BACKGROUND

Calcium removal from the medium promptly reduces human sperm motility and induces a Na⁺-dependent depolarization that is accompanied by an increase in intracellular sodium concentration ([Na⁺]_i) and a decrease in intracellular calcium concentration ([Ca²⁺]_i). Sodium loading activates a Na⁺/K⁺-ATPase.

METHODS

Membrane potential (Vm) and [Ca²⁺]_i were simultaneously detected in human sperm populations with the fluorescent probes diSC₃(5) and fura 2. [Na⁺]_i and was measured independently in a similar fashion using sodium-binding benzofuran isophthalate. Motility was determined in a CASA system, ATP was measured using the luciferin-luciferase assay, and cAMP was measured by radioimmunoassay.

RESULTS

Human sperm motility reduction after calcium removal is related to either Na⁺-loading or Na⁺-dependent depolarization, because, under conditions that inhibit the calcium removal-induced Na⁺-dependent depolarization and [Na⁺]_i increase, sperm motility was unaffected. By clamping sperm Vm with valinomycin, we found that the motility reduction associated with the calcium removal was related to sodium loading, and not to membrane potential depolarization. Mibefradil, a calcium channel blocker, markedly inhibited the Na⁺-dependent depolarization and sodium loading, and also preserved sperm motility. In the absence of calcium, both ATP and cAMP concentrations were decreased by 40%. However ATP levels were unchanged when calcium removal was performed under conditions that inhibit the calcium removal-induced Na⁺-dependent depolarization and [Na⁺]_i increase.

CONCLUSIONS

Human sperm motility arrest induced by external calcium removal is mediated principally by sodium loading, which would stimulate the Na⁺/K⁺-ATPase and in turn deplete the ATP content.

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BACKGROUND

A proportion of women with ‘unexplained’ infertility may present with subfertility because their pregnancies fail before they are clinically recognized. In order to test whether pre-clinical early pregnancy losses (EPL) occur more frequently in women with unexplained infertility, serial urinary hCG concentrations were measured to compare EPL per cycle rates following spontaneous conception in patients with unexplained infertility versus healthy volunteers.

METHODS

Sixty patients under 39 years of age with unexplained infertility and 60 healthy controls, who were trying to conceive spontaneously, participated in this study. All participants were asked to collect daily urine samples from cycle day 14 until menstruation for three consecutive cycles or until a positive pregnancy test was obtained.

Urinary hCG and creatinine levels were measured by immunoassay. Implantation was detected when urinary hCG levels rose above reference levels constructed from samples obtained from 12 women not attempting to conceive. EPL rates were determined by a linear mixed model using logarithmically transformed hCG/creatinine data.

RESULTS

In the 133 cycles of 60 women with unexplained infertility, just one implantation was detected, which became an ongoing pregnancy. In contrast, in 103 such cycles in 46 control patients, 30 implantations were detected (24 clinical pregnancies, 6 cases of EPL). The odds ratio for EPL/cycle in the unexplained versus control group was 0 (95% confidence interval: 0–0.795, P= 0.026).

CONCLUSIONS

Our data do not support the hypothesis that recurrent EPL may present as unexplained infertility. Post-implantation failure is therefore unlikely to contribute significantly to the presentation of subfertility.

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BACKGROUND

Debate exists regarding the effect of raised BMI on the outcome of pregnancies after assisted reproduction technology. We assessed the effect of BMI on the risk of miscarriage in women conceiving following single blastocyst transfer (SBT) after controlling for confounding factors.

METHODS

Fresh and cryo-thawed cycles of SBT that resulted in a pregnancy between January 2006 and March 2010 were included. Patients with BMI < 18.5 kg/m² or older than 40 years were excluded. Patients were grouped according to their BMI at the start of treatment cycle. The main outcome measure was the miscarriage rate before 23 weeks gestation. Confounding variables examined included female age, duration and cause of infertility, previous miscarriage, smoking status and quality of blastocyst replaced.

RESULTS

A total of 413 women conceived following SBT in fresh (n= 325) or cryo-thawed (n= 88) IVF cycles, of whom 244 had a normal BMI (18.5–24.9) and 169 had a raised BMI of ≥25. Overall, 27% (113/413) of women miscarried before 23 weeks gestation. Women with a BMI of ≥25 had more than double the risk of miscarriage compared with women who had normal BMI [38 versus 20%, odds ratio (OR): 2.4, 95% confidence interval (CI) 1.6–3.8, P < 0.001, respectively]. After adjusting for confounding variables, having a BMI of ≥25 significantly increased the risk of clinical miscarriage before 23 weeks gestation in both fresh (adjusted OR = 2.7, 95% CI 1.5–4.9, P= 0.001) and cryo-thawed IVF cycles (OR = 6.8, 95% CI 1.5–31.1, P= 0.012).

