I. E. Messinis

Prognostic value of follicular fluid 25-OH vitamin D and glucose levels in the IVF outcome

ObjectivesThe aim of the present study was to measure serum and follicular fluid 25-OH vitamin D and glucose levels in women who underwent IVF-ET treatment and to further investigate whether the circulating 25-OH vitamin D and glucose levels correlate with IVF success.MethodsThis prospective observational study included 101 consecutive women who underwent 101 IVF-ICSI ovarian stimulation cycles and were allocated to one of the three groups according to their follicular fluid 25-OH vitamin D concentrations. Group A (n = 31) with less than 20 ng/ml, group B (n = 49) with vitamin levels between 20.1 and 30 ng/ml and group C (n = 21) with more than 30 ng/ml vitamin concentration.ResultsFollicular fluid vitamin levels significantly correlated with the quality of embryos in total (r = -0.27, p = 0.027), while the quality of embryos of group C were of lower quality as compared to those of groups A and B (p = 0.009). Follicular fluid glucose levels were lower in women of group C as compared to the respective levels of groups A and B (p = 0.003). Clinical pregnancy rate demonstrated in 14.5% in women of group C and 32.3% and 32.7% in groups A and B, respectively (p = 0.047).ConclusionThe data suggests that excess serum and follicular fluid vitamin levels in combination with decreased follicular fluid glucose levels have a detrimental impact on the IVF outcome.

Stable expression of a neuronal dopaminergic progenitor phenotype in cell lines derived from human amniotic fluid cells

Cells from human amniotic fluid derived from the fetus are considered a source of multipotent cells. Their properties have not been fully exploited, partially because unlike other embryonic sources such as embryonic stem (ES) cells, cell lines from amniocentesis samples have not been generated. We have established and characterized the properties of eight individual cell lines. Flow cytometry using several cell surface markers showed that all cell lines generated consisted of homogeneous populations that lack HLAII antigenicity. Using a combination of immunocytochemistry, Western blotting, and RT‐PCR, we found weak expression of Oct4 and nestin and strong expression of tubulin‐βIII, MAP2, and tau. Specific markers for cholinergic, (nor)adrenergic, and GABAergic neurons or glia were weakly expressed or absent, whereas expression of factors implicated in early induction of dopaminergic neurons, TGF‐β3 and β‐catenin were present. Further analysis showed strong expression of EN‐1, c‐RET, PTX3, and NURR1 essential for induction and survival of midbrain dopaminergic neurons, TH, AADC, and VMAT2 components of dopamine synthesis and secretion, and syntaxin1A and SNAP‐25 necessary for neurotransmitter exocytosis. This phenotype was retained throughout passages and up to the current passage 36. Expression of neuronal and dopaminergic markers in individual AF cell lines was comparable to expression in neurons induced from ES cells and in IMR‐32 and SH‐SY5Y neuroblastomas. Our data show that cell lines can be derived from subcultures of amniocentesis, and are primarily composed of a population of progenitors with a phenotype similar to that of committed mesencephalic dopaminergic neurons.

Heterogeneity-based genome search meta-analysis for preeclampsia

Preeclampsia is a pregnancy-related disorder that causes maternal and fetal morbidity and mortality. Its exact inheritance pattern is still unknown, and genome searches for identifying susceptibility loci for preeclampsia have thus far produced inconclusive or inconsistent results. We performed a heterogeneity-based genome search meta-analysis (HEGESMA) that synthesized the available genome scan data on preeclampsia. HEGESMA identifies genetic regions (bins) that rank highly on average in terms of linkage statistics across genome scans (searches). The significance of each bin’s average rank and heterogeneity across scans was calculated using Monte Carlo tests. The meta-analysis involved four genome-scans on general preeclampsia and five scans on severe preeclampsia. In general preeclampsia, 13 bins had significantly high average rank (Prankxa0<xa00.05) by either unweighted or weighted analyses, while four of them (2p11.2–2q21.1, 9q21.32–9q31.2, 2p15–2p11.2, 2q32.1–2q35) were formally significant by both analyses. Heterogeneity of bin 2.8 (2q32.1–2q35) was significantly low in both unweighted and weighted analysis (PQxa0<xa00.01). In severe preeclampsia, 10 bins had significantly high average rank by either unweighted or weighted analyses and five of them (3q11.1–3q21.2, 2q37.1–2q37.3, 18p11.32–18p11.22, 2p15–2p11.2, 7q34–7q36.3) were significant by both analyses. Bin 2q37.1–2q37.3 showed marginal low heterogeneity in unweighted and weighted analysis (PQxa0=xa00.06). Results should be interpreted with caution as the p values were modest. Further investigation of these regions by genotyping with additional markers and families may help to direct the identification of candidate genes for preeclampsia.

