Ovarian reserve is determined by the number of primordial follicles in the ovary. Quiescent primordial follicles are activated for growth and pass through stages of development before they reach the antral stage. Then a cohort of antral follicles is recruited for further growth, dominance and ovulation under the cyclic stimulation of gonadotrophins. What triggers the initiation of growth in primordial follicles has remained a mystery for decades. However, recent studies on mutant mouse models have shown that primordial follicles are maintained in a dormant state by the actions of various inhibitory molecules to preserve the follicle pool, such as the transcription factor Foxo3a, PTEN (phosphotase and tensin homolog deleted on chromosome 10) and Tsc-1 (tumour suppressor tuberous schlerosis complex). Mice with deletions of these oocyte-specific genes exhibit premature activation of dormant primordial follicles, and all primordial follicles become depleted in early adulthood, causing premature ovarian failure. Other oocyte and somatic cell-derived growth factors are also involved in the early, gonadotrophin-independent phase of follicle growth via autocrine and paracrine interactions. Interestingly, some of these factors also play critical roles at later stages of follicle growth, such as the process of selecting the dominant follicle, by modifying the response of the follicles to gonadotrophins and inhibiting premature luteinization. Therefore, a thorough understanding of the molecular aspects of folliculogenesis is of paramount importance in the context of translational medicine and future clinical applications in human reproduction.

BACKGROUND

The Piwi subfamily of genes is involved in spermatogenesis for the maintenance and meiosis of germline stem cells. Mice bearing targeted mutations in Piwi genes (Miwi, Mili and Miwi2) are sterile with distinct defects in spermatogenesis. We hypothesized that Piwi gene polymorphisms could be a risk factor for spermatogenic failure.

METHODS

For this study, 490 patients with idiopathic azoospermia or oligozoospermia and 468 fertile controls were recruited from an infertility clinic. Nine single nucleotide polymorphisms (SNPs) of four Piwi genes (PIWIL1/HIWI, PIWIL2/HILI, PIWIL3/HIWI3 and PIWIL4/HIWI2) were genotyped using the SNPstream^® 12-plex platform and the Taqman method.

RESULTS

An SNP in the 3'untranslated region of HIWI2 and a non-synonymous SNP in HIWI3 were significantly associated with an altered risk of oligozoospermia. The variant-containing genotypes of HIWI2 rs508485 exhibited a significantly increased risk, with an odds ratios (OR) of 1.49 [95% confidence interval (CI), 1.02–2.18], and individuals with HIWI3 non-synonymous rs11703684 variant genotypes exhibited a significantly reduced oligozoospermia risk (OR = 0.70; 95% CI, 0.49–1.00). The haplotype analysis showed that a common haplotype of HIWI2 was associated with a significant reduction in the risk of oligozoospermia (OR = 0.73, 95% CI, 0.56–0.97). In addition, to assess the cumulative effects, we performed a combined unfavourable genotype analysis. A significant trend towards increased risk of oligozoospermia with an increasing number of unfavourable genotypes was observed (P for trend < 0.001).

CONCLUSIONS

We present the first epidemiologic evidence supporting the involvement of genetic polymorphisms in Piwi genes in spermatogenic failure.

BACKGROUND

Transforming growth factor-β cytokines have various biological effects in female reproductive tissue, including modulation of inflammatory response and induction of immune tolerance to seminal antigens in the reproductive tract. However, no studies have analyzed the presence of growth/differentiation factor-15 (GDF-15/macrophage inhibitory cytokine-1) in seminal fluid or demonstrated the quantity and form of GDF-15, its possible role or the relationship between its concentration and semen quality.

METHODS

The form and the concentration of GDF-15 were determined in 53 seminal plasma samples of both fertile and infertile men by ELISA and western blot. The sperm cells of three volunteers were treated with recombinant GDF-15, and cell viability and apoptosis were assessed by flow cytometry. The effect of GDF-15 on vaginal epithelial cells and peripheral blood mononuclear cells (PBMCs) was analyzed by quantitative RT-PCR.

RESULTS

The GDF-15 concentration in seminal plasma ranged from 0.2 to 6.6 µg/ml as determined by ELISA. Western blot analysis revealed that GDF-15 is present in the active form. In vitro cultivation of sperm cells with GDF-15 did not affect their viability or rates of apoptosis; however, it did inhibit proliferation of PBMCs and induce expression of FOXP3 in CD4+CD25+ cells.

