Treatment effect of oil-based contrast is related to experienced pain at HSG: a post-hoc analysis of the randomised H2Oil study.

STUDY QUESTION: Does pain or volume of used contrast medium impact the effectiveness of oil-based contrast during hysterosalpingography (HSG)? SUMMARY ANSWER: In women who report moderate to severe pain during HSG, the use of oil-based contrast resulted in more ongoing pregnancies compared to the use of water-based contrast, whereas in women who reported mild or no pain, no difference in ongoing pregnancies was found. WHAT IS KNOWN ALREADY: We recently showed that in infertile women undergoing HSG, the use of oil-based contrast results in more ongoing pregnancies within 6 months as compared to the use of water-based contrast. However, the underlying mechanism of this fertility-enhancing effect remains unclear. STUDY DESIGN, SIZE, DURATION: We performed a post-hoc analysis of the H2Oil study, a multicentre randomised controlled trial (RCT) evaluating the therapeutic effect of oil- and water-based contrast at HSG. Here, we evaluated the impact of pain experienced at HSG and volume of used contrast media during HSG on ongoing pregnancy. PARTICIPANTS/MATERIALS, SETTING, METHODS: In a subset of 400 participating women, pain during HSG by means of the Visual Analogue Scale (VAS) (range: 0.0-10.0 cm) was reported, while in 512 women, we registered the volume of used contrast (in millilitres). We used logistic regression analyses to assess whether pain and volume of used contrast media modified the effect of oil-based contrast on ongoing pregnancy rates. Data were analysed according to intention-to-treat principle. MAIN RESULTS AND THE ROLE OF CHANCE: In 400 women in whom pain scores were reported, the overall median pain score was 5.0 (Interquartile range (IQR) 3.0-6.8) (oil group (n = 199) 4.8 (IQR 3.0-6.4); water group (n = 201) 5.0 (IQR 3.0-6.7); P-value 0.28). There was a significant interaction between pain (VAS ≤5 versus VAS ≥6) and the primary outcome ongoing pregnancy (P-value 0.047). In women experiencing pain (VAS ≥6), HSG with oil-based contrast resulted in better 6-month ongoing pregnancy rates compared to HSG with water-based contrast (49.4% versus 29.6%; RR 1.7; 95% CI, 1.1-2.5), while in women with a pain score ≤5, 6-month ongoing pregnancy rates were not significantly different between the use of oil- (28.8%) versus water-based contrast (29.2%) (RR 0.99; 95% CI, 0.66-1.5). In the 512 women in whom we recorded contrast, median volume was 9.0 ml (IQR 5.7-15.0) in the oil group versus 8.0 ml (IQR 5.9-13.0) in the water group, respectively (P-value 0.72). Volume of used contrast was not found to modify the effect of oil-based contrast on ongoing pregnancy (P-value for interaction 0.23). LIMITATIONS, REASONS FOR CAUTION: This was a post-hoc analysis that should be considered as hypothesis generating. The RCT was restricted to infertile ovulatory women, younger than 39 years of age and with a low risk for tubal pathology. Therefore, our results should not be generalised to infertile women who do not share these features. WIDER IMPLICATIONS OF THE FINDINGS: The underlying mechanism of the fertility-enhancing effect induced by HSG with the use of oil-based contrast remains unclear. However, these findings suggest a possible mechanistic pathway, that is increasing intrauterine pressure occurring prior to dislodging pregnancy hindering debris or mucus plugs from the proximal part of otherwise normal fallopian tubes. This information might help in the search of the underlying fertility-enhancing mechanism found by using oil-based contrast during HSG. STUDY FUNDING/COMPETING INTEREST(S): The original H2Oil RCT was an investigator-initiated study that was funded by the two academic institutions (AMC and VUmc) of the Amsterdam UMC. The funders had no role in study design, collection, analysis and interpretation of the data. K.D. reports consultancy for Guerbet. H.V. reports consultancy fees from Ferring. C.B.L. reports speakers' fees from Ferring and research grants from Ferring, Merck and Guerbet. V.M. reports receiving travel and speakers fees as well as research grants from Guerbet. B.W.M. is supported by an NHMRC Practitioner Fellowship (GNT1082548). B.W.M. reports consultancy for ObsEva, Merck KGaA and Guerbet and travel and research grants from Merck KGaA and Guerbet. The other authors do not report conflict of interests. TRIAL REGISTRATION NUMBER: The H2Oil study was registered at the Netherlands Trial Registry (NTR 3270). TRIAL REGISTRATION DATE: 1 February 2012. DATE OF FIRST PATIENT'S ENROLMENT: 3 February 2012.

