Defective sperm–zona pellucida (ZP) binding (DSZPB) is a common cause of failure of fertilization in vitro. This study was to determine if DSZPB is caused by defective pathways upstream of protein kinase A (PKA) and C (PKC), or reduced protein tyrosine phosphorylation (TP).

Infertile men with DSZPB and either normal sperm morphology (NSM) ≥ 14% (n = 15) or ≤5% (n = 15) were studied. Sperm–ZP binding test was performed by incubation of motile sperm with oocytes for 2 h with or without dibutyryl cyclic AMP (dbcAMP, PKA activator) or phorbol myristate acetate (PMA, PKC activator). TP of capacitated sperm in medium was assessed by immunofluorescence with an anti-phosphotyrosine monoclonal antibody.

For normal sperm with normal sperm–ZP binding, both PMA and dbcAMP significantly enhanced sperm–ZP binding in a dose–response manner. Only dbcAMP, but not PMA, significantly increased TP of capacitated sperm. In DSZPB men with severe teratozoospermia (NSM ≤ 5%), neither PMA nor dbcAMP enhanced sperm–ZP binding, despite dbcAMP significantly increasing the TP of capacitated sperm for all samples. In contrast, for DSZPB with NSM ≥ 14%, PMA caused significantly increased sperm binding up to normal levels (≥40 sperm bound/ZP) in five men, and dbcAMP had a similar result in two men. Again TP was significantly enhanced only by dbcAMP, but not by PMA.

There is defective signalling in pathways upstream of PKC and PKA in some men with DSZPB and normal semen analysis. Stimulation of TP by dbcAMP does not enhance sperm–ZP binding capacity in DSZPB men with low TP, regardless of sperm morphology.

Sperm chromatin status and nuclear DNA damage can be detected using well-established assays. However, most techniques are time-consuming and/or involve elaborate protocols and equipment. We have recently developed a simple and fast method to monitor sperm chromatin status in field conditions using the Diff-Quik assay which is employed in fertility clinics to assess sperm morphology with standard bright field microscopy. In the present study, we demonstrate that any Diff-Quik-like stain can easily, reproducibly and routinely monitor human sperm chromatin status as well.

Different Diff-Quik-like stains were used to assess sperm morphology and the presence of abnormal dark nuclear staining in human sperm from four ART centres. The TUNEL assay was performed in the same samples, and fertility outcomes were assessed.

A significant correlation was found between TUNEL-positive sperm and dark sperm nuclei. Moreover, associations were also found between the percentage of dark sperm nuclei and seminal parameters, embryo development rate, embryo quality and clinical pregnancy, as well as with cryptorchidism, and there was a tendency towards an association with age. A value of 32% abnormal staining is suggested as a predictive threshold for embryo development and pregnancy.

Our results show that any Diff-Quik-like stain, already implemented in most laboratories to assess sperm morphology, can be adapted as an indicator for chromatin status in human sperm.

Sperm banking is a suitable procedure to prevent infertility after cancer therapy in male adolescents. We evaluated the feasibility of semen preservation in 156 adolescents aged between 13 and 20 years and then we assessed fertility outcome after treatment.

Age, urogenital history, indications for cryopreservation, histological diagnosis and semen parameters were recorded. Fertility status after treatment was assessed by a questionnaire addressed to those patients who had utilized sperm storage. Post-treatment semen analysis was performed for 22 patients.

Cryopreservation was possible in 88.5% of cases. Azoospermia was detected in 2.6% of the patients at the time of diagnosis. Malignant disease accounted for 84% of our male adolescents. In this type of disease, semen parameters were significantly altered only among patients with metastatic malignant bone tumour. After treatment, nine patients presented azoospermia, five patients achieved pregnancy spontaneously, two achieved it after assisted reproductive technique using fresh ejaculated spermatozoa and one following sperm donation. Three failed with cryopreserved sperm.

Semen cryopreservation is possible for most adolescents and, regardless of disease type, may be a means of preserving fertility prior to gonadotoxic treatment that might impair the spermatogenesis process.

Increased numbers of phenotypically unusual CD56^bright CD16– uterine natural killer (uNK) cells have been associated with recurrent reproductive failure. uNK cells produce angiogenic growth factors and are potential regulators of decidual angiogenesis in early pregnancy. The final common mechanism for early pregnancy loss is thought to be early onset of the maternal circulation and excessive placental oxidative stress. We tested the hypothesis that increased uNK cells in preimplantation endometrium are associated with altered angiogenesis.

Women with recurrent reproductive failure (n = 122) were investigated with uterine artery Doppler and endometrial biopsy. Immunohistochemistry was used to identify uNK, endothelial and vascular smooth muscle cells and image analysis was used to assess location, density and differentiation.

uNK cell density was positively correlated with the formation of blood (P = 0.005, r = 0.5) and lymphatic vessels (P = 0.0001, r = 0.6), spiral arteriole smooth muscle differentiation (P = 0.01, r = 0.5) and endometrial oedema (P = 0.004). The functional effect of this was a reduced uterine artery resistance to blood flow.

These data suggest that uNK cells may regulate angiogenesis in non-pregnant endometrium. The mechanisms of reproductive failure associated with increased uNK cell density appear to be increased angiogenesis and peri-implantation blood flow, which may lead to early maternal circulation and hence pregnancy failure due to excessive oxidative stress.

Previous studies have found that 1 in 10 in vitro fertilization (IVF) singletons originates from a twin gestation. First trimester Down's syndrome screening markers are altered in assisted reproductive techniques (ART) pregnancies compared with spontaneously conceived pregnancies. The presence of a perished embryo may further complicate prenatal screening among women pregnant after ART. The aim of this study was to assess the impact of a ‘vanishing twin’ on first trimester combined biochemical and ultrasound screening in pregnancies conceived after IVF and intracytoplasmatic sperm injection.

From a national prospective cohort study concerning first trimester combined screening among women pregnant after ART, 56 cases of pregnancies with a vanishing twin were identified. As control group 897 cases of ART singleton pregnancies were used. All women completed a first trimester combined ultrasound and biochemical screening programme comprising serum PAPP-A and free β-hCG together with nuchal translucency (NT) measurement.

There were no significant differences in geometric mean MoM free β-hCG and PAPP-A between pregnancies with an early (gestational week <9, EVT) or late vanishing twin (gestational week 9–13, LVT) or singleton pregnancies (0.98, 1.13 and 0.95 for free β-hCG and 0.84, 0.80 and 0.74 for PAPP-A, respectively). Likewise, no difference was seen for NT measurements. The gestational age at the time of blood sampling and NT scan was similar for the three groups. The proportion of EVT pregnancies with a PAPP-A and free β-hCG log₁₀MoM value below the 5th%iles and above the 95th%iles of the value in the singleton pregnancies were 4.3%, 4.3%, 6.4% and 8.5%, respectively, which did not constitute a significant difference from singletons. The corresponding values for LVT pregnancies were 0%, 22.2%, 0% and 11.1%, respectively; however, these numbers were too small to allow for statistical calculations.

First trimester biochemical screening markers in women pregnant after ART, and with a vanished twin diagnosed at early ultrasound, do not differ from those of other ART singleton pregnancies. In cases where the fetal demise was first diagnosed at the time of the NT scan, it is doubtful whether the serum risk assessment is as precise as it is in singleton ART pregnancies. No difference was seen for NT measurements.

During human preimplantation development, early blastomeres are believed to be totipotent. It is likely, however, that blastomeres are allocated to a specific lineage prior to any morphological differentiation. NANOG, SOX2 and SALL4 are transcription factors that play a key role in controlling stemness in embryonic stem cells (ESC) and are therefore candidate markers for developmental triggers in early embryos. KRT18, a trophoblast-determining gene, may mark early differentiation. Examining the expression pattern of these genes may inform us about when and in which cells totipotency is lost during early human development.

Thirtheen oocytes, 124 preimplantation embryos and 7 human embryonic stem cell (hESC) lines were examined for the presence of NANOG, SOX2, SALL4 or KRT18 proteins using immunostaining and confocal microscopy.

All stemness markers were expressed in the hESC, but none of them was specific for totipotent cells during human preimplantation development, and none of them seemed to mark cells allocated to the inner cell mass (ICM) or trophectoderm. After lineage specification, only the nuclear expression of NANOG and SOX2 became restricted to the ICM, at least to some cells because only a subpopulation expressed NANOG. KRT18 expression was seen for the first time during compaction in some outer cells. KRT18 was not expressed in hESC.

We conclude that the protein expression patterns of markers that define stemness in ESC do not identify the totipotent cells in human preimplantation embryos. Assessing the presence of KRT18 proteins implied that the outer cells of compacting embryos have probably lost their totipotent competence prior to any visible differentiation.