CONCLUSIONS

To the best of our knowledge, this is the first demonstration that GDF-15 is an abundant cytokine in seminal plasma, although its concentration is not associated with semen quality or the fertility/infertility status of the donors. Moreover, our data show that GDF-15 displays immunosuppressive characteristics.

BACKGROUND

Low-dose aspirin therapy could improve remodelling of maternal spiral arteries during early placentation and prevent subsequent pregnancy-related hypertensive disorders. We investigated whether low-dose aspirin therapy reduces the incidence of hypertensive pregnancy complications in unselected IVF and ICSI patients when medication was started prior to pregnancy.

METHODS

A total of 487 patients who underwent IVF/ICSI were randomized to receive 100 mg aspirin (n = 242) or placebo (n = 245) daily, starting on the first day of gonadotrophin stimulation. Pregnant women continued the medication until delivery. A total of 107 patients (52 with aspirin and 55 with placebo) experienced live birth and were included in this follow-up study. The main outcome measure was the incidence of hypertensive pregnancy complications.

RESULTS

Embryo transfer took place in 227 (94%) women in the aspirin group and in 229 (93%) women in the placebo group. The live birth rate between the aspirin (22.9%) and placebo (24.0%) groups did not differ significantly (P = 0.78). The overall incidence of hypertensive pregnancy complications was 15.4% (8/52) in the aspirin group and 18.2% (10/55) in the placebo group (P = 0.70, 95% confidence interval for the difference of proportions –17 to 11%). There were two cases of severe pre-eclampsia in the aspirin group and three cases in the placebo group.

CONCLUSIONS

In the present study, the incidence of hypertensive pregnancy complications did not differ statistically significantly between low-dose aspirin and placebo groups in unselected IVF/ICSI patients, when medication was started concomitantly with gonadotrophin stimulation and continued until delivery.

The study was registered at clinicaltrials.gov. NCT00683202.

BACKGROUND

The association among hereditary thrombophilia, recurrent pregnancy loss (RPL) and obstetric complications is yet uncertain. The objective of the study was to assess the prognostic value of the factor V Leiden (FVL) and prothrombin (PT) mutations for the subsequent chance of live birth for women with RPL.

METHODS

Pregnancy outcome was recorded in a retrospective cohort of 363 women with a minimum of three consecutive pregnancy losses (early miscarriage, late miscarriage or stillbirth/neonatal death) who were not treated with anticoagulation therapy.

RESULTS

Of the 363 women, 29 were FVL-mutation carriers and 6 were PT-mutation carriers. The unadjusted live birth rate was 45.7% in FVL/PT carriers versus 63.4% in FVL/PT non-carriers, P = 0.04. The adjusted odds ratio for live birth in FVL/PT carriers was 0.48 (95% CI = 0.23–1.01), P = 0.05. Among the obstetric complications, only excessive bleeding was found to be associated with FVL/PT mutations.

CONCLUSIONS

In the unadjusted analysis, FVL and PT mutations have a negative prognostic impact on the live birth rate in women with RPL; however, when adjusting for significant covariates, the results no longer reach statistical significance. Strong conclusions on the association between obstetric complications and hereditary thrombophilia cannot be drawn from this study. Whether anticoagulation therapy would improve the prognosis in women with RPL and FVL/PT mutations remains to be documented in large randomized controlled trials.

BACKGROUND

The retinoic acid metabolizing enzyme Cyp26a1 plays a pivotal role in vertebrate embryo development. Cyp26a1 was characterized previously as a differentially expressed gene in peri-implantation rat uteri via suppressive subtracted hybridization analysis. However, the role of Cyp26a1 in rat embryo implantation remained elusive.

METHODS

The expression of Cyp26a1 in the uteri of early pregnancy, pseudopregnancy and artificial decidualization was detected by northern blotting, real time-PCR, in situ hybridization, western blotting and immunofluorescent staining. The effect of Cyp26a1 on apoptosis of endometrial stromal cells (ESCs) isolated from rat uteri was determined by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) and Hoechst staining. Apoptosis-related proteins in ESCs were detected by western blotting.