Soluble HLA-G promotes Th1-type cytokine production by cytokine-activated uterine and peripheral natural killer cells.

Soluble forms of HLA-G (sHLA-G) have been implicated in immune regulation. Fetal trophoblast cells are a prime source of HLA-G. Hence, an interaction between sHLA-G and uterine lymphocytes in the decidual tissues can easily be envisaged. These lymphocytes, when properly activated, are implicated in successful trophoblast invasion, placental maturation and maintenance of pregnancy. However, so far, no data are available on the effect of sHLA-G on the function and phenotype of these cells. Herein, we used a recombinant sHLA-G construct to determine the effect of sHLA-G on uterine lymphocyte cells present in endometrium at the time that it is optimally receptive to trophoblast invasion. In addition, we ascertained the effect of sHLA-G on peripheral lymphocytes. We found that upon co-culture with sHLA-G, proliferation of unfractionated IL-15-stimulated uterine mononuclear cells (UMCs) was inhibited. However, sHLA-G increased both interferon (IFN)-gamma and tumour necrosis factor (TNF)-alpha production by these cells. Vascular endothelial growth factor (VEGF) production was reduced. Notably, in contrast to membrane-bound HLA-G, sHLA-G did not affect the natural cytolytic activity of UMCs. Similarly, sHLA-G inhibited proliferation but stimulated pro-inflammatory cytokine production by cytokine-activated, unfractionated peripheral blood mononuclear cells (PBMCs). In addition, we showed that the overall inhibitory effect of sHLA-G on proliferation of the whole cell population could be ascribed to selective inhibition of CD4(+) T cells. In contrast, sHLA-G induced proliferation and IFN-gamma production by both uterine and peripheral natural killer (NK) cells. In conclusion, our data show that the sHLA-G modulates both UMC and PBMC function. sHLA-G, by promoting IFN-gamma production by uterine NK cells, may contribute to vascular remodelling of spiral arteries to allow for successful embryo implantation.

Two cycles with single embryo transfer versus one cycle with double embryo transfer: a randomized controlled trial.

BACKGROUND: With the aim of reducing the number of multiple pregnancies after IVF we investigated the effectiveness of two cycles with single embryo transfer (SET) and one cycle with double embryo transfer (DET) after IVF and calculated the cost-effectiveness of both strategies. METHODS: A randomized controlled trial was performed in 107 women, aged <35 years, in their first IVF cycle, with at least one good quality embryo. They were randomized to the SET (n = 54) or DET (n = 53) group using a computer-generated random block number table, stratified for primary or secondary infertility. RESULTS: The cumulative live birth rates per woman randomized of two consecutive cycles of SET [41%; 95% confidence interval (CI) 27-54] versus one cycle of DET (36%; 95% CI 23-49) were comparable, whereas the multiple pregnancy rate was significantly higher: 37% (95% CI 15-59) in the DET and 0% in the in the SET group (P = 0.002). Combining the medical costs of the IVF treatments (where 1.5 more SET cycles were required to achieve each live birth) and of pregnancies up to 6 weeks after delivery, the total medical costs of DET per live birth were 13,680 and 13,438 for SET. CONCLUSIONS: Two cycles with SET were equally effective as one cycle with DET, and the medical costs per live birth up to 6 weeks after delivery were the same. However, if lifetime costs for severe handicaps are included, more than 7000 per live birth will be saved after implementing SET. Because of the high probability of multiple pregnancies in this group of IVF patients, only SET should be performed.

Cost analysis of singleton versus twin pregnancies after in vitro fertilization.