The impact of cigarette smoking and alcohol consumption on sperm parameters and sperm DNA fragmentation (SDF) measured by Halosperm(

PurposeBoth cigarette smoking and alcohol consumption are somehow implicated in sperm function, but the impact of these two lifestyle factors on sperm parameters remains controversial. The present study is focused on the impact of cigarette smoking and alcohol consumption separately and combined on sperm parameters and sperm DNA fragmentation (SDF).MethodsThe study included 207 consecutive semen samples derived from men who were seeking semen analysis for fertility purposes in our IVF Unit.ResultsSemen volume, percent of degenerated spermatozoa and SDF were significantly correlated with the various smoking status. The percent of spermatozoa with small halos significantly correlated with the alcohol status. The smoking status of the men was correlated with the alcohol status.ConclusionsCigarette smoking and alcohol consumption separately and combined were found to have deleterious effect on sperm parameters and SDF. It is suggested that both habits may contribute to infertility problems.

Molecular and Cellular Mechanisms of Sperm-Oocyte Interactions Opinions Relative to in Vitro Fertilization (IVF)

One of the biggest prerequisites for pregnancy is the fertilization step, where a human haploid spermatozoon interacts and penetrates one haploid oocyte in order to produce the diploid zygote. Although fertilization is defined by the presence of two pronuclei and the extraction of the second polar body the process itself requires preparation of both gametes for fertilization to take place at a specific time. These preparations include a number of consecutive biochemical and molecular events with the help of specific molecules and with the consequential interaction between the two gametes. These events take place at three different levels and in a precise order, where the moving spermatozoon penetrates (a) the outer vestments of the oocyte, known as the cumulus cell layer; (b) the zona pellucida (ZP); where exocytosis of the acrosome contents take place and (c) direct interaction of the spermatozoon with the plasma membrane of the oocyte, which involves a firm adhesion of the head of the spermatozoon with the oocyte plasma membrane that culminates with the fusion of both sperm and oocyte membranes (Part I). After the above interactions, a cascade of molecular signal transductions is initiated which results in oocyte activation. Soon after the entry of the first spermatozoon into the oocyte and oocyte activation, the oocyte’s coat (the ZP) and the oocyte’s plasma membrane seem to change quickly in order to initiate a fast block to a second spermatozoon (Part II). Sometimes, two spermatozoa fuse with one oocyte, an incidence of 1%–2%, resulting in polyploid fetuses that account for up to 10%–20% of spontaneously aborted human conceptuses. The present review aims to focus on the first part of the human sperm and oocyte interactions, emphasizing the latest molecular and cellular mechanisms controlling this process.

Changes in gonadotrophin response to gonadotrophin releasing hormone in normal women following bilateral ovariectomy

Pituitary responsiveness to GnRH varies throughout the normal menstrual cycle. We have investigated whether there are differences in the ovarian mechanisms which regulate gonadotrophin secretion between the follicular and the luteal phase of the cycle.

Effects of guaiazulene on in vitro bovine embryo production and on mRNA transcripts related to embryo quality.

Reactive oxygen species (ROS) are between the major contributors for the reduced rate of in vitro bovine embryo production. It is believed that they can cause abnormal meiosis of oocytes, developmental arrest or cell death of embryos. Reports on the effectiveness of various antioxidants on embryo yield are rather conflicting mainly due to the nature and the concentration of the substances used. Here we report the effects of guaiazulene--an exogenous antioxidant, without known properties that could interfere with the biological process of IVF--on embryo development and on the quality of the produced blastocysts. Bovine cumulus oocyte complexes (COCs) were aspirated from abattoir ovaries and COCs were matured in TCM199 with FCS and EGF at 39 °C under an atmosphere of 5% CO(2) in air, with maximum humidity. After 24 h oocytes were inseminated with frozen/thawed semen and co-incubated for further 24 h. Zygotes were cultured in groups of 25 in 25 μl of SOF with 5% FCS at 39 °C under an atmosphere of 5% CO(2) , 5% O(2) in air with maximum humidity. In the first experiment the maturation medium was modified with addition of 0.1 mM of G (n = 497), or 0.01 mM of guaiazulene (n = 468), 0.05% DMSO--the guaiazulene diluent (Control(+), n = 467), and 459 oocytes were used as Control(-). In the second experiment, the culture medium was modified with the addition of 0.1 mM of guaiazulene (n = 344), 0.01 mM of guaiazulene (n = 345), 0.05% DMSO (Control(+), n = 347) and 355 were the Control(-). Blastocyst yield was recorded on days 6, 7, 8 and 9. Day 7 blastocysts from each experiment and group were snap frozen and stored for mRNA extraction. Quantification of transcripts for mRNA of genes related to metabolism (AKR1B1, PTGS2, GADPH, SLC2A5, G6PD); oxidation (GPX1); and implantation (PLAC8) was carried out by real time quantitative RT-PCR. Data for embryo development and on transcript abundance were analysed by χ(2) and anova respectively. In the first experiment no differences were found between groups in terms of cleavage rate (Control(-): 74.20%; Control(+): 74.58%; 0.01 mM: 71.61%; 0.1 mM: 71.63%) or day 9 blastocyst yield (Control(-): 28.26%; Control(+): 25.80%; 0.01 mM: 25.86%; 0.1 mM: 25.25%). In the second experiment, cleavage rate tended to be higher in 0.01 mM group than in Control(-) (77.87% vs 71.41% respectively, p = 0.07). No other differences were detected in cleavage rate (Control(+): 71.32%; 0.1mM: 72.75%) or in the overall blastocyst yield on day 9 (Control(-): 25.50%; Control(+): 26.71%; 0.01 mm: 29.58%; 0.1 mM: 25.75%). In both experiments the relative abundance of genes studied varied between groups but these differences were not statistically significant. Our results imply that oxidation has minimal effect on the in vitro embryo production. Guaiazulene, a compound possessing no biological properties other than those of a strong antioxidant, while it increased cleavage rate, it failed to improve either the blastocyst formation rate, or the quality of the produced embryos under 5% O(2) .

