Cryopreservation of ovarian tissue for fertility preservation is based on the ovarian cortex that contains the vast majority of the follicular reserve, while the remaining tissue, the medulla is discarded. The present study describes the development of a gentle method for isolating pre-antral follicles from human ovarian medulla and evaluating its follicular content.
METHODS
Medulla was collected from 40 girls/women aged 3–35 years undergoing cryopreservation of the ovarian cortex. Follicle density was assessed for all patients and pre-antral follicles were isolated from 22 patients. On the basis of the neutral red (NR) staining of follicles and enzymatic digestion with a mixture of Collagenase IV and Liberase Thermolysin Medium, viable pre-antral follicles were isolated.
RESULTS
NR accumulated in follicles resulting in a distinct red staining within the medulla. Follicle density of the medulla varied from 0 to 9824 follicles/gram of medulla and was significantly higher (P< 0.001) in the 3–9-year age group when compared with older groups (10–35 years). Enzymatic digestion combined with follicle identification by NR yielded a high output of isolated and viable pre-antral follicles from medulla, of which, 3607 follicles were collected and classified. The percentage of primordial and growing follicles decreased and increased, respectively, with age (P< 0.0001 and <0.0007).
CONCLUSIONS
Discarded medulla contained a considerable pool of pre-antral follicles, especially in young girls. Our new method allowed the isolation of viable pre-antral follicles from human ovarian medulla and provides a unique opportunity for basic scientific studies and for culture and grafting purposes.
PGD has been described in previous cross-sectional and retrospective studies as a stressful experience. No prospective studies of the psychological impact of PGD are currently available.
METHODS
Using a prospective study design, validated measures exploring anxiety and depression were used to assess women using PGD prior to treatment, following embryo transfer, following the pregnancy test result and at 24 weeks of pregnancy. Maternal-fetal attachment was also assessed during pregnancy.
RESULTS
The prospective design revealed the cyclical pathway through PGD for many women, often comprising repeated cycles of ovarian stimulations and IVF and frozen embryo transfers. As predicted, there were significant fluctuations in women's anxiety scores, with increases observed following embryo transfer and pregnancy testing. Women's anxiety scores returned to baseline levels during pregnancy as assessed at 24 weeks gestation. Depression scores did not significantly fluctuate during PGD. Maternal-fetal attachment scores in this sample did not differ from the normative Australian data.
CONCLUSIONS
For some women, the PGD pathway is convoluted and requires multiple IVF cycles and embryo transfers to achieve pregnancy. A subset of women experience significant emotional burden during PGD treatment, and it is these women who require closer attention and support. In this sample, emotional adjustment in pregnancy following PGD appears to be sound.
Hepatitis C virus (HCV) carriers are often accepted into the assisted reproduction technique programme of fertility centres. Studies showed that HCV RNA was detected in the follicular fluid of HCV PCR positive females. The objective of this study was to assess the impact of HCV active on the outcome of ICSI.
METHODS
This study was conducted on 40 women who proved to be positive for HCV, using RT–PCR. Two control groups (both n = 40), who were negative for HCV by PCR were also included. The first control group was HCV sero-positive and the second was HCV sero-negative. We compared the three groups regarding the ovarian response to stimulation, embryo quality and pregnancy rates.
RESULTS
The number of failed cycles (lack of ovarian response to stimulation) was higher in HCV RT–PCR positive and sero-positive females than sero-negative controls (P = 0.0001). There were no differences in embryo cleavage or morphology between the study and control groups. The pregnancy rate was significantly reduced in the HCV–PCR-positive group compared with the PCR negative/HCV sero-positive and HCV sero-negative control groups (5, 3 and 48%, respectively; P = 0.001). There was a negative correlation between number of oocytes and viral load (0.419; P = 0.007).
CONCLUSIONS
Our results suggest that HCV infection in females undergoing ICSI has a negative impact on the outcome, and the impact is higher in PCR positive cases: this might be attributed to hormonal disturbance associated with viral liver cirrhosis coinciding with active viral replication.
Anti-Mullerian hormone (AMH) is a marker of ovarian reserve status and represents a good predictor of ovarian response to ovarian hyperstimulation. The aim of this study was to assess the accuracy of AMH and antral follicle count (AFC) as predictors of an excessive response in IVF/ICSI treatment.
METHODS
A systematic review and meta-analysis of the existing literature was performed. Studies were included if 2 x 2 tables for the outcome excessive response in IVF patients in relation to AMH/AFC could be constructed. Using a bivariate meta-analytic model, both summary point estimates for sensitivity and specificity were calculated, as well as summary ROC curves. Clinical value was analysed by calculating post-test probabilities of excessive response at optimal cut-off levels, as well as the corresponding abnormal test rates.
RESULTS
Nine studies reporting on AMH and five reporting on AFC were found. Summary estimates of sensitivity and specificity for AMH were 82 and 76%, respectively, and 82 and 80%, respectively, for AFC. Comparison of the summary estimates and ROC curves for AMH and AFC showed no statistical difference. Abnormal test rates for AMH and AFC amounted to ~14 and 16%, respectively, at cut-off levels where test performance is optimal [likelihood ratio for a positive result (LR + )>8], with a post-test probability of ±70%.
CONCLUSIONS
Both AMH and AFC are accurate predictors of excessive response to ovarian hyperstimulation. Moreover, both tests appear to have clinical value. This opens ways to explore the potential of individualized FSH dose regimens based on ovarian reserve testing.
Non-invasive selection of developmentally competent human oocytes may increase the overall efficiency of human assisted reproduction and is regarded as crucial in countries where legal, social or religious factors restrict the production of supernumerary embryos. The purpose of this study was to summarize the predictive value for IVF success of morphological features of the oocyte that can be obtained by light or polarized microscopic investigations.