OBJECTIVE: To determine the difference in costs between singleton and twin pregnancies after IVF treatment from pregnancy to 6 weeks after delivery from a health care perspective. DESIGN: Retrospective cost analysis. SETTING: IVF department at the University Medical Center Nijmegen, The Netherlands. PATIENT(S): A representative sample of singleton and twin pregnancies after IVF treatment between 1995 and 2001 at the University Medical Center Nijmegen. INTERVENTION(S): IVF with or without intracytoplasmic sperm injection and with or without cryopreservation. MAIN OUTCOME MEASURE(S): Medical costs per singleton and twin pregnancy after IVF. RESULT(S): In patients pregnant with twins, the incidence of hospital antenatal care, complicated vaginal deliveries, and cesarean sections was higher and was associated with more frequent and longer maternal and neonatal hospital admissions. Maternal and neonatal hospital admissions were the major cost drivers. The medical cost per twin pregnancy was found to be more than five times higher than per singleton pregnancy, 13,469 and 2,550, respectively. CONCLUSION(S): The medical cost per twin pregnancy was more than 10,000 higher than per singleton pregnancy. A reduction in the number of twin pregnancies by elective single ET will save substantial amounts of money. This money might be used for the additional IVF cycles that will probably be needed to achieve similar success rates between single ET and two-embryo transfer.

Hormonal stimulation for IVF treatment positively affects the CD56bright/CD56dim NK cell ratio of the endometrium during the window of implantation.

The effects of hormone stimulation for IVF treatment on endometrial receptivity remain controversial. Since CD56(bright) natural killer (NK) cells in the endometrium positively contribute to implantation and decidualization whereas CD56(dim) NK cells are negatively associated with reproduction, shifts in the balance between those cells will affect receptivity. Therefore, we compared the leukocyte composition in the endometrium of IVF women (n=20) with non-pregnant women (n=18) in a natural cycle, as a parameter for endometrial quality. Biopsies were obtained 7 days after ovulation. Histological dating of the endometrium showed no increased endometrial advancement after IVF treatment as compared to the control group. Flow cytometric analysis of leukocyte subsets showed that hormonal stimulation positively affected the CD56(bright)/CD56(dim) ratio in the endometrium by a relative decrease in the cytotoxic CD56(dim)CD16(+) NK cell numbers. The relative number of T-cells remained unaffected, while the number of non-T and non-NK cells (i.e. B-cells and macrophages) was higher in the IVF group. These effects were restricted to the endometrium and not observed in peripheral blood. Within the CD56(bright) population we could identify a distinct subset of NK cells (CD56(superbright)) that was unique for the endometrium. We conclude that hormonal stimulation for IVF treatment positively affects the CD56(bright)/CD56(dim) ratio of the endometrium during the window of implantation and does not affect endometrial advancement.

Membrane-bound HLA-G activates proliferation and interferon-gamma production by uterine natural killer cells.

The expression of HLA-G by invading trophoblasts suggests a role for this molecule in embryo implantation. Putative targets for HLA-G are the uterine natural killer cells (uNK) that are abundantly present at the time of implantation. Since NK cells are potent producers of a variety of cytokines, interaction with HLA-G may result in the production of cytokines involved in trophoblast differentiation or tissue remodelling. In the present study we investigated the effect of membrane-bound HLA-G (mHLA-G) on the uterine mononuclear cell population (UMC) as a whole and on uNK cells in particular by measuring proliferation and cytokine production [interferon-gamma (IFN-gamma)/vascular endothelial growth factor (VEGF)/leukaemia inhibitory factor (LIF)/interleukin-3 (IL-3)]. Uterine cells were isolated from endometrium of non-pregnant women at the time that the endometrium is thought to be receptive to implantation, and then co-cultured with HLA-class I(-)/HLA-class II(+) 721.221 B-LCL cells transfected with mHLA-G. HLA-G suppressed the alloproliferative response of unfractionated UMC to 721.221 cells. Also, IFN-gamma and IL-3 production was strongly reduced. In contrast, purified uNK cells were stimulated by mHLA-G. Proliferation as well as IFN-gamma production was increased after co-culture with mHLA-G transfected 721.221 cells. HLA-G stimulated VEGF production by UMC as well as purified uNK cells. LIF-levels were below the detection level of our enzyme-linked immunosorbent assay. In conclusion, our data show that mHLA-G stimulates proliferation and cytokine production by NK cells, while down-modulating the response of unfractionated UMC.