CONCLUSIONS

Raised BMI is independently associated with higher miscarriage rate after IVF treatment.

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OBJECTIVE

To evaluate the contribution of referent pathologists (RPs) to the quality of diagnosis of trophoblastic diseases and to study the level of diagnostic agreement between the initial pathologists and the RPs.

METHODS

This observational retrospective study was carried between 1 November 1999 and 11 January 2011 using the database of the French Trophoblastic Disease Reference Centre in Lyon. All files for hydatiform moles (HMs), trophoblastic tumours and non-molar pregnancies for which there was an initial suspicion of trophoblastic disease were included, whenever there was rereading of the slides by an RP. A total of 1851 HMs and 150 gestational trophoblastic tumours were analysed.

RESULTS

When the initial pathologist diagnosed a complete mole, the RP confirmed the diagnosis in 96% of cases. When the initial pathologist diagnosed a partial mole, the RP confirmed the diagnosis in only 64% of cases. For trophoblastic tumours, when the initial pathologist diagnosed a choriocarcinoma, the RP confirmed the diagnosis in 86% of cases. When the initial anatomopathology suggested an invasive mole, the diagnosis was confirmed in 96% of cases. Finally, when the initial diagnosis was a placental site trophoblastic tumour or an epithelioid trophoblastic tumour, the RP confirmed the diagnosis in 60 and 100% of cases, respectively.

CONCLUSION

A systematic policy of rereading of slides for all suspicious moles improves the quality of management of trophoblastic diseases at a national level.

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BACKGROUND

Time-lapse observation presents an opportunity for optimizing embryo selection based on morphological grading as well as providing novel kinetic parameters, which may further improve accurate selection of viable embryos. The objective of this retrospective study was to identify the morphokinetic parameters specific to embryos that were capable of implanting. In order to compare a large number of embryos, with minimal variation in culture conditions, we have used an automatic embryo monitoring system.

METHODS

Using a tri-gas IVF incubator with a built-in camera designed to automatically acquire images at defined time points, we have simultaneously monitored up to 72 individual embryos without removing the embryos from the controlled environment. Images were acquired every 15 min in five different focal planes for at least 64 h for each embryo. We have monitored the development of transferred embryos from 285 couples undergoing their first ICSI cycle. The total number of transferred embryos was 522, of which 247 either failed to implant or fully implanted, with full implantation meaning that all transferred embryos in a treatment implanted.

RESULTS

A detailed retrospective analysis of cleavage times, blastomere size and multinucleation was made for the 247 transferred embryos with either failed or full implantation. We found that several parameters were significantly correlated with subsequent implantation (e.g. time of first and subsequent cleavages as well as the time between cleavages). The most predictive parameters were: (i) time of division to 5 cells, t5 (48.8–56.6 h after ICSI); (ii) time between division to 3 cells and subsequent division to 4 cells, s2 (≤0.76 h) and (iii) duration of cell cycle two, i.e. time between division to 2 cells and division to 3 cells, cc2 (≤11.9 h). We also observed aberrant behavior such as multinucleation at the 4 cell stage, uneven blastomere size at the 2 cell stage and abrupt cell division to three or more cells, which appeared to largely preclude implantation.

CONCLUSIONS

The image acquisition and time-lapse analysis system makes it possible to determine exact timing of embryo cleavages in a clinical setting. We propose a multivariable model based on our findings to classify embryos according to their probability of implantation. The efficacy of this classification will be evaluated in a prospective randomized study that ultimately will determine if implantation rates can be improved by time-lapse analysis.

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BACKGROUND

The aim of this study was to examine the effect of the cryopreservation procedure (slow freezing or vitrification) and cryoprotectants (1,2-propanediol or dimethylsulphoxide) on mouse blastocyst gene expression.

METHODS

Cultured mouse blastocysts were cryopreserved with different protocols. Following thawing/warming, total RNA from re-expanded blastocysts was isolated, amplified and then analyzed using mouse whole-genome microarrays.

RESULTS

Compared with non-cryopresevered control blastocysts, gene expression was only significantly altered by slow freezing. Slow freezing affected the expression of 115 genes (P < 0.05). Of these, 100 genes exhibited down-regulation and 15 genes were up-regulated. Gene ontology revealed that the majority of these genes are involved in protein metabolism, transcription, cell organization, signal transduction, intracellular transport, macromolecule biosynthesis and development. Neither of the vitrification treatment groups showed statistically different gene expression from the non-cryopreserved control embryos. Hierarchical cluster analysis, did however, reveal that vitrification using 1,2-propanediol could result in a gene expression profile closest to that of non-cryopreserved blastocysts.