Human embryos grow naturally in vivo in lower oxygen (O₂) tension environments than atmospheric O₂ tension. Therefore, human embryonic stem cells (hESC), a derivative of embryos, will likely grow more favorably in a reduced O₂ tension. This study aimed to compare the behavior of hESC under reduced O₂ tension (5%) versus normoxia (21%).

hESC lines were cultured in different O₂ tensions and then examined for morphology, apoptosis and gene expression profiles.

hESC grown in 5% O₂ tension were not morphologically different from hESC grown in normoxia on day 7 of the first and fourth passages. However, after prolonged culture without splitting (10–14 days), hESC colonies were thinner and looked better morphologically in 5% O₂, but the cells proliferated more slowly and their sizes were larger. At most time points, the gene expression profiles in both O₂ tensions showed no major difference in representative stemness genes (Oct-3/4, Nanog and Cripto), differentiation genes (Desmin, Nestin, -fetoprotein and GDF-9) and hypoxia-related genes (HIF-1 and VEGF). A lower level of cyclin-D1 mRNA (suggestive of less Wnt pathway signaling on day 7 of the fourth passage) and a higher level of Desmin (suggestive of more differentiation to mesoderm, at day 7 of the first passage) were detected in 5% O₂.

This study suggests that for routine culture of hESC with a short splitting interval (7 days), a low O₂ tension (5% O₂) probably does not provide significant advantages over the standard 21% O₂ tension for the maintenance of an undifferentiated state by the criteria used in this study.

Embryos with greater viability have a lower or ‘quieter’ amino acid metabolism than those which arrest. We have hypothesized this is due to non-viable embryos possessing greater cellular/molecular damage and consuming more nutrients, such as amino acids for repair processes. We have tested this proposition by measuring physical damage to DNA in bovine, porcine and human embryos at the blastocyst stage and relating the data to amino acid profiles during embryo development.

Amino acid profiles of in vitro-derived porcine and bovine blastocysts were measured by high-performance liquid chromatography and the data related retrospectively to DNA damage in each individual blastomere using a modified alkaline comet assay. Amino acid profiles of spare human embryos on Day 2–3 were related to DNA damage at the blastocyst stage.

A positive correlation between amino acid turnover and DNA damage was apparent when each embryo was examined individually; a relationship exhibited by all three species. There was no relationship between DNA damage and embryo grade.

Amino acid profiling of single embryos can provide a non-invasive marker of DNA damage at the blastocyst stage. The data are consistent with the quiet embryo hypothesis with viable embryos (lowest DNA damage) having the lowest amino acid turnover. Moreover, these data support the notion that metabolic profiling, in terms of amino acids, might be used to select single embryos for transfer in clinical IVF.

Ovarian tissue cryopreservation is a promising technique to safeguard fertility in cancer patients. However, in some types of cancer, there is a risk of transmitting malignant cells present in the cryopreserved tissue. To avoid such a risk, pre-antral follicles could be isolated from ovarian tissue and grown in vitro. On the basis of this assumption, the aim of our study was to investigate in vitro survival and growth of pre-antral follicles after cryopreservation of ovarian tissue and follicular isolation, followed by encapsulation in alginate beads.

Ovarian biopsies from four patients were frozen and thawed. Pre-antral follicles were then isolated and embedded in an alginate matrix before in vitro culture for 7 days.

Small pre-antral follicles (42.98 ± 9.06 µm) from frozen–thawed tissue can survive and develop after enzymatic isolation and in vitro culture. A total of 159 follicles were incubated in a three-dimensional system (alginate hydrogel) and, after 7 days, all of them showed an increase in size (final size 56.73 ± 13.10 µm). The survival rate of the follicles was 90% (oocyte and all granulosa cells viable).

Our preliminary results indicate that alginate hydrogels may be a suitable system for in vitro culture of isolated human pre-antral follicles. However, more studies are required to establish whether follicular morphology and functionality can be maintained using this matrix.

In studies of twin and triplet pregnancies, molecular genetic techniques have rarely been used to confirm zygosity yet this is the most accurate approach. The aim of this study was to present the cross-distribution of chorionicity and zygosity in triplet pregnancies as a function of their mode of conception using such techniques. Rates of monozygosity were studied simultaneously.

Forty-nine consecutive sets of triplets were observed in the study, including 18 sets of spontaneously conceived (SC) triplet pregnancies and 31 sets resulting from assisted reproduction technologies (ARTs). Zygosity was determined through PCR-amplified microsatellite analysis. Chorionicity was determined by placental analysis in our department of fetopathology. Sets of triplets were considered as twin pairs in order to determine the rate of monozygosity.

For SC triplet pregnancies, the rate of monozygotic (MZ) twin pairs was 48%; 30% of dichorionic (DC) triplet pregnancies were MZ and 70% dizygotic (DZ); 20% of trichorionic (TC) triplet pregnancies were DZ and 80% trizygotic (TZ). For triplet pregnancies conceived using ART, the rate of MZ twin pairs was 6.5%; 100% of DC triplet pregnancies were DZ; 4% of TC triplet pregnancies were DZ and 96% TZ.

This study is the first report to present the cross-distribution of chorionicity and zygosity in triplet pregnancies as a function of their mode of conception. In triplet pregnancies conceived using ART, DC triplets are always DZ, and TC triplets are almost always TZ.

To identify an effective misoprostol-only regimen for the termination of second trimester pregnancy, we compared sublingual and vaginal administration of multiple doses of misoprostol in a randomized, placebo-controlled equivalence trial.

Six hundred and eighty-one healthy pregnant women requesting medical abortion at 13–20 weeks' gestation were randomly assigned within 11 gynaecological centres in seven countries into two treatment groups: 400 µg of misoprostol administered either sublingually or vaginally every 3 h up to five doses, followed by sublingual administration of 400 µg misoprostol every 3 h up to five doses if abortion had not occurred at 24 h after the start of treatment. We chose 10% as the margin of equivalence. The primary end-point was the efficacy of the treatments to terminate pregnancy in 24 h. Successful abortion within 48 h was also considered as an outcome along with the induction-to-abortion-interval, side effects and women's perceptions on these treatments.

At 24 h, the success (complete or incomplete abortion) rate was 85.9% in the vaginal administration group and 79.8% in the sublingual group (difference: 6.1%, 95% CI: 0.5 to 11.8). Thus, equivalence could not be concluded overall; the difference, however, was driven by the nulliparous women, among whom vaginal administration was clearly superior to sublingual administration (87.3% versus 68.5%), whereas no significant difference was observed between vaginal and sublingual treatments among parous women (84.7% versus 88.5%). The rates of side effects were similar in both groups except for fever, which was more common in the vaginal group. About 70% of women in both groups preferred sublingual administration.

Equivalence between vaginal and sublingual administration could not be demonstrated overall. Vaginal administration showed a higher effectiveness than sublingual administration in terminating second trimester pregnancies, but this result was mainly driven by nulliparous women. Fever was more prevalent with vaginal administration. Registered with International Standard Randomized Controlled Trial number ISRCTN72965671.

The levonorgestrel-releasing intrauterine system (LNG-IUS) is an effective contraceptive and has many non-contraceptive health benefits. However, it is commonly associated with irregular endometrial bleeding. Metalloproteinases contribute to extracellular matrix (ECM) remodelling and regulate bleeding during the menstrual cycle. Enhanced metalloproteinase expression participates in the pathogenesis of breakthrough bleeding. Thus the objective of this study was to compare matrix metalloproteinase (MMP) expression in endometrium during luteal phase and in short-term (1 month) and long-term (≥6 months) LNG-IUS users.

MMP expression was analysed by semi-quantitative RT-PCR and immunohistochemistry. Gelatinase activity was determined by gelatin zymography.

MMP-1, -2, -3, -7, -9 and -12 mRNAs levels were increased, whereas that of MMP-26 was decreased in the endometrium of LNG-IUS users. MMP-1, -2, -3, -7 and -9 were localized by immunohistochemistry in all biopsies in the short-term group but in only 0–27% in the control group. The incidence of positive immunostaining for MMP-2 and -3 decreased significantly in the long-term compared with short-term LNG-IUS users. MMP-26 was localized in all biopsies from the control group but in only 14 and 25% from the short- and long-term LNG-IUS groups, respectively. In both LNG groups, the numbers of macrophages (the major source of MMP-12) was increased.

MMP-1, active MMP-2, MMP-3, MMP-7, MMP-9 and MMP-12 are more prevalent in the short-term LNG-IUS group, suggesting their important contribution to ECM breakdown and transient bleeding. The decrease in the percentage of women expressing MMP-2 and -3 might contribute to the decreased occurrence of unwanted spotting and bleeding in long-term LNG-IUS users.

Greater use of single embryo transfer (SET) to reduce twin rates associated with IVF requires good information on prognostic factors and appropriate models of treatment outcomes.

Using data from a cohort of 1198 IVF cycles, we have developed a statistical model of live birth and twin outcomes in terms of routinely measured clinical parameters. From this model, we predict potential outcomes if those who had two embryos transferred had actually received SET.

Embryo quality, age, FSH level, idiopathic diagnosis, sperm count, smoking and alcohol consumption are all significant factors predicting outcome. Couples with good embryos and good prognosis have a much greater risk of producing twins. In this cohort, to achieve a 10% twin rate would require 55% SET which, without selection of appropriate cycles, would lead to a reduction in success rate from ca. 21% to 17%. Selecting on the basis of twin risk can partially mitigate this reduction to give a success rate of 18.5%.