RESULTS

Cyp26a1 showed distinctive expression patterns in embryos and uteri during the peri-implantation period, with a remarkable increase (P < 0.01 versus Days 4–5) in mRNA and protein in the implantation phase (Days 5.5–6.5 of pregnancy). CYP26A1 was specifically localized in glandular epithelium, luminal epithelium and decidua basalis. The level of CYP26A1 protein was significantly increased in uteri of artificial decidualization (P < 0.01 versus control). Forced Cyp26a1 overexpression significantly reduced the sensitivity of ESCs to etoposide-induced apoptosis, with reductions in p53 (P < 0.01) and Fas (P < 0.05) proteins versus control, while in contrast, FasL (P < 0.01) and proliferating cell nuclear antigen (P < 0.05) proteins increased.

CONCLUSIONS

Cyp26a1 is spatiotemporally expressed in the uterus during embryo implantation and decidualization. Overexpression of Cyp26a1 attenuates the process of uterine stromal cell apoptosis, probably via down-regulating the expression of p53 and FasL.

BACKGROUND

Oocyte in vitro maturation (IVM) reduces the need for gonadotrophin-induced ovarian hyperstimulation and its associated health risks but the unacceptably low conception/pregnancy rates have limited its clinical uptake. We report the development of a novel in vitro simulated physiological oocyte maturation (SPOM) system.

METHODS AND RESULTS

Bovine or mouse cumulus–oocyte complexes (COCs) were treated with cAMP modulators for the first 1–2 h in vitro (pre-IVM), increasing COC cAMP levels ~100-fold. To maintain oocyte cAMP levels and prevent precocious oocyte maturation, COCs were treated during IVM with an oocyte-specific phosphodiesterase inhibitor and simultaneously induced to mature with FSH. Using SPOM, the pre-IVM and IVM treatments synergized to increase bovine COC gap-junctional communication and slow meiotic progression (both P < 0.05 versus control), extending the normal IVM interval by 6 h in bovine and 4 h in mouse. FSH was required to complete maturation and this required epidermal growth factor signalling. These effects on COC had profound consequences for oocyte developmental potential. In serum-free conditions, SPOM increased bovine blastocyst yield (69 versus 27%) and improved blastocyst quality (184 versus 132 blastomeres; both P < 0.05 versus standard IVM). In mice, SPOM increased (all P < 0.05) blastocyst rate (86 versus 55%; SPOM versus control), implantation rate (53 versus 28%), fetal yield (26 versus 8%) and fetal weight (0.9 versus 0.5 g) to levels matching those of in vivo matured oocytes (conventional IVF).

CONCLUSIONS

SPOM is a new approach to IVM, mimicing some characteristics of oocyte maturation in vivo and substantially improving oocyte developmental outcomes. Adaption of SPOM for clinical application should have significant implications for infertility management and bring important benefits to patients.

BACKGROUND

In vitro culture (IVC) and IVF of preimplantation mouse embryos are associated with changes in gene expression. It is however not known whether ICSI has additional effects on the transcriptome of mouse blastocysts.

METHODS

We compared gene expression and development of mouse blastocysts produced by ICSI and cultured in Whitten's medium (ICSI_WM) or KSOM medium with amino acids (ICSI_KSOMaa) with control blastocysts flushed out of the uterus on post coital Day 3.5 (in vivo). In addition, we compared gene expression in embryos generated by IVF or ICSI using WM. Global pattern of gene expression was assessed using the Affymetrix 430 2.0 chip.

RESULTS

Blastocysts from ICSI fertilization have a reduction in the number of trophoblastic and inner cell mass cells compared with embryos generated in vivo. Approximately 1000 genes are differentially expressed between ICSI blastocyst and in vivo blastocysts; proliferation, apoptosis and morphogenetic pathways are the most common pathways altered after IVC. Unexpectedly, expression of only 41 genes was significantly different between embryo cultured in suboptimal conditions (WM) or optimal conditions (KSOM_aa).

CONCLUSIONS

Our results suggest that fertilization by ICSI may play a more important role in shaping the transcriptome of the developing mouse embryo than the culture media used.

BACKGROUND

Cryopreservation of follicles for culture and oocyte growth and maturation in vitro provides an option to increase the number of fertilizable oocytes and restore fertility in cases where transplantation of ovarian tissue poses a risk for malignant cell contamination. Vitrification for cryopreservation is fast and avoids ice crystal formation. However, the influences of exposure to high concentrations of cryoprotectants on follicle development, oocyte growth and maturation, and particularly, on the DNA integrity and methylation imprinting has not been studied systematically.