CONCLUSIONS

Investigating the effects of cryopreservation on cellular biology, such as gene expression, is fundamental to improving techniques and protocols. This study demonstrates that of the cryopreservation regimens employed, slow freezing induced the most changes in gene expression compared with controls.

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BACKGROUND

Prior studies have documented increased risks to the offspring of IVF singletons that result from a vanished twin pregnancy. We aim to investigate the effect on perinatal outcomes of having an early vanished triplet in IVF twins.

METHODS

This is a retrospective cohort study of twins from a large academic IVF practice. Multivariate analysis was performed to examine the perinatal risks—including small for gestational age (SGA), low-birthweight (LBW), preterm delivery and early preterm delivery—in twins that resulted from an early vanished triplet compared with twins without a vanished embryo.

RESULTS

Of 829 IVF twin deliveries, 59 were a result of vanished triplet pregnancies (7.1%). There was no significant increase in SGA, LBW or delivery <37 weeks in the vanished triplets compared with other twins; however, the risk of early preterm birth (<32 weeks) was significantly higher (OR 3.09, 95% CI 1.63–5.87) and the length of gestation of these pregnancies was on average 1.5 weeks shorter (P< 0.01). In addition, the unadjusted mean birthweight was lower by nearly 200 g in the vanished triplet pregnancies (P< 0.01).

CONCLUSIONS

IVF twin pregnancies with a vanished triplet are at an increased risk for early preterm birth compared with other twin pregnancies. These pregnancies should be recognized at higher risk for early preterm birth and considered for increased obstetrical monitoring. A significant limitation of this study is that the cause for preterm birth was unknown.

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BACKGROUND

The aim of this study was to assess the diagnostic accuracy of three-dimensional ultrasound (3D-US) for determining the position of Essure microinserts and the success of sterilization by the Essure method.

METHODS

This retrospective observational study examined the case records of 311 women who underwent hysteroscopic sterilization from October 2002 through October 2008. Imaging with 3D-US or pelvic X-radiography or both was performed 3 months after the procedure to verify device position. Hysterosalpingography (HSG) was performed when a bilateral procedure was not completed because of a history of salpingectomy or blocked tube, when doubt persisted after 3D-US or pelvic radiography, or for comparative purposes in a prospective study. The positions seen on 3D-US were classified in four categories according to a specific scale we devised.

RESULTS

The insertion procedure was completed in 94.2% patients. Only 90.5% underwent imaging verification of the device 3 months afterwards. In all, 227 3D-US, 175 pelvic radiography and 64 HSG imaging procedures were performed. Visualization of the device was possible in 99.6% of the 3D-US images. According to our classification, 3D-US was appropriate for assessing device position for 195 (85.9%) patients. The need for HSG confirmation was significantly lower with 3D-US than radiographic imaging (14.1 versus 26.8%, P = 0.001). 3D-US examinations, compared with the results of HSG as the reference test, had a sensitivity of 100% and a specificity of 76.6%. Neither pregnancy nor early expulsion occurred when 3D-US found that the devices were correctly placed.

CONCLUSIONS

3D-US is a simple technique for assessing the position of Essure^® microinserts, even after concomitant endometrial surgery. The 3D-US classification presented here appears to make it possible to use HSG for back-up confirmation only when the microinsert is found in a very distal position on 3D-US and thus to protect the majority of women from the negative effects of pelvic radiography and HSG.

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BACKGROUND

The recommended time interval between mifepristone and misoprostol in medical second trimester termination of pregnancy (TOP) has been 36–48 h. However, a more flexible interval would be of value. The aim of this investigation was to compare one- and two-day intervals in second trimester medical TOP. The main outcome measures were induction-to-abortion time and the rate of surgical evacuation.

METHODS

This open randomized trial included 227 women undergoing TOP between gestational weeks 13–24. Mifepristone (200 mg) was followed by misoprostol (400 mcg) after one (17–28 h) or two (41– 45 h) days.

RESULTS

In intention-to-treat analysis, the median induction-to-abortion interval was 1h longer in the one-day group (8.5 versus 7.2 h, P= 0.038), but in per-protocol analysis, the rate of surgical evacuation was higher in the 2-day group [30/115 (25%) versus 40/112 (37%); 95% confidence interval 0.3–24.1, P= 0.044]. A subgroup analysis showed that the median induction-to-abortion interval was 3h longer in the one-day group, amongst women without previous vaginal deliveries (10.1 versus 7.6, P= 0.013) and when gestation exceeded 16 weeks (10.8 versus 7.2, P= 0.024).