The use of SET to reduce twin rates will lead to a significant reduction in treatment success. Around half this reduction could be mitigated with careful selection of patients and cycles, including embryo quality.

Ovarian epithelial dysplasia was first described after prophylactic oophorectomies for genetic risk. Ovarian stimulation has been considered as a risk factor of ovarian cancer by Fathalla's incessant ovulation theory. In this study, we have investigated the risk of ovarian dysplasia after ovulation induction.

We reviewed 99 oophorectomies or cystectomies between 1990 and 2005 divided them into two groups: previous in vitro fertilization (n = 37) and a panel of fertile controls (n = 62). Eleven epithelial cytological and architectural features were defined and an ovarian epithelial dysplasia score was calculated to quantify the degree of ovarian epithelial abnormalities.

All the ovaries were macroscopically non-cancerous except in two patients (one endometrioid cancer and one borderline tumour). The mean ovarian dysplasia score was significantly higher in the ovulation induction group than in the control group (7.64 versus 3.62, P = 0.0002). We also found a relationship between the number of ovulation-inducted cycles and the severity of ovarian dysplasia (‘dose-effect’) and a relationship between time after the end of ovulation induction (over 7 years) and the severity of ovarian dysplasia (‘time-effect’).

There is probably a relationship between ovarian epithelial dysplasia and either ovulation inducing drugs or infertility. By Fathalla’s incessant ovulation theory, ‘the dose effect and the time effect’ of ovarian stimulation may explain ovarian dysplasia formation.

There has been little interest in the research literature on public opinions regarding assisted conception and surrogacy, particularly in European countries, despite the growing evidence showing that problems in adaptation and coping may be related to perceived normative values. This study investigated British women's attitudes to surrogacy using components of the theory of planned behaviour (TPB).

Questionnaires on attitudes to surrogacy and reasons for parenthood were completed by 187 women from the general public.

Significant socio-demographic differences were found between women who were possibly willing (n = 76) and those who were unwilling (n = 111) to become surrogate mothers. General attitudes to surrogacy also differed between groups (P = 0.000). This study supported the predictive utility of components of the TPB, and differentiated adequately between groups on attitudes to recruitment for surrogacy (P = 0.000), the consequences of surrogacy (P = 0.000), factors that induce people to become surrogates (P = 0.000), social support (P = 0.000), having personal control (P = 0.002) and reasons for parenthood (P = 0.000). Age (P = 0.000), attitudes to advertising (P = 0.02) and the consequences of surrogacy (P = 0.05) predicted (un)willingness to become a potential surrogate mother.

Further research is needed with larger sample sizes of potential surrogates to determine whether the predictive attitudes reported here translate to actual behaviours. The larger group which was not interested in considering becoming a surrogate scored significantly more negatively on all attitudes towards surrogacy. The negative attitudes reported by the ‘unwilling to consider being a surrogate’ group may reflect attitudes held by the majority of the population and are likely to be influenced by reports of stigma associated with surrogacy.

The impact of prenatal depression on pregnancy outcomes is largely unknown.

We conducted a population-based prospective cohort study among pregnant women of the Kaiser Permanente Medical Care Program to examine the impact of prenatal depression on the risk of preterm delivery. We interviewed pregnant women in their early pregnancy. Women's depressive symptoms were ascertained using the standard Center for Epidemiological Studies Depression Scale (CESD). The presence of significant prenatal depressive symptoms and severe depressive symptoms was determined by CESD scores ≥16 and ≥22, respectively.

Among the 791 participants who answered CESD questions and delivered a live birth, after controlling for potential confounders using the Cox proportional hazard regression, women with CESD scores ≥16 had almost twice the risk of preterm delivery compared with women without depressive symptoms: adjusted hazard ratio (aHR) = 1.9, 95% confidence interval (CI) 1.0–3.7. The risk of preterm delivery increased with increasing severity of depression: aHR = 1.6 (CI 0.7–3.6) for CESD 16–21 and aHR = 2.2 (CI 1.1–4.7) for CESD ≥22. The risk of preterm delivery associated with prenatal depression appears to be exacerbated by low educational level, a history of fertility problems and the presence of obesity and stressful events. The observed associations were not confounded by the use of antidepressants, although some of the associations did not reach statistical significance.

Our findings show that pregnant women with depressive symptoms are at increased risk of preterm delivery and, in addition, provide preliminary evidence that social and reproductive risk factors as well as obesity and stressful events may exacerbate the effect.

Human placental mesenchymal stem cells (hPMCs) are thought to be multipotent, but their fate after in utero transplantation is not known.

hPMCs isolated from term placenta were assessed for their phenotype markers, mutilineage capacity, and immunomodulatory properties. Their engraftment potential was analyzed in a pregnant rat model after in utero transplantation at embryonic day 17. Immunohistochemistry, tracing of labeled cells, fluorescence in situ hybridization and real-time PCR were used to assess post-transplant chimerism.

In vitro, lineage-negative, CD34-negative hPMCs differentiated into osteocytes, adipocytes, hepatocytes and endothelial cells with tube formation, and actively suppressed the rat lymphocyte proliferative response to allogeneic lymphocyte stimulation (P < 0.0001). After in utero transplantation into pregnant rats, a low level of engraftment was achieved in various fetal tissues. Engraftment occurred in more than 60% of the fetal rats. Cells persisted for at least 12 weeks after delivery and evidence was obtained to suggest differentiation into specific lineages, including hepatocytes and hematopoietic cells. However, a greater number of hPMCs migrated to the placenta than to the fetus, thus limiting the degree of cell engraftment in fetal organs.

We conclude that hPMCs are mutipotent cells that can be engrafted long-term in immunocompetent rats after in utero transplantation.

Successful implantation is followed by a local pro-inflammatory and Th1 response, subsequently controlled by Th2. Regulated upon activation, normal T cell expressed and secreted (RANTES) promotes a Th1 response and is implicated as a physiologic tolerogenic factor; therefore, we studied its potential role in the trophoblast–maternal leukocyte dialog.

We performed co-cultures of immortalized trophoblast cell line (Swan 71) and peripheral blood mononuclear cells (PBMCs) from fertile women (n = 23) or with recurrent spontaneous abortions (n = 18, RSA). After 24 and 48 h, supernatant and cells were analyzed by enzyme-linked immunosorbent assay, fluorescence-activated cell sorting, Western blot and apoptosis assay. To investigate the physiological effects at peripheral level, we co-cultured maternal and paternal PBMCs with conditioned media from Swan cells and progesterone.

Following interaction of maternal PBMCs and trophoblast cells, RANTES production increased (P < 0.05) and was accompanied by low levels of interferon , interleukin-12 p70 and high levels of tumor necrosis factor-, nitrites and leukemia-inhibitory factor. RANTES production resulted in elevated apoptosis of potentially deleterious maternal CD3+ lymphocytes, accompanied by a decrease in the proliferative maternal response. During fetal–maternal dialog, the anti-RANTES antibody significantly reduced the frequency of CD4+CD25+Foxp3+ cells (P < 0.05) and was associated with trophoblast cell survival. However, co-cultures of Swan cells and RSA-PBMCs displayed a differential RANTES kinetics, lower levels of regulatory T cells (Tregs) and CD3+annexin-V+cells, accompanied by higher levels of apoptotic trophoblast cells.

RANTES promotes an adequate pro-implantatory microenvironment that influences trophoblast cell survival and modulates the balance of maternal Treg/T effector lymphocytes in favor of maternal tolerance.

This study aims to investigate the role of epidermal growth factor-like ligands, amphiregulin (Ar) and epiregulin (Ep), in regulation of apoptosis in luteinized human granulosa cells.

Luteinized human granulosa cells were obtained from women undergoing IVF treatment. Ar and Ep mRNA levels were measured by real-time RT–PCR. The rate of apoptosis was measured by TUNEL. Progesterone levels were measured using radioimmunoassay. Ar- and Ep-induced activation of signaling cascades and Ar protein levels were detected by western blotting.

LH stimulation of luteinized human granulosa cells induced biosynthesis of Ar and Ep mRNA in a time-dependent manner. The blockade of MEK (by U0126) reduced the expression of LH-induced Ar and Ep biosynthesis. Incubation of the cells with Ar and Ep completely abolished the increase in apoptosis rate induced by serum starvation, and concomitantly caused a pronounced increase in progesterone production. Stimulation of the cells with Ar and Ep also activated the ERK and AKT signaling cascades. Finally, we demonstrated that the pro-survival effect of Ar and Ep is partially dependent on their ability to induce progesterone production.

Ar and Ep serve as pro-survival LH mediators in the human corpus luteum.

Decidual vascular development is important for implantation. This study analysed decidual vascular adaptation to implantation in correlation with miscarriage in decidual secretory endometrium (DSE), decidua parietalis (DP) and decidua basalis (DB) of miscarriage patients and matched controls.

Decidua was obtained during first trimester termination of pregnancy (controls) and vacuum aspiration in case of missed abortion (cases). Vascularization and the expression of VEGF-A, placental growth factor, Flt-1, KDR, angiopoietin (Ang)-1, Ang-2, TIE-2, and membrane-type matrix metalloproteinases MT1-, MT2-, MT3- and MT5-MMP were determined at mRNA and protein level. Uterine natural killer cells (CD56), macrophages (CD68), proliferation (Ki67) and apoptosis (activated caspase-3) were evaluated in consecutive sections.