Effect of follicle stimulating hormone or human chorionic gonadotrophin treatment on the production of gonadotrophin surge attenuating factor (GnSAF) during the luteal phase of the human menstrual cycle

OBJECTIVEu2003Although there is much in‐vivo evidence for the existence of a gonadotrophin surge attenuating factor (GnSAF), its source and identity remain unknown. We have studied the control of GnSAF production by FSH and hCG during the luteal phase of the cycle.

Ghrelin accelerates in vitro maturation of bovine oocytes.

Ghrelin, apart from its metabolic role, is nowadays considered as a basic regulator of reproductive functions of mammals, acting at central and gonadal levels. Here, we investigated for possible direct actions of ghrelin on in vitro maturation of bovine oocytes and for its effects on blastocyst yield and quality. In experiment 1, cumulus oocyte complexes (COCs) were matured in the presence of four different concentrations of ghrelin (0, 200, 800 and 2000 pg/ml). In vitro fertilization and embryo culture were carried out in the absence of ghrelin, and blastocyst formation rates were examined on days 7, 8 and 9. In experiment 2, only the 800 pg/ml dose of ghrelin was used. Four groups of COCs were matured for 18 or 24 h (C18, Ghr18, C24 and Ghr24), and subsequently, they were examined for oocyte nuclear maturation and cumulus layer expansion; blastocysts were produced as in experiment 1. The relative mRNA abundance of various genes related to metabolism, oxidation, developmental competence and apoptosis was examined in snap-frozen cumulus cells, oocytes and day-7 blastocysts. In experiment 1, ghrelin significantly suppressed blastocyst formation rates. In experiment 2, more ghrelin-treated oocytes matured for 18 h reached MII compared with controls, while no difference was observed when maturation lasted for 24 h. At 18 and 24 h, the cumulus layer was more expanded in ghrelin-treated COCs than in the controls. The blastocyst formation rate was higher in Ghr18 (27.7 ± 2.4%) compared with Ghr24 (17.5 ± 2.4%). Differences were detected in various genes expression, indicating that in the presence of ghrelin, incubation of COCs for 24 h caused over-maturation (induced ageing) of oocytes, but formed blastocysts had a higher hatching rate compared with the controls. We infer that ghrelin exerts a specific and direct role on the oocyte, accelerating its maturational process.

Daily supplementation with ghrelin improves in vitro bovine blastocysts formation rate and alters gene expression related to embryo quality

Ghrelin is a gastric peptide having regulatory role in the reproductive system functionality, acting mainly at central level. Because the expression of ghrelin system (ghrelin and its receptor) has been detected in the bovine ovary, the objectives of the present study were to investigate whether ghrelin can affect the developmental potential of in vitro-produced embryos, and to test their quality in terms of relative abundance of various genes related to metabolism, apoptosis and oxidation. In the first experiment, in vitro-produced zygotes were cultured in the absence (control [C]) and in the presence of three concentrations of acylated ghrelin (200 pg/mL [Ghr200], 800 pg/mL [Ghr800]; and 2000 pg/mL [Ghr2000]); blastocyst formation rates were examined on Days 7, 8, and 9. In the second experiment, only the 800 pg/mL dose of ghrelin was used. Zygotes were produced as in experiment 1 and 24 hours post insemination they were divided into 4 groups; in two groups (C; without ghrelin; Ghr800 with ghrelin), embryos were cultured without medium replacement; in the remaining two groups (Control N and GhrN), the culture medium was daily renewed. A pool of Day-7 blastocysts were snap frozen for relative mRNA abundance of various genes related to metabolism, oxidation, implantation, and apoptosis. In experiment 3, embryos were produced as in experiment 2, but in the absence of serum (semi-defined culture medium). In experiment 1, no differences were detected between C, Ghr200, and Ghr2000, although fewer blastocysts were produced in group Ghr800 compared with C. In experiment 2, the lowest blastocysts yield was found in Ghr800, whereas daily renewal of ghrelin (Ghr800N) resulted to increased blastocysts formation rate, which on Day 7 was the highest among groups (P < 0.05). In experiment 3, ghrelin significantly suppressed blastocysts yield. Significant differences were detected in various relative mRNA abundance, giving an overall final notion that embryos produced in the presence of ghrelin were of better quality than controls. Our results imply a specific role of ghrelin in early embryonic development; however, the specific mode of its action needs further investigation.