The proportion of follicular fluid CD16+CD56DIM NK cells is increased in IVF patients with idiopathic infertility.

One-fifth of all in-vitro fertilization (IVF) patients suffer from idiopathic infertility. A low fertilization rate is one of the most characteristic features of IVF in this group, probably caused by oocyte dysfunction. We speculate that an altered lymphocyte profile in follicular fluid (FF) may affect oocyte function and thus play a role in idiopathic infertility. Therefore, we compared levels of lymphocyte populations present in FF of 11 patients with idiopathic infertility (study group) with 29 patients in the control group, i.e. severe male factor infertility (n=17) or tubal factor infertility (n=12). Triple color flow cytometry was used to discriminate between T cells and NK cell subpopulations. In the idiopathic infertility group, a shift from T to NK cells was observed in FF as compared to the control group, caused mainly by a significant higher level of NK cells--20.3 and 13.6% (P<0.05), respectively. This high level of NK cells was due to a rise of the CD16+CD56dim NK cell subset. In peripheral blood, the NK cell levels showed a similar although not significant trend (P=0.08). As the CD16+CD56dim NK cell subpopulation is known for its cytotoxic properties, this subpopulation may negatively affect folliculogenesis and oocyte maturation, reflected by a diminished fertilization rate in the idiopathic infertility group. An altered lymphocyte profile in FF could therefore influence fertility in these patients.

Detection of HLA-G by a specific sandwich ELISA using monoclonal antibodies G233 and 56B.

Human leukocyte antigen (HLA)-G, which is mainly expressed at the maternal-fetal interface, may play a role in the immune tolerance of the semi-allogenic fetus by the mother. Functional studies have shown that HLA-G is indeed a potential modulator of different immune responses. Therefore, it is of interest to study the level of expression of soluble HLA-G in several biological fluids derived from women with and without fertility problems. In order to measure soluble HLA-G, a reliable and sensitive HLA-G specific sandwich ELISA is required. Here, we describe such an ELISA in which G233 is used as the coating antibody and 56B as the detecting antibody. In comparison with two other assays, this assay shows highest responses to recombinant HLA-G and native HLA-G in primary trophoblast culture supernatant and high responses to HLA-G in amniotic fluid. No HLA-G in follicular fluid or preimplantation embryo culture supernatant could be detected.

House dust mite avoidance measures improve peak flow and symptoms in patients with allergy but without asthma: a possible delay in the manifestation of clinical asthma?

BACKGROUND: Asthma caused by allergy to house dust mite is a growing problem. Patients with allergy who do not have asthma (yet) might develop asthma depending on exposure to precipitating factors. OBJECTIVE: We sought to determine whether house dust mite avoidance measures have an effect on the development of asthma. METHODS: Patients with allergy (n = 29) who had no diagnosis of asthma (FEV1 of 99.1% +/- 10.6% of predicted, peak flow variability of 5.21% +/- 3.41%, reversibility of FEV1 after 400 microg salbutamol of 3.92% +/- 3.75% according to the reference values) were randomly allocated (subjects blinded) to a treatment (n = 16) and a placebo group (n = 13). House dust mite avoidance treatment consisted of applying Acarosan (Allergopharma, J. Ganzer KG, Hamburg, Germany) (the placebo group used water) to the floors (living room, bedroom), and the use of covers for mattresses and bedding that were impermeable to house dust mite (the placebo group used cotton covers for mattresses only). We tested whether the intervention had an effect on peak flow parameters and asthma symptom scores during 6 weeks of treatment. RESULTS: Significant improvements were seen in the treatment group in symptom scores (Borg score) for disturbed sleep, breathlessness, wheeze, and overall symptom score. Slight but statistically significant improvements in peak flow (morning, evening, and variability) were seen in the treatment group also. No significant changes were seen in the placebo group. CONCLUSIONS: Although this study is not long enough to study the development of asthma, the results indicates that house dust mite avoidance measures had an effect on peak flow parameters and asthma symptoms in patients with allergy but without asthma. These findings might implicate that a shift in developing clinically manifest asthma could be achieved with house dust mite avoidance measures. To give a better answer to whether preventing the development of asthma is possible, larger studies with a longer follow-up period are necessary.