Decidual vascularization showed differences between cases and controls, i.e. fewer vessels with larger circumference in cases. This correlated with the differential expressions of various factors at mRNA/antigen level and with increased endothelial flt1, KDR, MT2- and MT5-MMP expression in miscarriage patients. The differences between cases and controls were probably not based on altered proliferation and/or apoptosis, since Ki67 and active Caspase-3 showed comparable expression levels in both groups. Although DB of cases and controls showed similar amounts of CD56- and CD68-positive cells, the case group did show elevated levels of CD56 in DSE (P < 0.05) and of CD68 in DP compared with the control group (P < 0.05).

The differences in vascularization and in the expression of angiogenic factors and proteases between groups suggest a correlation between decidual vascularization and the occurrence of miscarriages.

Identification of new markers assessing endometrial receptivity may help in improving the clinical outcome of IVF. This study aimed at identifying genes expressed in human endometrium during the implantation window that could be used as such markers.

A series of normoresponder patients (n = 31) underwent endometrial biopsies (n = 62, 2 per patient) during the early secretory phase, 2 days after the LH surge (LH + 2) and the mid-secretory phase (LH + 7) of the same natural cycle that preceded a new ICSI attempt for male infertility factor. Samples were analyzed using DNA microarrays and gene expression profiles at the time of the implantation window were computed. Systems biology analysis allowed the identification of biological pathways that were over-represented in this signature. A new approach for class prediction applied to microarray experiments was then used to identify biomarkers putatively involved in endometrial receptiveness.

Five genes expressed during the implantation window were all up-regulated in the LH + 7 samples compared with LH + 2 [laminin β3 (P = 0.002), microfibril-associated protein 5 (P = 0.009), angiopoietin-like 1 (P = 0.005), endocrine gland-derived vascular endothelial growth factor (P = 0.049) and nuclear localized factor 2 (P = 0.007)]. Increased expression was validated by quantitative RT–PCR.

Five genes have been identified for the first time as being up-regulated during the implantation window and are proposed as new biomarkers for exploration of endometrial receptiveness. As the endometrial biopsy procedure can be performed during a natural cycle, it would be worth testing this approach as a novel strategy in patients with poor implantation after IVF or ICSI.

Storage of embryos for fertility preservation before chemotherapy is widely practiced. For multiple oocyte collection, the ovaries are hyperstimulated with gonadotrophins that significantly alter ovarian physiology. The effects of ovarian stimulation prior to chemotherapy on future ovarian reserve were investigated in an animal model.

Cyclophosphamide (Cy) in doses of 0, 50 or 100 mg/kg was administered to 38 adult mice (control, unstimulated). A second group of 12 mice were superovulated with equine chorionic gonadotrophin (eCG, 10 IU on Day 0) before Cy administration; hCG (10 IU) was administered (Day 2) followed by 0, 50 or 100 mg/kg Cy (Day 4). In both groups ovaries were removed, serially sectioned (7-day post-Cy), primordial follicles were counted and differences between groups evaluated.

Follicle number dropped from 469 ± 24 (mean ± SE) to 307 ± 27 and 234 ± 19 with 50 or 100 mg/kg Cy, respectively (P < 0.0001). In the eCG pretreated group, follicle count dropped from 480 ± 31 to 345 ± 16 and 211 ± 26 when 50 or 100 mg/kg Cy were administered (P < 0.0001). There were no significant differences in follicle count between the pretreated eCG group and controls for each chemotherapy dose.

This animal study indicates that ovarian stimulation before administration of Cy does not adversely affect ovarian reserve post-treatment. These results provide support for the safety of fertility preservation using ovarian stimulation and IVF–embryo cryopreservation procedures prior to chemotherapy.

Hemoglobin is a precursor of antibacterial peptides. Our aim was to identify an antibacterial peptide in human endometrium. We tested the antimicrobial activities of hemoglobin and a derived peptide in vitro and in vivo in rats.

Samples (n = 3) were scraped from the surface of endometrium. Acid-soluble proteins underwent electrophoresis followed by gel overlay assay and reversed-phase high-performance liquid chromatography. Antibacterial activities were determined by agar radial diffusion assay. Purified peptides were further characterized by electrophoresis, mass spectrometry, N-terminal amino acid (AA) sequencing and protein structure analysis. A rat model was used to test the inhibitory activity of human hemoglobin on vaginal infection with Escherichia coli, using one experimental group (intravaginal hemoglobin, n = 9) and three control groups (n = 14). Vaginal histology was studied.

The purified peptide exhibited potent antibacterial activities against E. coli ML-35P. The N-terminal AA sequence was F L S F P T T K T Y, identical to AA 32–41 of the human hemoglobin chain, and it had the same mass (m/z = 6776.8) as the chain 32–93 AA fragment, with at least three -helices. Histology indicated that the hemoglobin group changed significantly compared with the matrix control group (no treatment after infection): the surface layer of stratified squamous epithelium was smoother, inflammatory cell infiltration was relieved in the lamina propria and congestion pattern was decreased.

These results suggest that erythrocytes from endometrium are another source of the antimicrobial molecules. Hemoglobin and its derived peptides may play a role in the host defense against pathogens in human vagina.

Laparoscopic ovarian diathermy (LOD) is currently accepted as a successful second-line treatment for ovulation induction (OI) in clomiphene citrate (CC)-resistant women with polycystic ovary syndrome (PCOS). The aim of this study was to test the hypothesis that LOD may be superior to CC as a first-line treatment.

The study included 72 anovulatory women with PCOS who were randomized to LOD (n = 36) or CC (n = 36). Women who remained anovulatory after LOD were offered CC. Similarly, women receiving CC who failed to ovulate or conceive were offered LOD. Pregnancy rates were compared between the two groups using ² and odds ratio with 95% confidence interval (OR, 95% CI).

After randomization, six women conceived before starting treatment and another patient postponed treatment. The remaining 65 women received the treatment (33 underwent LOD and 32 received CC). After the primary treatment, more pregnancies (44%) occurred in women receiving CC than in those undergoing LOD (27%), although the difference did not reach statistical significance [P = 0.13, OR 2.1 (0.7 – 5.8)]. After adding the second treatment, the pregnancy rate was still higher, but to a less extent, in the CC group [63% versus 52%, P = 0.2, OR 1.6 (0.6 – 4.2)].

LOD is not superior to CC as a first-line method of OI in women with PCOS. The trial is registered with ClinicalTrials.gov with an identifier number NCT00220545.

An association between a woman’s own birthweight and her fecundity has been suggested, but no empirical data have been published on the association between maternal birthweight and waiting time to pregnancy (TTP).

In the Danish National Birth Cohort (1996–2002), which is an ongoing study of 92 274 women and their pregnancies, information about TTP and prepregnancy BMI was collected during pregnancy. At the 7-year follow-up of the children, 21 786 mothers reported their own birthweight and whether they were born at term or preterm. The association between maternal birthweight and TTP is presented as adjusted odds ratios with 95% confidence intervals.

Low maternal birthweight (≤2500 g for term and ≤1500 g for preterm birth) was associated with an increased risk of TTP of >1 year [term: 1.2 (1.0–1.5); preterm: 1.8 (1.1–3.1)]. The latter association was strongest in women with a BMI < 25 kg/m² [2.6 (1.4–4.7)]. High maternal birthweight (>4500 g for term and >3500 g for preterm) was also associated with an increased risk of TTP of >1 year [1.5 (1.0–2.0) and 1.3 (0.7–2.4), respectively], especially in women with a BMI ≥ 25 kg/m² [1.8 (1.1–3.1) and 2.5 (1.0–6.4), respectively].

High or low maternal birthweight was associated with TTP > 1 year. Longer waiting times in women with very low birthweight may reflect an effect of being born very preterm. Subfecundity may partly be programmed in foetal life by factors that cause or correlate with foetal growth.

Over the last three decades, technological developments facilitating assisted reproductive techniques (ART) have revolutionized the treatment of subfertile couples, including men suffering from severe oligospermia or azoospermia. In parallel with the advent of these technologies, there is a great concern about the biological safety of ART. This concern is supported by the clinical observation that the frequency of congenital malformations is slightly elevated among ART-conceived children.

In this explorative study, we have used tiling-resolution BAC array-mediated comparative genomic hybridization to investigate the incidence of de novo genomic copy number changes in a group of 12 ICSI children, compared with a control group of 30 naturally conceived children.

In 6 of the 12 ICSI children, we found 10 apparently de novo ‘same direction genomic copy number changes’ [i.e. simultaneous copy number gain (or loss) with respect to both biological parents], notably losses. In statistically significant contrast, similar observations were encountered only six times in the control group in 5 of the 30 children. However, our study group was small, so a larger group is needed to confirm these findings.

Loci at which we found de novo alterations are known from the human genome database to be prone to large DNA segment copy number changes. As discussed, various molecular mechanisms, including the consequences of delayed male meiotic synapsis and replication fork stalling at early embryonic cell cycles, might trigger these copy number changes.

