rec-LH together exerted a synergistic effect on the recovery of preembryos.Conclusions:Optimal follicular development required a combination of 20 IU follicle stimulating hormone and 1–10 IU hCG. The potency of hCG was higher than that of rec-LH, but a synergistic effect of rec-LH and hCG was observed. The results may be pertinent for the development of strategies for ovarian stimulation of women with low levels of endogenous LH.

Does assisted reproductive treatment increase the risk of birth defects in the offspring

In a recent paper published in the New England Journal of Medicine based on an Australian cohort of 6163 in vitro fertilization/intracytoplasmic sperm injection (IVF/ICSI) children (1407 after ICSI), Davies et al. (1) conclude that “the risk of birth defects associated with ICSI remained increased after multivariate adjustment, although the possibility of residual confounding cannot be excluded”. This has raised a lot of media attention and concerns among couples treated or pregnant after IVF treatment. Davies et al. presented an elegant study based on a cohort of assisted reproductive technologies (ART) infants born from 1986 to 2002. However, the study did not refer to a recent Swedish population-based study on 15 570 ART infants (9372 conceived after ICSI) born between 2001 and 2007 (2). In the Australian study an overall increased adjusted risk of birth defects was demonstrated for ART vs. the general population with an adjusted odds ratio (aOR 1.28; 95%CI 1.16–1.41). Among singleton newborns from fresh-embryo IVF compared with the general population, the adjusted risk was 1.06 (95%CI 0.87–1.30), thus showing no increased overall risk for birth defects after IVF. Among singleton newborns after ICSI based on fresh embryo transfer, the study demonstrated a raised adjusted risk (aOR1.55; 95%CI 1.24–1.94). On the other hand, the Swedish study demonstrated that the adjusted risk of birth defects for the ICSI vs. IVF cohort (2001–2007) had an OR of 0.90 (95%CI 0.78–1.04). Thus there did not seem to be any apparent risk difference associated with the IVF method. The aOR for malformations among IVF/ICSI infants compared with the general population was 1.15 (95%CI 1.07–1.24) and the adjusted risks decreased in the younger cohort of women undergoing ART (2001–2007) compared with the older ART cohort (children born in 1982–2000) published earlier (3). Moreover, the risk of hypospadiasis, which had previously been associated with ICSI, was no longer significantly different from the risk in the general population. A recent meta-analysis looking at ICSI vs. IVF reaches the same conclusion as the Swedish study, i.e. there is no additional risk connected to ICSI (4). Some differences between the Australian and the Swedish study may explain the different findings regarding ICSI. The follow-up period was five years for all children in the Australian study, but shorter for some of the children in the Swedish study. The Australian study included medical termination of pregnancies due to malformations after week 20, whereas the Swedish only included births after week 20. However, the distribution of birth defects in the medical terminations after IVF and ICSI treatments is not available either in the article itself or in its supplementary files. Furthermore, the Australian study also included those cases of cerebral palsy (CP) that were not acquired after the perinatal period. As CP is closely related to preterm birth, one can question whether it is appropriate to define cerebral palsy as a birth defect. It would have been intriguing to see the distribution of CP between IVF and ICSI children and the total risk of birth defects excluding CP in the Australian study. The largest article so far on malformations among ART children and the differences between the studies ought to have been discussed by Davies et al. The reassuring finding that the younger or more recent ART populations may have a lower risk of malformations should have been mentioned. This finding may be due to changes in the subfertile parent populations; the IVF parent populations are healthier in a reproductive sense than a decade ago, i.e. ICSI is performed on broader indications than just very severe male infertility. The improved perinatal outcomes may also be due to improvements in the ART stimulation regimens and laboratory techniques. Moreover, the Australian study included a little more than half the number of pregnancies that were in the Swedish study. By enlarging the cohorts, the “effect” of a higher birth defect incidence observed after ICSI may simply vanish – the known effect related to a larger sample size which is less liable to be confounded by various methodological aspects, such as inclusion or exclusion criteria. Earlier studies have demonstrated that ICSI children carry a higher risk of chromosomal aberrations related to the severity of the male infertility (5); however, Davies et al. do not disentangle data to determine whether this was the case in the Australian figures.

Competence Classification of Cumulus and Granulosa Cell Transcriptome in Embryos Matched by Morphology and Female Age

Objective By focussing on differences in the mural granulosa cell (MGC) and cumulus cell (CC) transcriptomes from follicles resulting in competent (live birth) and non-competent (no pregnancy) oocytes the study aims on defining a competence classifier expression profile in the two cellular compartments. Design: A case-control study. Setting: University based facilities for clinical services and research. Patients: MGC and CC samples from 60 women undergoing IVF treatment following the long GnRH-agonist protocol were collected. Samples from 16 oocytes where live birth was achieved and 16 age- and embryo morphology matched incompetent oocytes were included in the study. Methods MGC and CC were isolated immediately after oocyte retrieval. From the 16 competent and non-competent follicles, mRNA was extracted and expression profile generated on the Human Gene 1.0 ST Affymetrix array. Live birth prediction analysis using machine learning algorithms (support vector machines) with performance estimation by leave-one-out cross validation and independent validation on an external data set. Results We defined a signature of 30 genes expressed in CC predictive of live birth. This live birth prediction model had an accuracy of 81%, a sensitivity of 0.83, a specificity of 0.80, a positive predictive value of 0.77, and a negative predictive value of 0.86. Receiver operating characteristic analysis found an area under the curve of 0.86, significantly greater than random chance. When applied on 3 external data sets with the end-point outcome measure of blastocyst formation, the signature resulted in 62%, 75% and 88% accuracy, respectively. The genes in the classifier are primarily connected to apoptosis and involvement in formation of extracellular matrix. We were not able to define a robust MGC classifier signature that could classify live birth with accuracy above random chance level. Conclusion We have developed a cumulus cell classifier, which showed a promising performance on external data. This suggests that the gene signature at least partly include genes that relates to competence in the developing blastocyst.