METHODS

Follicle survival and development, DNA damage, oocyte growth patterns, maturation, spindle formation and chromosomal constitution were studied after Cryo-Top vitrification of mouse pre-antral follicles cultured to the antral stage and induced to ovulate in vitro. Methylation of differentially methylated regions (DMRs) of two maternally (Snrpn and Igf2r) and one paternally (H19) imprinted genes was studied by bisulfite pyrosequencing.

RESULTS

Vitrification results in partial or total loss of oocyte–granulosa cell apposition and actin-rich transzonal projections, a transient increase in DNA breaks and a delay in follicle development. However, the oocyte growth pattern, maturation, spindle and chromosomal constitution are not significantly different between the vitrified and the control groups. Vitrification is not associated with elevated levels of imprinting mutations (aberrant methylation of the entire DMR), although the distribution of sporadic CpG methylation errors in the Snrpn DMR appears to differ slightly between control and vitrified oocytes.

CONCLUSIONS

DNA breaks appear to be rapidly repaired and vitrification of oocytes inside pre-antral follicles by the Cryo-Top method does not appear to increase risks of abnormal imprinting or disturbances in spindle formation and chromosome segregation.

BACKGROUND

Non-steroidal anti-inflammatory drug (NSAID)-activated gene-1 (NAG-1) is involved in cellular processes such as inflammation, apoptosis and tumorigenesis. However, little is known about the expression and function of NAG-1 in the endometrium. This study aimed to evaluate the expression of NAG-1 in the endometrium and in the absence or presence of endometriosis and to investigate the effect of celecoxib, a selective cyclooxygenase (COX)-2 inhibitor, on NAG-1 mRNA levels and apoptosis in human endometrial stromal cells (HESCs).

METHODS

Eutopic endometrial samples were obtained during surgery from 40 patients with, and 40 patients without, endometriosis. Real-time PCR was used to quantify NAG-1 mRNA levels and immunohistochemistry was used to localize NAG-1 protein in the endometrium. To investigate the effects of celecoxib, HESCs were isolated and cultured with different concentrations of celecoxib or with 100 µM celecoxib at different times. Apoptosis was assessed by flow cytometry.

RESULTS

NAG-1 mRNA levels and immunoreactivity showed cyclical changes through the menstrual cycle, increasing during the late secretory and menstrual phases. NAG-1 mRNA and protein levels were significantly lower in patients with endometriosis, compared with the control group. Celecoxib induced NAG-1 mRNA levels and apoptosis in cultured HESCs, with the effects dependent on drug concentrations and duration of treatment. Celecoxib treatment had no effect on prostaglandin E₂ levels in the culture supernatants.

CONCLUSIONS

NAG-1 may be important in maintaining homeostasis in the normal endometrium and alterations in NAG-1 expression may be associated with the establishment of endometriosis. NAG-1 might be a therapeutic target for endometriosis.

BACKGROUND

Currently, no standard post-operative medical therapy has been shown to be superior in reducing the recurrence of endometrioma. This retrospective study was performed to evaluate the efficacy of post-operative cyclic oral contraceptive (OC) use after GnRH agonist (GnRHa) treatment for the prevention of endometrioma recurrence.

METHODS

This retrospective study included 362 reproductive-aged patients who had undergone pelviscopic surgery for endometrioma. Patients were divided into two groups: a group that received cyclic, low-dose, monophasic OCs after GnRHa treatment (n = 175) and a group that received only GnRHa treatment (n = 187). Endometrioma recurrence was evaluated by an ultrasound.

RESULTS

During the follow-up period (median, 35 months; range, 12–114 months), recurrent endometriomas were detected in 67 patients (18.5%). Patients receiving cyclic OCs after GnRHa treatment showed a significantly reduced recurrence risk of endometrioma, when compared with patients receiving GnRHa alone (odds ratio = 0.20; 95% confidence interval, 0.10–0.38). The cumulative proportion of recurrent endometrioma after 60 months was significantly lower in OC users than in non-users (6.1 versus 43.3%; P < 0.001), and a statistically significant difference was first detected at 30 months of follow-up (5.1 versus 17.1%; P = 0.018). The number of GnRHa injections (3 versus 6) did not affect the recurrence rate for up to 60 months in OC non-users or users.