Polycystic ovaries display an increased number of pre-antral and antral follicles compared with normal ovaries, suggesting that early and late follicle development are disturbed. The pathophysiology of this process is poorly understood. Since the transforming growth factor β family members, anti-Müllerian hormone (AMH) and bone morphogenetic proteins (BMPs), inhibit FSH sensitivity, their signalling may contribute to the aberrant follicle development in these women. Here, we investigated the role of ALK2, a type I receptor for AMH/BMP signalling, in PCOS using a genetic approach.

Seven single nucleotide polymorphisms in the ACVR1 gene, encoding ALK2, were genotyped in 359 PCOS patients and 30 normo-ovulatory and 3543 population-based control women, and haplotypes were determined. Subsequently, the association of ACVR1 variants with ovarian parameters and hormone levels was investigated.

The polymorphisms rs1220134, rs10497189 and rs2033962 and their corresponding haplotypes did not show different frequencies from controls, but were associated with AMH levels in PCOS women (P = 0.001, P = 0.002 and P = 0.007, respectively). Adjustment for follicle number revealed that the association with AMH levels was, in part, independent from follicle number, suggesting that variants in ACVR1 also influence AMH production per follicle.

Genetic variation within ACVR1 is associated with AMH levels and follicle number in PCOS women, suggesting that ALK2 signalling contributes to the disturbed folliculogenesis in PCOS patients.

A membrane-based electrophoretic filtration system, known as the Cell Sorter-10 (CS-10), that preferentially isolates spermatozoa with very low levels of DNA damage has recently been developed. However, it remains to be proven whether spermatozoa prepared in this way are capable of achieving fertilization in assisted conception. Therefore, this clinical trial was designed to answer this question.

A split-sample split-cohort study design was employed to control for differences in semen and oocyte quality between 28 couples undergoing either intracytoplasmic sperm injection (ICSI) or IVF in this clinical trial. Each semen sample was split between preparation using the CS-10 and preparation by standard density gradient centrifugation (DGC) and each cohort of oocytes was split for insemination using either CS-10 (n = 197) or DGC (n = 195) prepared spermatozoa.

Both methods of sperm preparation yielded comparable rates of sperm recovery, motility and DNA fragmentation. There was no significant difference between the ability of CS-10 and DGC prepared spermatozoa to produce fertilization (62.4% versus 63.6%), cleavage (99.0% versus 88.5%) and high-quality embryos (27.4% versus 26.1%).

This pilot study demonstrates that membrane-based electrophoresis is as effective as DGC in preparing sperm for IVF and ICSI, although it takes only a fraction of the time.

Protein tyrosine phosphorylation is one of the main processes associated with sperm activation. Although this process and its targets have been well characterized, only few tyrosine kinases have been identified so far and their roles in spermatozoa are still largely unknown. In this study, we report the presence and localization of Src kinase in ejaculated human spermatozoa and investigate its role in regulating the processes underlying sperm activation.

Specific anti-Src antibodies, against different epitopes of the protein, identified a single band of ~70 kDa relating to a protein which is mainly localized in the post-acrosomal region of the head, neck and midpiece. By immunoprecipitation and immunofluorescence techniques performed with antibodies against Src phosphorylated at Tyr416, which identifies the active kinase, we showed an increased phosphorylation during sperm capacitation. Blocking Src activity with SU6656 resulted in a significant reduction in the protein tyrosine phosphorylation. Moreover, this inhibitor also blocked the progesterone-induced acrosome reaction and interfered with the calcium response to progesterone evaluated in fura-2-loaded spermatozoa. No effect on sperm motility and hyperactivation resulted from incubation with SU6656.

We identified a novel Src isoform in human spermatozoa, which appears to be involved in regulating sperm capacitation, calcium fluxes, tyrosine phosphorylation and acrosome reaction.

Sperm DNA damage is common amongst infertile men and may adversely impact natural reproduction, IUI-assisted reproduction and to a lesser degree IVF pregnancy. The aim of this study was to examine the influence of sperm DNA damage on the risk of spontaneous pregnancy loss after IVF and ICSI.

We conducted a systematic review and meta-analysis of studies on sperm DNA damage and pregnancy loss after an IVF and/or ICSI pregnancy.

Two by two tables were constructed and odds ratios (ORs) were derived from 11 estimates of pregnancy loss (five IVF and six ICSI studies from seven reports). These 11 studies involved 1549 cycles of treatment (808 IVF and 741 ICSI cycles) with 640 pregnancies (345 IVF and 295 ICSI) and 122 pregnancy losses. The combined OR of 2.48 (95% CI 1.52, 4.04, P < 0.0001) indicates that sperm DNA damage is predictive of pregnancy loss after IVF and ICSI.

In conclusion, sperm DNA damage is associated with a significantly increased risk of pregnancy loss after IVF and ICSI. These data provide a clinical indication for the evaluation of sperm DNA damage prior to IVF or ICSI and a rationale for further investigating the association between sperm DNA damage and pregnancy loss.

The precise mechanisms in the materno-fetal dialogue still remain unclear. The aim of this study was to investigate the role of the chemokine CXCL12 and its receptor CXCR4 in the interaction of trophoblasts and decidual stromal cells (DSCs).

Expression of CXCL12/CXCR4 in trophoblasts and DSCs was detected by reverse transcription–polymerase chain reaction and immunochemical staining. The secretion of CXCL12 by trophoblasts was determined by enzyme-linked immunosorbent assay. The effects of CXCL12 on the biological functions of trophoblasts and DSCs were analyzed using a cell viability assay, matrigel invasion assay and zymography. Finally, a co-culture model was established to investigate the modulation of CXCL12/CXCR4 in the interaction of trophoblasts and DSCs.

CXCR4 was transcribed and translated by both human trophoblasts and DSCs. Human trophoblasts secreted CXCL12 spontaneously in vitro, but DSCs did not. CXCL12 induced an apparent increase in the invasiveness of trophoblasts (P < 0.01), and up-regulated matrix metalloproteinase (MMP) 9 and MMP2 activity of both trophoblasts and DSCs (both P < 0.01) in an autocrine and paracrine manner. The invasiveness and MMP9 and MMP2 activity of trophoblasts in co-culture with DSCs increased significantly (P < 0.01), and these could be inhibited by anti-CXCR4 neutralizing antibody.

CXCL12 secreted by human trophoblasts enhances the coordination between trophoblasts and DSCs, via the regulation of MMP9 and MMP2, which may improve the functional materno-fetal interface.

The efficiency of in vitro maturation (IVM) techniques is suboptimal compared with controlled ovarian stimulation combined with IVF cycles, and studies are needed to identify factors that predispose IVM cycles to success or failure. We compared the outcome of IVM cycles with different dominant follicle (DF) size at oocyte retrieval following hCG priming.

IVM was performed in 160 patients with polycystic ovaries (171 cycles). We administered 10 000 IU hCG s.c. 35–38 h before oocyte collection when endometrial thickness reached at least 6 mm. IVM cycles were retrospectively analyzed according to DF diameter as follows; Group 1: DF diameter ≤10 mm, Group 2: between 10 and 14 mm, Group 3: >14 mm.

A positive correlation was observed between DF size and number of in vivo matured oocytes collected (Group 1, 2 and 3 = 6.9, 10.6 and 15.1%, respectively). The rates of IVM, fertilization and embryo development were similar among the sibling immature oocytes collected from the three groups. However, clinical pregnancy rate in Group 2 (40.3%) was higher than Group 3 (17.1%) (P < 0.05). Moreover, implantation rates in Groups 1 (13.6%) and 2 (14.3%) were higher than Group 3 (4.9%) (P < 0.01).

Our results suggest that oocyte collection in IVM cycles should be performed when the DF is 14 mm diameter or less. Sibling immature oocytes may be affected detrimentally if a DF >14 mm is present at oocyte collection.

In this study, we characterized the fibromuscular (FM) tissue, typical of deeply infiltrating endometriosis, investigated which cells are responsible for the FM reaction and evaluated whether transforming growth factor-β (TGF-β) signaling is involved in this process.

FM differentiation and TGF-β signaling were assessed in deeply infiltrating endometriosis lesions (n = 20) and a nude mouse model of endometriosis 1, 2, 3 and 4 weeks post-transplantation. The FM reaction was evaluated by immunohistochemistry using different markers of FM and smooth muscle cell differentiation (vimentin, desmin, alpha-smooth muscle actin, smooth muscle myosin heavy chain). TGF-β signaling was assessed by immunostaining for its receptors and phosphorylated Smad.

Deeply infiltrating endometriosis lesions contain myofibroblast-like cells that express multiple markers of FM differentiation. Expression of TGF-β receptors and phospho-Smad was more pronounced in the endometrial component of the lesions than in the FM component. In the nude mouse model, alpha-smooth muscle actin expression was observed in murine fibroblasts surrounding the lesion, but not in human endometrial stroma.

FM differentiation in deeply infiltrating endometriosis is the result of a reaction of the local environment to the presence of ectopic endometrium. It shares characteristics with pathological wound healing, but cannot be explained by TGF-β signaling alone.