CONCLUSIONS

Both one- and two-day dosing intervals seem to be suitable for second trimester medical TOP, but women with no previous deliveries and those whose gestation exceeds 16 weeks may benefit from the longer interval. However, evaluated on the basis of surgical evacuation, the one-day interval could be supported as an option for second trimester medical TOP. Effective use of both one- and two-day dosing intervals is important when optimizing clinical service.

Trial Registration: ISRCTN09944151.

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BACKGROUND

This double-blind trial investigated the efficacy and safety of estradiol valerate/dienogest (E₂V/DNG) for the treatment of heavy menstrual bleeding without recognizable organic pathology.

METHODS

Otherwise healthy women with idiopathic heavy, prolonged or frequent menstrual bleeding, confirmed during a 90-day run-in phase, were randomized (2:1) according to a permuted-block, computer-generated schedule to E₂V/DNG or placebo for 196 days at 34 centres in Europe and Australia. The primary efficacy end-point was the proportion of women with a ‘complete’ response (i.e. a return to ‘menstrual normality’) during a 90-day efficacy phase. Secondary end-points included changes in measured menstrual blood loss (MBL) and iron metabolism parameters.

RESULTS

The intention-to-treat population comprised 231 women. The E₂V/DNG response rate was much higher than with placebo (P < 0.0001). The mean reduction in MBL volume in E₂V/DNG recipients was 69.4% (median 79.2%) versus 5.8% (median 7.4%) in placebo recipients. The between-treatment difference in MBL volume was 373 ml in favour of E₂V/DNG (95% confidence interval 490, 255 ml; P < 0.0001). Significant improvements in iron metabolism parameters were observed with E₂V/DNG but not placebo. Overall, 14 women (9.7%) treated with E₂V/DNG and 5 (6.2%) treated with placebo prematurely discontinued treatment because of adverse events, headache being the most prevalent. Serious adverse events occurred in both the E₂V/DNG and placebo groups (each n = 2).

CONCLUSIONS

E₂V/DNG is an effective treatment in women with heavy and/or prolonged menstrual bleeding without organic pathology. Further study of E₂V/DNG compared with an active comparator is warranted.

ClinicalTrials.gov identifier: NCT00307801.

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BACKGROUND

In patients diagnosed with deep infiltrating endometriosis (DIE), foci of endometriosis are detected in mesorectal lymph nodes (LNs) after segmental bowel resection and in pelvic sentinel LNs. Lymph vessels (LVs) seem to be the possible routes for the dissemination of endometriotic cells from DIE-lesions to LN. Therefore, we conducted a study to investigate the occurrence and density of LV and lymphangiogenic growth factors in DIE.

METHODS

Included in this study were 38 premenopausal women who underwent surgery due to symptomatic rectovaginal DIE. In order to identify LV, immunohistochemical analysis with anti-Podoplanin (D2-40), LYVE-1 and Prox-1 was performed. Furthermore, the expression of VEGF-C and VEGF-D in endometriotic tissue was investigated.

RESULTS

LV density (LVD) of DIE lesions was significantly higher compared with healthy corresponding tissue. All LV makers could be detected, and the density of LYVE-1- or Prox-1-positive LV was significantly higher than that of D2-40-positive LV. Endometriotic epithelial cells and stromal cells showed a moderate to strong VEGF-C and VEDF-D expression.

CONCLUSIONS

DIE lesions have lymphangiogenic properties, probably leading to endometriosis-like cells in lymphatic vessels and LNs featuring a loco-regional disease.

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BACKGROUND

Smooth muscle cells (SMC) are common components of endometriotic lesions. SMC have been characterized previously in peritoneal, ovarian and deep infiltrating endometriotic lesions and adenomyosis. The aim of this retrospective study was to investigate the extent of differentiation in endometriosis-associated SMC (EMaSMC) in peritoneal endometriotic lesions.

METHODS

We obtained biopsies from peritoneal endometriotic lesions (n= 60) and peritoneal sites distant from the endometriotic lesion (n= 60), as well as healthy peritoneum from patients without endometriosis (control tissue, n= 10). These controls were hysterectomy specimens from patients without endometriosis or adenomyosis. Histopathological examination of peritoneal specimens using antibodies against oxytocin receptor (OTR), vasopressin receptor (VPR), smooth muscle myosin heavy chain (SM-MHC), estrogen receptor (ER) or progesterone receptor (PR) was performed. To identify SMC and their level of differentiation, antibodies for smooth muscle actin desmin and caldesmon were used.

