Category Archives: Human Reproduction

BACKGROUND

The precise assessment of embryo viability is an extremely important factor for the optimization of IVF treatments. In order to assess embryo viability, several embryo scoring systems have been developed. However, they rely mostly on a subjective visual analysis of embryo morphological features and thus are subject to inter- and intra-observer variation. In this paper, we propose a method for image segmentation (the dividing of an image into its meaningful constituent regions) and classification of human blastocyst images with the aim of automating embryo grading.

METHODS

The delineation of the boundaries (segmentation) of the zona pellucida, trophectoderm (TE) and inner cell mass (ICM) were performed using advanced image analysis techniques (level set, phase congruency and fitting of ellipse methods). The fractal dimension and mean thickness of TE and ICM image texture descriptors (texture spectrum and grey-level run lengths) were calculated to characterize the main morphological features of the blastocyst with the aim of automatic grading using Support Vector Machine classifiers.

RESULTS

The fractal dimension calculated from the delineated TE boundary provided a good indication of cell number (presented a 0.81 Pearson correlation coefficient with the number of cells), a feature closely associated with blastocyst quality. The classifiers showed different accuracy levels for each grade. They presented accuracy ranges from 0.67 to 0.92 for the embryo development classification, 0.67–0.82 for the ICM classification and 0.53–0.92 for the TE classification. The value 0.92 was the highest accuracy achieved in the tests with 73 blastocysts.

CONCLUSIONS

Semi-automatic grading of human blastocysts by a computer is feasible and may offer a more precise comparison of embryos, reducing subjectivity and allowing embryos with apparently identical morphological scores to be distinguished.

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STUDY QUESTION

Do different human ART culture protocols prepare embryos differently for post-implantation development?

SUMMARY ANSWER

The type of ART culture protocol results in distinct cellular and molecular phenotypes in vitro at the blastocyst stage as well as subsequently during in vivo development.

WHAT IS KNOWN ALREADY

It has been reported that ART culture medium affects human development as measured by gestation rates and birthweights. However, due to individual variation across ART patients, it is not possible as yet to pinpoint a cause–effect relationship between choice of culture medium and developmental outcome.

STUDY DESIGN, SIZE, DURATION

In a prospective study, 13 human ART culture protocols were compared two at a time against in vivo and in vitro controls. Superovulated mouse oocytes were fertilized in vivo using outbred and inbred mating schemes. Zygotes were cultured in medium or in the oviduct and scored for developmental parameters 96h later. Blastocysts were either analyzed or transferred into fosters to measure implantation rates and fetal development. In total, 5735 fertilized mouse oocytes, 1732 blastocysts, 605 fetuses and 178 newborns were examined during the course of the study (December 2010–December 2011).

PARTICIPANTS/MATERIALS, SETTING, METHODS

Mice of the B6C3F1, C57Bl/6 and CD1 strains were used as oocyte donors, sperm donors and recipients for embryo transfer, respectively. In vivo fertilized B6C3F1 oocytes were allowed to cleave in 13 human ART culture protocols compared with mouse oviduct and optimized mouse medium (KSOM(aa)). Cell lineage composition of resultant blastocysts was analyzed by immunostaining and confocal microscopy (trophectoderm, Cdx2; primitive ectoderm, Nanog; primitive endoderm, Sox17), global gene expression by microarray analysis, and rates of development to midgestation and to term.

MAIN RESULTS AND THE ROLE OF CHANCE

Mouse zygotes show profound variation in blastocyst (49.9–91.9%) and fetal (15.7–62.0%) development rates across the 13 ART culture protocols tested (R²= 0.337). Two opposite protocols, human tubal fluid/multiblast (high fetal rate) and ISM1/ISM2 (low fetal rate), were analyzed in depth using outbred and inbred fertilization schemes. Resultant blastocysts show imbalances of cell lineage composition; culture medium-specific deviation of gene expression (38 genes, ≥4-fold) compared with the in vivo pattern; and produce different litter sizes (P ≤ 0.0076) after transfer into fosters. Confounding effects of subfertility, life style and genetic heterogeneity are reduced to a minimum in the mouse model compared with ART patients.

LIMITATIONS, REASONS FOR CAUTION

This is an animal model study. Mouse embryo responses to human ART media are not transferable 1-to-1 to human development due to structural and physiologic differences between oocytes of the two species.

WIDER IMPLICATIONS OF THE FINDINGS

Our data promote awareness that human ART culture media affect embryo development. Effects reported here in the mouse may apply also in human, because no ART medium presently available on the market has been optimized for human embryo development. The mouse embryo assay (MEA), which requires ART media to support at least 80% blastocyst formation, is in need of reform and should be extended to include post-implantation development.