Celecoxib, a selective cyclooxygenase (COX)-2 inhibitor, also has anti-proliferative properties and pro-apoptotic effects on different in vivo and in vitro models, two actions that may be efficacious in therapy for endometriosis. We evaluated the effects of celecoxib on apoptosis and proliferation, and vascular endothelial growth factor (VEGF) production and COX-2 expression and activity in endometrial epithelial cells (EECs).

Thirty-two endometriosis and 13 control women were included in the study. EECs from eutopic endometrium and control biopsies were cultured with different doses of celecoxib. Celecoxib at 50, 75 and 100 µM (versus vehicle control) inhibited EEC proliferation in cultures from controls (P < 0.05, P < 0.01 and P < 0.01, respectively) and patients with endometriosis (P < 0.05, P < 0.01 and P < 0.01), as assessed by ³H-thymidine uptake. Celecoxib at 50, 75 and 100 µM induced apoptosis in EEC from controls (P < 0.05, P < 0.001 and P < 0.001) and patients with endometriosis (P < 0.001, P < 0.001 and P < 0.01), as revealed by the Acridine Orange–Ethidium Bromide technique. Western blot analysis showed that celecoxib was effective at increasing COX-2 protein at 100 µM in EEC from endometriosis patients (P < 0.05). In EEC from endometriosis patients, celecoxib at 25, 50 and 100 µM was also effective in reducing COX-2 activity, reflected in the reduction of prostaglandin E₂ (PGE₂) synthesis (P < 0.001), and VEGF secretion (P < 0.001; P < 0.05 and P < 0.001), assessed by enzyme-linked immunosorbent assay. Exogenous PGE₂ did not reverse celecoxib-induced growth inhibition.

This study suggests a direct effect of celecoxib on reduction of endometrial growth and supports further research on selective COX-2 inhibition as a novel therapeutic modality in endometriosis.

Despite interest in ovarian tissue transplantation (OTT) as a promising procedure for fertility preservation, to date, no precise data are available about its effectiveness. We systematically reviewed reproductive function after OTT for fertility preservation in women at high risk of premature ovarian failure (POF).

We searched the MEDLINE, EMBASE, Cochrane Systematic Reviews, CENTRAL, Web of Science and Scopus databases for studies on the reproductive outcomes after OTT in humans up to June 2007. Women with follicle-stimulating hormone (FSH) >30 IU/l at the time of OTT were included in a meta-analysis of individual-patient data to evaluate the time to re-establishment of ovarian function (ROF). Secondary outcomes included short-term (<12 months) and long-term (>12 months) ovarian function (OVF) and pregnancy after OTT.

We identified 25 reports including 46 unique cases. OTT was performed to treat POF in 27 women, to prevent POF in 15, to treat infertility in 2 and accidentally in 1. In 23 women with FSH >30 at the time of OTT, OVF was re-established with a median time to ROF of 120 days (range 60–244). Within 6 months after ROF, four women had recurrent ovarian failure. There are insufficient data to evaluate the long-term OVF (>12 months). Fresh grafts had an increased likelihood of return of OVF and a decreased likelihood for recurrent ovarian failure compared with cryopreserved grafts [HR of 2.44 (95% CI 0.92, 6.49) and 0.47 (95% CI 0.18, 1.12), respectively]. In 25 women who sought pregnancy, eight women had nine pregnancies at 12 months, giving a cumulative pregnancy rate of 37% (95% CI 19, 60).

Transplantation of ovarian tissue can re-establish OVF after POF; however, the efficacy of OTT using cryopreserved tissues is not yet equivalent to that of fresh grafts. A controlled multicenter trial with sufficient follow-up would provide valid evidence of the potential benefit of this procedure.

After initial years of improvement, the multiple pregnancy rate after in vitro fertilization (IVF) in Europe now remains stable at 23% with single embryo transfer (SET) constituting 19% of all IVF cycles. Although elective SET prevents multiple pregnancies after IVF, couples and professionals apparently often decide to transfer more embryos. Previous qualitative research has identified factors that impede the use of elective SET. The aim of this study was to quantify those barriers among IVF professionals and to identify predictors of professionals’ willingness to perform elective SET.

A national survey among all Dutch IVF professionals quantified the barriers suggested by a previous qualitative study and assessed characteristics of the professionals and clinics. Multivariate analysis identified predictors related to the willingness of IVF professionals to perform elective SET.

In total, 107 professionals participated. The most frequently mentioned barriers to elective SET use were suboptimal success rates associated with cryopreservation (96%), not seeing twin pregnancies as a complication (79%) and lack of a SET protocol (78%). Two variables seem to predict the professionals’ willingness to perform elective SET: university hospital of the initial fertility training (P< 0.01) and high scores of perceived barriers, e.g. professionals’ attitudes and skills (P < 0.01). The explained variance of these two variables was 25%.

This study has identified the main barriers to elective SET use and predictors for willingness of professionals to perform elective SET. This insight into the decision-making process could be critical in terms of increasing the use of elective SET.

A follow-up study was conducted in mid-adolescence on parenting and the child's psychosocial development after in vitro fertilization (IVF). The first phase of the study had compared 31 IVF families and 31 families with a naturally conceived child when the children were 2 years old (Colpin et al., 1995). Of these, 24 IVF families and 21 control families participated again when the children were 15–16 years old.

Fathers, mothers and adolescents completed questionnaires assessing parenting style and stress, and adolescent psychosocial adjustment.

No significant differences were found in self- or adolescent-reported parenting style, or in parenting stress between IVF mothers and mothers in the control group, nor between IVF fathers and fathers in the control group. Neither did we find significant differences in self- or parent-reported behavioural problems between adolescents conceived by IVF and those conceived naturally. Comparison of behavioural problems between IVF adolescents informed or not informed about the IVF conception did not reveal significant differences.

Parenting and 15–16-year-old adolescents' psychosocial adjustment did not differ significantly between IVF families and control families. This study is, to the best of our knowledge, the first psychosocial follow-up in mid-adolescence, and adds to the evidence that IVF children and their parents are well-adjusted. Large-scale studies in adolescence are needed to support these findings.

MicroSort, a sperm-sorting technology for sex selection, may eventually be approved by the Food and Drug Administration and marketed to the public. Data on US public attitudes about the morally appropriate uses and regulation of this technology are lacking.

We conducted 20 focus groups in April 2003 with participants from five major US cities to identify the values that shape Americans’ attitudes about the use and regulation of preconception sex selection (PSS) technology. One hundred and seventy-six individuals between the ages of 18 and 68 were assigned to groups ranging from 6 to 11 participants based on their location, sex, race/ethnicity, religion, age, education and parental status. Qualitative analysis of focus group transcripts was conducted using NVivo 2.0 software to determine beliefs and values that shape participants’ opinions about the appropriate use and regulation of PSS.

Most participants strongly favor using PSS to avoid X-linked genetic diseases. Although some participants were uncomfortable with the use of PSS for non-medical sex selection, believing it to be ‘selfish’ and inconsistent with parental love, they did not perceive the potential harms to be significant enough to warrant governmental intrusion into reproductive decisions.

PSS should face little public opposition in the US if widely marketed.

The purpose of this study was to determine positive and negative social interactions experienced by infertile Japanese women.

Semi-structured interviews were conducted with 24 infertile women. The informants were asked about their experiences of positive (helpful) and negative (unhelpful) social interactions with members of their social networks, excluding their partners, with regard to their infertility.

Nine positive social interaction categories were clarified, including listening closely to the distress experienced in infertility and treatment, not prying or interfering with the topic of children and respecting the women's decision regarding fertility treatment and taking a wait-and-see attitude. Nine negative social interaction categories were also identified, including prying with the topic of children, showing a negative attitude toward infertility or reproductive medicine, being criticized for not having children and avoiding contact.

The present findings systematically and qualitatively determined the positive and negative social interactions experienced by infertile Japanese women within their social networks. This is essential knowledge for medical staff to counsel patients and their family members. To form a supportive social environment for infertile women, we recommend practical measures for health workers and helpful advice with regard to interactions between infertile women and their social networks.

Human embryonic stem cells (hESCs) have potential use in clinical therapy and regenerative medicine. One of the major challenges regarding the application of these cells is the development of an efficient cryopreservation protocol, since current methods, which include slow-freezing–rapid thawing and vitrification of colonies in suspension, present poor viability and high differentiation rates. Dissociated hESC suspensions do not survive cryopreservation because they are susceptible to apoptosis upon cell detachment and dissociation. A selective Rho-associated kinase (ROCK) inhibitor has been reported to increase the survival of dissociated hESCs and their cloning efficiency.

Here, we describe a novel method for dissociated hESCs cryopreservation in the presence of the ROCK inhibitor Y-27632. The addition of this inhibitor to the freezing and post-thawing medium significantly increased the survival rate and efficiency of colony formation. Moreover, the hESC colonies obtained after the cryopreservation in the presence of the ROCK inhibitor showed a very low rate of differentiation and a reduced time of recovery. After prolonged culture of frozen–thawed dissociated hESCs, the characteristic properties of pluripotent cells were observed, including normal karyotype, morphological features, marker expression (SSEA-4, TRA-1-60, TRA-1-81 and Oct-4) and the potential to differentiate into derivatives of all three germ layers after embryoid bodies formation.