RESULTS

SMC were detected in all endometriotic lesions. SMC were more abundant in unaffected peritoneum of women with endometriosis (38%) compared with women without endometriosis (6%; P < 0.0001). Depending on the level of differentiation, SMC stained for SM-MHC, OTR, VPR, ER and PR. OTR was only detected in fully differentiated SMC.

CONCLUSIONS

Identification of OTR, VPR, ER and PR leads to the hypothesis that the EMaSMC might be functionally active and possibly involved in the generation of pain associated with endometriosis.

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BACKGROUND

The roles of cell proliferation and genomic instability in endometriosis are highly debated aspects of the pathogenesis of this disease. Aurora A and B kinases play different important roles in cell cycle control and genomic instability and have never been studied in endometriosis. The aim of this study was to compare the expression levels of Aurora kinases, Ki-67 and hormone receptor in endometriotic tissue (ET) and normal endometrium.

METHODS

We retrospectively analysed 438 samples obtained from 194 patients affected by endometriosis and 28 samples from 28 patients with normal endometrium, which were all collected by the Pathology Department and Gynecologic Clinic of the University Hospital of Udine. A tissue microarray model was constructed to use immunohistochemistry to analyse the expression of Aurora A and B kinases, Ki-67 and the estrogen and progesterone receptors in ET and normal endometrium.

RESULTS

Aurora A and B kinases were expressed at a very low level in the majority of endometriosis core biopsies. Aurora A and B kinases, Ki-67 and the estrogen and progesterone receptors were expressed at a higher level in the proliferative endometrium than in the secretory endometrium and in ovarian and non-ovarian ET (P< 0.05). Additionally, Aurora B kinase, Ki-67 and the estrogen and progesterone receptors were more highly expressed in non-ovarian than ovarian ET (P< 0.05).

CONCLUSIONS

Considering the low expression levels of Aurora A and B kinases in the majority of endometriosis core biopsies, the growth and survival of endometrial tissue outside the uterus cannot be explained by deregulation of this pathway. The analysed ectopic endometrium protein expression pattern resembled that of the secretory endometrium, and markers of proliferation and hormone receptors were expressed at lower levels in ovarian than in non-ovarian ET. The low level of hormone receptors and the consequent low levels of proliferation markers in ovarian ETs may be due to down-regulation by the ovary's hormone milieu.

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BACKGROUND

In view of the discrepancies about the GnRH antagonist (GnRH-ant) ovarian stimulation protocols having some potential advantages compared with the GnRH agonist (GnRH-a) protocols in poor ovarian responders IVF/ICSI, a meta-analysis of the published data was performed to compare the efficacy of GnRH-ant versus GnRH-a protocols for ovarian stimulation in IVF poor response patients.

METHODS

We searched for all published articles indexed in MEDLINE (1950–2010), EMBASE (1974–2010) and China National Knowledge Infrastructure (CNKI, 1994–2010). Any randomized controlled study that compared the GnRH-ant with GnRH-a in ovarian stimulation protocols for poor responders undergoing IVF/ICSI was included, and data were extracted independently by two reviewers. The searches yielded 64 articles, from which 14 studies met the inclusion criteria. We performed this meta-analysis involving 566 IVF patients in a GnRH-ant protocol group and 561 patients in a GnRH-a protocol group with Review Manager 4.2 software. Odds ratio (OR) and weighted mean difference (WMD) with 95% confidence intervals (CIs) were used to evaluate dichotomous and continuous data, respectively.

RESULTS

Fourteen eligible studies were included in this meta-analysis. GnRH-ant protocols resulted in a statistically significantly lower duration of stimulation compared with GnRH-a protocols (P = 0.04; WMD: –1.88, 95% CI: –3.64, –0.12), but there was no significant difference in the number of oocytes retrieved (P = 0.51; WMD: –0.17, 95% CI –0.69, 0.34) or the number of mature oocytes retrieved (P = 0.99; WMD: –0.01, 95% CI: –1.14, 1.12). Moreover, no significant difference was found in the cycle cancellation rate (CCR, P = 0.67; OR: 1.01, 95% CI: 0.71–1.42) or clinical pregnancy rate (CPR, P = 0.16; OR: 1.23, 95% CI: 0.92, 1.66).

CONCLUSIONS

Clear advantage was gained in duration of stimulation with GnRH-ant in poor ovarian responders undergoing IVF, although there was no statistical difference in the number of oocytes retrieved, the number of mature oocytes retrieved, the CCR and CPR between GnRH-ant and GnRH-a protocols. These results may be helpful to our clinical practice. However, further controlled randomized prospective studies with larger sample sizes are needed.

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