STUDY FUNDING/COMPETING INTEREST(S)

This work was supported by the Deutsche Forschungsgemeinschaft (BO 2540/4-1 to M.B. and SCHL 394/9 to S.S.) and by the Nederlandse Organisatie voor Wetenschappelijk Onderzoek (no. 63-258 to S.L.G.). No competing interests.

TRIAL REGISTRATION NUMBER

Not applicable.

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STUDY QUESTION

Does the type of medium used to culture fresh and frozen–thawed embryos influence neonatal birthweight after single embryo transfer (SET) in IVF?

SUMMARY ANSWER

A comparison of two commercially available culture media showed no significant influence on mean birthweight and mean birthweight adjusted for gestational age, gender and parity (z-scores) of singletons born after a fresh or frozen–thawed SET. Furthermore, we show that embryo freezing and thawing cycles may lead to a significantly higher mean birthweight.

WHAT IS KNOWN AND WHAT THIS PAPER ADDS

Animal studies have shown that culture media constituents are responsible for changes in birthweight of offspring. In human IVF, there is still little knowledge of the effect of medium type on birthweight. Until now, only a small number of commercially available culture media have been investigated (Vitrolife, Cook^® Medical and IVF online medium). Our study adds new information: it has a larger population of singleton births compared with the previously published studies, it includes outcomes of other media types (HTF and Sage^®), not previously analysed, and it includes data on frozen–thawed SETs.

DESIGN

This study was a retrospective analysis of birthweights of singleton newborns after fresh (Day 3) or frozen–thawed (Day 5) SET cycles, using embryos cultured in either of two different types of commercially available culture media, between 2008 and 2011.

PARTICIPANTS AND SETTING

Before January 2009, a single-step culture medium was used: human tubal fluid (HTF) with 4 mg/ml human serum albumin. From January 2009 onwards, a commercially available sequential medium was introduced: Sage^®, Quinn's advantage protein plus medium. Singletons born after a fresh SET (99 embryos cultured in HTF and 259 in Sage^®) and singletons born after a frozen–thawed SET (32 embryos cultured in HTF only, 41 in HTF and Sage^® and 86 in Sage^® only) were analysed. Only patients using autologous gametes without the use of a gestational carrier were considered. Also excluded were (vanishing) twins, triplets, babies with congenital or chromosomal abnormalities and babies born before 22 weeks of gestation.

MAIN RESULTS AND THE ROLE OF CHANCE

Analysis of 358 singletons born after a fresh SET and 159 singletons born after a frozen–thawed SET showed no significant difference between the HTF and Sage^® groups in terms of birthweight. Gestational age, parity and gender of the baby were significantly related to birthweight in multiple linear regression analyses, and other possible confounding factors included maternal age, BMI and smoking, the number of blastomeres in the transferred embryo and the type of culture medium. Maternal age, BMI and smoking, gestational age at birth, gender of the baby and the percentage of firstborns did not differ significantly between the HTF and Sage^® groups; however, among the fresh embryos, those cultured in Sage^® had significantly more blastomeres at the time of embryo transfer compared with the embryos cultured in HTF. Birthweights adjusted for gestational age and gender or gestational age and parity (z-scores) were not significantly different between the HTF and Sage^® groups for fresh or frozen–thawed SETs. Mean birthweight, as well as the mean birthweight among firstborns and the mean birthweights adjusted for gestational age and gender or parity (z-scores) were significantly higher in the cryopreservation group compared with the fresh embryo transfer group.

BIAS, CONFOUNDING AND OTHER REASONS FOR CAUTION

Our study is limited by its retrospective design and only two commercially available types of culture media were tested. More research is necessary to investigate the potential influence of culture media on gene expression.

GENERALIZABILITY TO OTHER POPULATIONS

Although our data do not indicate the major influences of the HTF and Sage^® culture media on birthweight, our results cannot be extrapolated to other culture media types. Furthermore, there remains a potential influence of embryo culture environment on epigenetic variation not represented by birthweight differences but by more subtle features.

STUDY FUNDING/COMPETING INTEREST(S)

No external funding was obtained for this study.

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BACKGROUND

More than half of recurrent pregnancy loss (RPL) remains unexplained. We hypothesized that women with a history of unexplained RPL (URPL) have low venous reserve.

METHODS

Case–control study in 12 women with a history of URPL, 11 healthy nulliparous controls and 12 primiparous controls with a history of uncomplicated pregnancy. To quantify venous reserve, we measured plasma volume (PV, ml/m²) and venous compliance in forearm and calf (VC_arm, VC_calf, (ml/dl)/mmHg) during the follicular phase of the menstrual cycle. Mean arterial blood pressure (mmHg) was measured by oscillometry. Arterial demand was evaluated by cardiac index (CI, (l/min)/m²).