This novel method for the cryopreservation of dissociated hESCs may reduce the time required to amplify frozen stocks, and facilitate not only the storage of large numbers of hESCs but also the widespread use of these cells in regenerative medicine.

Testicular germ cell tumours (TGCT) are thought to originate from fetal germ cells that fail to differentiate normally, but no animal model for these events has been described. We evaluated the marmoset (Callithrix jacchus) as a model by comparing perinatal germ cell differentiation with that in humans.

Immunohistochemical profiling was used to investigate germ cell differentiation (OCT4, NANOG, AP-2, MAGE-A4, VASA, NANOS-1) and proliferation (Ki67) in fetal and neonatal marmoset testes in comparison with the human and, to a lesser extent, the rat.

In marmosets and humans, differentiation of gonocytes into spermatogonia is associated with the gradual loss of pluripotency markers such as OCT4 and NANOG, and the expression of germ cell-specific proteins such as VASA. This differentiation occurs asynchronously within individual cords during fetal and early postnatal life. This contrasts with rapid and synchronous germ cell differentiation within and between cords in the rat. Similarly, germ cell proliferation in the marmoset and human occurs throughout perinatal life, in contrast to rats in which proliferation ceases during this period.

The marmoset provides a good model for normal human germ cell differentiation and proliferation. The perinatal marmoset may be a useful model in which to establish factors that lead to failure of normal germ cell differentiation and the origins of TGCT.

The microtubule-destabilizing protein stathmin is expressed by the villous cytotrophoblasts and invasive extravillous trophoblasts (EVTs) in the first-trimester human placenta. Here, we evaluated the significance of stathmin expression in terms of the functions of trophoblasts.

We employed two choriocarcinoma cell lines (BeWo and JEG-3), an EVT cell line (HTR-8/SVneo) and isolated first-trimester trophoblast cells. The effects of small-interfering (si) RNA-mediated stathmin knockdown on trophoblast proliferation and migration were measured by WST-1 and Transwell assays, respectively. Trophoblast differentiation was induced by dibutyryl (db)-cAMP treatment and evaluated by measuring human chorionic gonadotrophin β (hCGβ) and syncytin expression and cell fusion. We examined the effect of knockdown and induced stathmin expression on db-cAMP-induced differentiation.

siRNA-induced silencing of stathmin expression had a marked inhibitory effect on BeWo, JEG-3 and HTR-8/SVneo cell migration and also suppressed their proliferation, albeit to a lesser extent. db-cAMP-enhanced hCGβ and syncytin expression and cell fusion in BeWo cells was inhibited by stathmin knockdown. However, induced expression of stathmin reversed the hCGβ and syncytin expression and cell fusion in the Tet-On BeWo cells. Suppression of stathmin expression also inhibited the migration of and hCGβ production by first-trimester trophoblasts.

Stathmin expression may be closely associated with early trophoblast migration and differentiation into syncytiotrophoblasts during placentation.

During early pregnancy, the most important task of the corpus luteum (CL) is to produce sufficient progesterone until the luteoplacental shift occurs. Progesterone production is closely related to the extensive vasculature surrounding and supplying the CL. The synthesis of both progesterone and factors controlling the vasculature in the CL is regulated by hCG, which is released initially at rising levels from the placenta. The primary aim of this research was to evaluate changes in the CL vasculature during early pregnancy.

Twenty naturally conceived pregnancies were examined weekly from weeks 5 to 11. At each visit, blood samples were obtained to determine the concentrations of hCG, progesterone and 17-OH progesterone (17-OHP). The vasculature in the ovaries was assessed using three-dimensional power Doppler ultrasonography.

The vascular supply in the ovary containing the CL was greatest at week 5, and thereafter, declined continuously until week 11. The decrease in the vasculature correlated with the decrease in 17-OHP. Mean hCG levels reached a maximum at week 8, progesterone levels reached the nadir at week 7 and increased after that.

Vasculature in the CL appears to be created already by the fifth week of pregnancy and it does not enlarge despite rising hCG levels. The activity of the CL during pregnancy may be measured non-invasively by assessing its vasculature with three-dimensional ultrasonography.

Estrogen receptor related beta (ERRβ, ESRRB/NR3B2) is an orphan receptor that shares significant sequence homology with estrogen receptors ER and ERβ. ERR family members are reported to exhibit constitutive transcriptional activity; however, little is known about the biological function of ERRβ. In an attempt to delineate its role, we examined expression of ERRβ in normal human endometrium, a tissue that undergoes cyclic remodelling under the influence of estrogen and progesterone.

Well-characterized endometrial tissue (n = 31), including full-thickness biopsies, was obtained from women with regular menstrual cycles. RT–PCR was used to measure mRNA encoding ERRβ, the peroxisome proliferator activated receptor gamma coactivators (PGC)-1 and β and to determine whether ERRβ splice variant mRNAs were expressed. ERRβ was immunolocalized using both single and double antibody immunohistochemistry.

Total ERRβ mRNA appeared higher in proliferative phase samples but results did not reach significance. Transcripts corresponding to the long- and short-splice variants of ERRβ as well as PGC1 and β were detected but ERRβ10 was absent. ERRβ protein was localized to cell nuclei within multiple endometrial cell types including the glands, stroma, endothelium and immune cells, including uterine natural killer (uNK) cells and macrophages. Fluorescent immunohistochemistry revealed that some cells co-expressed ERRβ and ER or ERβ, for example, endothelial and uNK cells were ERRβ+/ERβ+.

ERRβ mRNA and protein are expressed in healthy human endometrium. Further studies are warranted to characterize the functional impact of ERRβ on endometrial biology.

Previous studies demonstrated a link between adverse conditions during prenatal life and the development of diseases in adult life. It is still unclear whether IVF conception could permanently affect early prenatal development in humans, with post-natal health consequences. The objective of the present study is to examine pubertal development in 8–18-year-old IVF singletons and controls born from subfertile parents who attended one Dutch fertility clinic were included.

IVF singletons and controls born from subfertile parents who attended one clinic in the Dutch OMEGA study were included. Pubertal stage by Tanner’s classification, age at menarche and menstrual cycle characteristics were studied in the total population (n = 233: 115 IVF-conceived boys and 118 IVF-conceived girls, each with age-matched comparison groups). Bone age and sex hormone levels were examined in two distinct pubertal subpopulations.

Pubertal stage and age at menarche were not significantly different between IVF and control children. In the pubertal subpopulation, a higher bone age–chronological age (BA–CA) ratio and a larger BA–CA difference were observed in IVF-conceived girls compared with controls (1.04 ± 0.07 versus 1.02 ± 0.08, P = 0.022; 0.54 ± 0.82 versus 0.18 ± 1.00 year, P = 0.021, respectively). Furthermore, dehydroepiandrosterone sulphate (DHEAS) and LH levels were significantly higher in IVF-conceived girls than in control subjects (2.5 versus 1.9 µmol/l, P = 0.017, and 1.5 versus 0.6 U/l, P = 0.031, respectively).

Bone age appeared to be advanced in pubertal IVF-conceived girls, but not in boys, compared with controls. Increased DHEAS and LH concentrations were found among IVF girls.

A few studies have investigated the association between male caffeine consumption in adult life and semen quality with conflicting results, but so far no studies have explored the effect of prenatal coffee exposure. We studied the association between prenatal coffee and current caffeine exposure and semen quality and levels of reproductive hormones.

From a Danish pregnancy cohort established in 1984–1987, 347 sons out of 5109 were selected for a follow-up study conducted 2005–2006. Semen and blood samples were analyzed for conventional semen characteristics and reproductive hormones and were related to information on maternal coffee consumption during pregnancy and present caffeine consumption. Data were available for 343 men.

There was a tendency toward decreasing crude median semen volume (P = 0.06) and adjusted mean testosterone (P = 0.06) and inhibin B (P = 0.09) concentrations with increasing maternal coffee consumption during pregnancy. Sons of mothers drinking 4–7 cups/day had lower testosterone levels than sons of mothers drinking 0–3 cups/day (P = 0.04). Current male caffeine intake was associated with increasing testosterone levels (P = 0.007). Men with a high caffeine intake had ~14% higher concentration of testosterone than those with a low caffeine intake (P = 0.008).

The results observed in this study are only tentative, but they do not exclude a small to moderate effect of prenatal coffee exposure on semen volume and levels of reproductive hormones. Present adult caffeine intake did not show any clear associations with semen quality, but high caffeine intake was associated with a higher testosterone concentration.

Advanced maternal age (AMA) is an important parameter that negatively influences the clinical pregnancy rate in IVF, in particular owing to the increased embryo aneuploidy rate. It has thus been suggested that only transferring euploid embryos in this patient group would improve the pregnancy rate. The purpose of this study was to test whether employing preimplantation genetic screening (PGS) in AMA patients would increase the clinical pregnancy rate.