CONCLUSIONS

Post-operative cyclic OC use after GnRHa treatment effectively reduces the recurrence of endometrioma.

BACKGROUND

This paper concerns the requirements of the EU Tissue and Cells Directives with regard to the biological screening of donors of reproductive cells which are to be used for partner donation.

METHODS

We review the evidence regarding the risks of transmission of blood-borne viruses [hepatitis B (HBV), hepatitis C (HCV) and human immunodeficiency virus (HIV)] in the assisted reproductive technology (ART) setting. We document the experience in seven Irish ART clinics since the introduction of the legislation.

RESULTS

Even among those known to be HBV-, HCV- or HIV-positive, when current best practice ART procedures are employed for gamete and embryo processing, cross-contamination in the ART facility or horizontal or vertical transmission to a partner or neonate has never been documented. When samples are processed and high-security straws are used for cryopreservation, transmission of virus and cross-contamination in storage have not been reported.

CONCLUSIONS

While initial screening of those about to embark on ART treatment is good practice, we can find no medical or scientific evidence to support re-screening prior to each treatment cycle for individuals undergoing partner donation in ART. It would seem more appropriate to focus on risk reduction using a combination of initial baseline screening (with a reduced frequency of re-testing), appropriate sample processing and best possible containment systems for cryostorage.

BACKGROUND

Monitoring assisted reproductive technology (ART) is essential to evaluate the performance of fertility treatment and its impact on birth rates. In Europe, there are two kinds of ART registers: voluntary and mandatory. The validity of register data is very important with respect to the quality of register-based observational studies. The aim of this paper is to determine the degree of agreement between voluntary and mandatory ART registers.

METHODS

The two sources for the data compared in this study (referring to 2005 and 2006) were FIVCAT.NET (an official compulsory Assisted Reproduction Registry within the Health Ministry of the Regional Government of Catalonia, to which all authorized clinics, both public and private, performing assisted reproduction in the region are obliged to report) and the register of the Spanish Fertility Society (SEF), to which data are provided on a voluntary basis. The SEF register data were divided into two groups: (i) data from clinics in Catalonia (SEF-CAT); (ii) data from the rest of Spain, excluding Catalonia (SEF-wCAT). The techniques compared were IVF cycle using patients’ own eggs (IVF cycle) versus donor egg cycles.

RESULTS

For IVF cycles, the voluntary ART register reflected 77.2% of those on the official one, but the corresponding figure was only 34.4% with respect to donated eggs. The variables analysed in the IVF cycle (insemination technique used, patients’ age, number of embryos transferred, pregnancy rates, multiple pregnancies and deliveries) were similar in the three groups studied. However, we observed significant differences in donor egg cycles with regard to the insemination technique used, pregnancy rates and multiple pregnancies between the voluntary and the official register.

CONCLUSIONS

Data from the voluntary ART register for IVF cycles are valid, but those for donor egg cycles are not. Further study is necessary to determine the reasons for this difference.

BACKGROUND

So far, research on the patients’ perspective on fertility care has mainly focused on women. Our primary aim was to explore what is important to men with respect to care related to testicular sperm extraction (TESE) and to identify strengths and weaknesses of that care.

METHODS

This was a mixed-method study including phenomenology on interviews with 17 ‘interview participants’ (a purposive sample with diversification for the TESE result) who received a TESE treatment at a tertiary university clinic. Strengths and weaknesses of our TESE-related quality of care were identified. Additionally, a telephone questionnaire was answered by 15 ‘rating participants’ not willing to be interviewed and the questionnaire was analyzed quantitatively.

RESULTS

Interview participants wanted more than effective treatment and attached importance to the attitude of fertility clinic staff, information, time flow, personalized care, ‘all that is necessary’, coaching, a homely atmosphere, continuity, privacy and separate accommodation. The satisfaction of rating participants (independent of the TESE result) was problematic for ‘overall experience’, ‘physician at the day clinic’ and ‘gynecologist at discussion of the result’. The attitude of fertility clinic staff and information were the most obvious strengths of our TESE-related care. Weaknesses were lack of practical information on post-surgical recovery and waiting times in the waiting room.

CONCLUSIONS

TESE patients focus not only on clinical effectiveness but also on patient-centeredness of care, and this has led to organizational changes and a new patient information brochure in our center. Qualitative research is useful to examine, understand and improve the patient-centeredness of care.