RESULTS

Baseline characteristics were comparable between groups. All groups had similar CI. Women with a history of RPL had 14% and 9% lower mean PV compared with nulliparous and primiparous controls (P < 0.01 and P = 0.04, respectively). In women with URPL, the mean VC_arm was 25% and 32% lower compared with nulliparous and primiparous controls (P = 0.04 and P < 0.01, respectively), while the mean VC_calf was 29 and 22% lower compared with the two control groups (P < 0.01 and P = 0.03, respectively).

CONCLUSIONS

Women with URPL have lower venous reserves when compared with controls at comparable arterial demand. Interventions that increase venous reserve may improve pregnancy outcome.

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BACKGROUND

Fetal cells (microchimerism) are acquired by women during pregnancy. Fetal microchimerism persists decades later and includes cells with pluripotent capacity. Persistent microchimerism has the capacity for both beneficial and detrimental maternal health consequences. Both miscarriage and termination of pregnancy can result in fetal microchimerism. We sought to determine whether cellular fetal microchimerism is acquired during management of pregnancy loss and further explored factors that could influence fetal cell transfer, including viability of fetal tissue, surgical versus medical management and gestational age.

METHODS

Pregnant women (n= 150 samples from 75 women) with singleton pregnancies undergoing a TOP (n= 63) or treatment for embryonic or fetal demise (miscarriage, n= 12) were enrolled. Mononuclear cells were isolated from blood samples drawn before, and 30 min after, treatment. Fetal cellular microchimerism concentrations were determined using quantitative PCR for a Y chromosome-specific sequence, expressed as genome equivalents of fetal DNA per 100 000 maternal cell equivalents (gEq/10⁵). Detection rate ratios were determined according to clinical characteristics.

RESULTS

Cellular fetal microchimerism was found more often in post- compared with pretreatment samples, 24 versus 5% (P= 0.004) and at higher concentrations, 0–36 versus 0–0.7 gEq/10⁵ (P< 0.001). Likelihood of microchimerism was higher in surgical than medical management, detection rate ratio 24.7 (P= 0.02). The detection rate ratio for TOP versus miscarriage was 16.7 for known male fetuses (P= 0.02). Microchimerism did not vary with gestational age.

CONCLUSIONS

Significant fetal cell transfer occurs during miscarriage and TOP. Exploratory analyses support relationships between obstetric clinical factors and acquisition of fetal cellular microchimerism; however, our limited sample size precludes definitive analysis of these relationships, and confirmation is needed. In addition, the long-term persistence and potential consequences of fetal microchimerism on maternal health merit further investigation.

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BACKGROUND

Dendritic cells (DCs), which are biased toward a tolerogenic profile, play a pivotal role in tissue-remodeling processes and angiogenesis at the maternal–fetal interface. Here, we analyzed the effect of trophoblast cells on the functional profile of DCs to gain insight on the tolerogenic mechanisms underlying the human placental–maternal dialog at early stages of gestation.

METHODS

DCs were differentiated from peripheral blood monocytes obtained from fertile women (n = 21), in the presence of interleukin (IL)-4 and granulocyte-macrophage colony-stimulating factor during 5 days in culture. Then, DCs were cultured with trophoblast cells (Swan-71 cell line obtained from normal cytotrophoblast, at 7 weeks) for 24 h and for an additional 24 h in the absence or presence of lipopolysaccharide (LPS) from Escherichia coli. DCs were recovered and used for flow cytometry, enzyme-linked immunosorbent assay, RT–PCR and suppression and migration assays.

RESULTS

Trophoblast cells significantly prevented the increase in CD83 expression induced by LPS without affecting the expression of CD86, CD40 and human leukocyte antigen-DR (P < 0.05). Trophoblast cells signifinatly decreased the production of IL-12p70 and tumor necrosis factor-α, while it increased the production of IL-10 (P < 0.05). No changes were observed in the production of IL-6 and monocyte chemotactic protein-1. The culture of DCs with trophoblast cells, also suppressed the stimulation of the allogeneic response triggered by LPS (P < 0.05). Conditioned DCs were able to increase the frequency of CD4 + CD25 + Foxp3 cells and this effect was accompanied by an increase in indoleamine 2, 3-dioxygenase expression in DCs (P < 0.05).

CONCLUSIONS

The interaction of DCs with trophoblast cells promotes the differentiation of DCs into cells with a predominantly tolerogenic profile that could contribute to a tolerogenic microenvironment at the maternal–fetal interface.

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