We conducted a two-center, randomized controlled trial (RCT) to analyze the outcome of embryo transfers in AMA patients (≥38 years of age) after PGS using FISH analysis for chromosomes X, Y, 13, 16, 18, 21 and 22. The PGS group was compared with a control group. The primary outcome measure was clinical pregnancy rate after 6–7 weeks of gestation per randomized patient.

The study was terminated early as an interim analysis showed a very low conditional power of superiority for the primary outcome. Of the 320 patients calculated to be included in the study, 56 and 53 patients were randomized into the PGS and control groups, respectively. The clinical pregnancy rate in the PGS group was 8.9% (95% CI, 2.9–19.6%) compared with 24.5% (95% CI, 13.8–38.3%) in the control group, giving a difference of 15.6% (95% CI, 1.8–29.4%, P = 0.039).

Although the study was terminated early, this RCT study provides evidence against the use of PGS for AMA patients when performing IVF. Trial registration number: ISRCTN38014610.

Human preimplantation embryos generated through in vitro fertilization (IVF) or intracytoplasmic sperm injection (ICSI) treatments show a variable rate of numerical chromosome abnormalities or aneuploidies. Preimplantation genetic screening (PGS) has been designed to screen for aneuploidies in high risk patients, with the aim of improving live birth rates in IVF/ICSI. We assessed whether the effect of PGS on live births rates differs in women of advanced maternal age with variable risks for embryonic aneuploidy, and weighed these effects against the results obtained after IVF/ICSI without PGS.

The effect of PGS on live birth rates was compared between groups defined by maternal age, number of previous miscarriages, semen quality, total amount of recombinant FSH (rFSH) administered during ovarian stimulation and total number of top-quality embryos, using data from a randomized controlled trial among women of advanced maternal age (35–41 years).

There was no significant differential effect of PGS in groups based on maternal age (P-value of interaction 0.16), the number of previous miscarriages (P-value of interaction 0.93), semen quality (P-value of interaction 0.26), rFSH dose (P-value of interaction 0.15) or the number of top-quality embryos (P-value of interaction 0.59). Live birth rates after IVF/ICSI with PGS were lower in all groups when compared with live birth rates after IVF/ICSI without PGS.

The paradigm that the effect of PGS is determined by a woman's risk for embryonic aneuploidy seems incorrect. In fact, PGS has no clinical benefit over standard IVF/ICSI in women of advanced maternal age regardless of their risk for embryonic aneuploidy.

Single-embryo transfer is a well-accepted strategy to avoid multiple pregnancies in an assisted reproductive technology (ART) programme. Besides the morphological quality and embryo kinetics up to the blastocyst stage, preimplantation genetic screening (PGS) of aneuploidy has been advocated as an adjuvant approach to select the embryo.

Couples with a female partner younger than 36 were randomly assigned to undergo transfer of a single blastocyst in a cycle with or without PGS using FISH for the chromosomes X, Y, 13, 16, 18, 21, 22.

After the enrolment of 120 of the projected 447 patients in each group, study recruitment was terminated prematurely on the basis of futility. The observed live birth delivery rates after ART were 30.8 versus 30.8% per randomized patient, 34.6 versus 34.6% per cycle initiated, 37.8 versus 37.0% per aspirated cycle and 41.6 versus 43.5% per embryo transfer for the control versus the PGS group, respectively, with absolute between-group differences (95% CI; P value) of 0% (–11.7 to 11.7; P = 1.00), 0% (–12.7 to 12.7; P = 1.00), –0.8% (–14.2 to 12.7; P = 0.91) and 2.1% (–12.7 to 16.7; P = 0.79), respectively. Even in this younger age group, only 61% of the embryos had a normal diploid status.

The absence of a beneficial treatment effect in this randomized clinical trial provides no arguments in favour of PGS to improve live birth delivery rate following single-embryo transfer in women under the age 36. Clinical Trials.gov: NCT00670059.

Premature ovarian failure (POF) is characterized by elevated gonadotrophins and amenorrhea before the age of 40 years and occurs approximately in 1% of women. POF etiology is highly heterogeneous with a wide spectrum of etiological pathogenic mechanisms including genetic causes. These mostly involve numerical, structural or monogenic defects on the X-chromosome. Mutations in a small number of autosomal genes (such as FOXL2 and NOBOX) have been identified as a cause of POF. However, in most cases, the disease underlying mechanisms are largely unknown.

We performed a genome-wide linkage analysis in a relatively large Dutch family with seven patients suffering from POF, showing a dominant pattern of inheritance. A genome-wide analysis, using 50K single nucleotide polymorphism arrays, was combined with conventional parametric linkage analysis.

We identified three genomic regions on chromosomes 5, 14 and 18 yielding suggestive linkage (multipoint LOD score of 2.4 for each region). After inclusion of one elder unaffected family member, only the region on chromosome 5 remains as a putative POF locus. In addition, we investigated a second family (three living patients over three generations) for the regions on chromosome 5, 14 and 18. Haplotype analysis supported only the locus on chromosome 5q14.1–q15.

We performed the first genome-wide linkage search in familial POF and identified a region on chromosome 5q14.1–q15, which may harbor a novel POF susceptibility gene.

Human homologs (FEM1A, FEM1B, FEM1C) of nematode sex determination genes are candidate genes for polycystic ovary syndrome (PCOS). We previously identified a FEM1A mutation (H500Y) in a woman with PCOS; FEM1B has been implicated in insulin secretion.

Women with and without PCOS (287 cases, 187 controls) were genotyped for H500Y and six FEM1A single nucleotide polymorphisms (SNPs), five FEM1B SNPs and five FEM1C SNPs. SNPs and haplotypes were determined and tested for association with PCOS and component phenotypes.

No subject carried the FEM1A H500Y mutation. FEM1A SNPs rs8111933 (P = 0.001) and rs12460989 (P = 0.046) were associated with an increased likelihood of PCOS whereas FEM1A SNP rs1044386 was associated with a reduced probability of PCOS (P = 0.013). FEM1B SNP rs10152450 and a linked SNP were associated with a reduced likelihood of PCOS (P = 0.005), and lower homeostasis model assessment (HOMA) for beta-cell function (HOMA-%B, P = 0.011) and lower HOMA for insulin resistance (HOMA-IR, P = 0.018). FEM1B SNP rs12909277 was associated with lower HOMA-%B (P = 0.008) and lower HOMA-IR (P = 0.037). Haplotype associations were consistent with SNP results, and also revealed association of FEM1B haplotype TGAGG with increased HOMA-%B (P = 0.007) and HOMA-IR (P = 0.024). FEM1C variants were not associated with PCOS.

This study presents evidence suggesting a role for FEM1A and FEM1B in the pathogenesis of PCOS. Only FEM1B variants were associated with insulin-related traits in PCOS women, consistent with prior evidence linking this gene to insulin secretion. Replication of these associations and mechanistic studies will be necessary to establish the role of these genes in PCOS.

Post-transcriptional modification by SUMOylation is involved in numerous cellular processes including human spermatogenesis. For human male meiosis, we previously showed that the small ubiquitin-related modifier-1 (SUMO-1) protein localizes to chromatin axes in early pachytene spermatocytes, then to kinetochores as meiosis progresses. Here, we delineate possible functional roles based on subcellular localization for SUMO-1 and SUMO-2/3.

Western and immunoprecipitation analyses were conducted on proteins isolated from the testis of two normal adult fertile men. Combinatorial immunofluorescence and chromosome-specific fluorescence in situ hybridization analyses were performed on male meiocytes obtained during testicular biopsy from four patients undergoing testicular sperm extraction for assisted reproduction technologies.

The synaptonemal complex (SC) and SC proteins (SCP)-1 and SCP2, but not SCP3, are SUMOylated by SUMO-1 during the pachytene substage. Likewise, two distinct localization patterns for SUMO-1 are identified: a linear pattern co-localized with autosomal SCs and isolated SUMO-1 near the centromeric heterochromatin of chromosomes 9 and 1. In contrast to SUMO-1, which is not detectable prior to pachytene in normal tissue, SUMO-2/3 is identified as early as leptotene and zygotene and in some, but not all, pachytene cells; no linear patterns were detected. Similar to SUMO-1, SUMO-2/3 localizes in two predominant subnuclear patterns: a single, dense signal near the centromere of human chromosome 9 and small, individual foci co-localized with autosomal centromeres.

Our data suggest that SUMO-1 may be involved in maintenance and/or protection of the autosomal SC. SUMO-2/3, though expressed similarly, may function separately and independently during pachytene in men.

One of the most well-documented cytokines suspected as a hazard to male fertility is tumor necrosis factor- (TNF). Genetic factors such as single-nucleotide polymorphisms (SNPs) in the TNF gene cluster impact TNF levels. Our objective was to establish the potential involvement of –308 TNF SNP in male infertility risk.

In 684 infertile male patients undergoing an intracytoplasmic sperm injection procedure, we used allele-specific polymerase chain reaction (PCR) and PCR-RFLP to investigate the distribution of the guanine (G)-to-adenosine (A) substitution at position –308 in the promoter region of the TNF gene.

An increased frequency of the –308 TNF A allele was found in patients with low sperm count of testicular origin [P = 0.002; odds ratio (OR) = 2.93] or with norm