METHODS
Studies about oocyte morphology and IVF/ICSI outcomes were identified by using a systematic literature search.
RESULTS
Fifty relevant articles were identified: 33 analysed a single feature, 9 observed multiple features and investigated the effect of these features individually, 8 summarized the effect of individual features. Investigated structures were the following: meiotic spindle (15 papers), zona pellucida (15 papers), vacuoles or refractile bodies (14 papers), polar body shape (12 papers), oocyte shape (10 papers), dark cytoplasm or diffuse granulation (12 papers), perivitelline space (11 papers), central cytoplasmic granulation (8 papers), cumulus–oocyte complex (6 papers) and cytoplasm viscosity and membrane resistance characteristics (2 papers). None of these features were unanimously evaluated to have prognostic value for further developmental competence of oocytes.
CONCLUSIONS
No clear tendency in recent publications to a general increase in predictive value of morphological features was found. These contradicting data underline the importance of more intensive and coordinated research to reach a consensus and fully exploit the predictive potential of morphological examination of human oocytes.
Despite extensive research, the pathogenesis of polycystic ovary syndrome (PCOS) remains unclear. Putatively, an elevated circulating concentration of insulin inhibits the production of insulin-like growth factor binding protein-1 (IGFBP-1), thus increasing the level of free IGF-I in serum and stimulating ovarian androgen production. Decreased IGFBP-1 has been reported in PCOS and in obesity; however, there are inconsistencies in the evidence. This systematic review and meta-analysis aimed to determine whether IGFBP-1 is decreased in PCOS when controlling for the influence of BMI.
METHODS
Articles published between 1988 and 2008 were searched using MEDLINE, PubMed, SCOPUS and Web of Knowledge. Unpublished literature, trials in progress, and recent reviews were also searched. Original articles were selected by two investigators. To be included, the study must have compared serum IGFBP-1 in two populations: either PCOS versus controls, or an overweight subgroup versus the normal weight subgroup in either population. From 617 identified articles, 12 were included in the meta-analysis. Data were abstracted by two reviewers independently and standardized for errors.
RESULTS
The population difference is presented as the Weighted Mean Difference (95% CI). PCOS subjects had a significantly lower serum concentrations of IGFBP-1 compared with controls [P< 0.00001; –36.6 (–52.0, –21.2) µg/l]. Overweight PCOS subjects also had lower IGFBP-1 levels compared with normal weight PCOS subjects [P < 0.006; –30.6 (–52.3, –8.8) µg/l]. No significant difference was found between overweight PCOS patients and overweight controls [P = 0.23; –5.1 (–13.5, 3.2) µg/l] or between normal weight PCOS patients and normal weight controls [P = 0.50; –3.8 (–14.9, 7.3) µg/l]. Overweight controls had significantly lower IGFBP-1 concentrations than normal weight controls [P = 0.03; –18.0 (–34.4, – 1.5) µg/l].
CONCLUSION
These data indicate that a decreased serum level of IGFBP-1 is unlikely to be a mechanism for ovarian hyperandrogenism in PCOS. BMI may be the major determinant of serum IGFBP-1.
It has been hypothesized that certain obstetrical populations, including women who conceive using assisted reproductive technologies (ART) and women with multiple births, may be at increased risk for postpartum depression. In this systematic literature review, we examine the published evidence for this hypothesis.
METHODS
The databases Medline, CINAHL, EMBASE, PsycINFO and the Cochrane Library were searched from their start dates through to April 1, 2009 using relevant keywords. All published, peer-reviewed articles in English, Spanish or French including a standardized assessment of depression administered between 2 and 52 weeks postpartum were considered for inclusion. Two independent reviewers abstracted and critically appraised a total of 13 eligible articles.
RESULTS
The data indicate little or no increased risk for postpartum depression among women who use ART to conceive. In contrast, most studies of adequate quality indicate that mothers of multiples may be at elevated risk for symptoms of depression. However, existing data do not permit differentiation between transient maternal distress and clinically significant postpartum depression.
CONCLUSIONS
Studies included in this review were often limited by small samples and lack of appropriate comparison groups, making further research in this area essential. In particular, lack of control for maternal psychiatric history and other important sociodemographic predictors of depression is a serious limitation of existing research on this topic. Further, the use of reproductive technologies and multiple births often co-occur, and few study designs enabled separation of the effects of these two variables. However, evidence of increased risk for symptoms of postpartum depression among women with multiple births, if confirmed, may warrant targeted interventions for this population.
Cigarette smoking is associated with lower fecundity rates, adverse reproductive outcomes and a higher risk of IVF failures. Over the last few decades, prevalence of smoking among women of reproductive age has increased. This review focuses on current knowledge of the potential effects of smoke toxicants on all reproductive stages and the consequences of smoke exposure on reproductive functions.
METHODS
We conducted a systematic review of the scientific literature on the impact of cigarette smoking and smoke constituents on the different stages of reproductive function, including epidemiological, clinical and experimental studies. We attempted to create hypotheses and find explanations for the deleterious effects of cigarette smoke observed in experimental studies.
RESULTS
Cigarette smoke contains several thousand components (e.g. nicotine, polycyclic aromatic hydrocarbons and cadmium) with diverse effects. Each stage of reproductive function, folliculogenesis, steroidogenesis, embryo transport, endometrial receptivity, endometrial angiogenesis, uterine blood flow and uterine myometrium is a target for cigarette smoke components. The effects of cigarette smoke are dose-dependent and are influenced by the presence of other toxic substances and hormonal status. Individual sensitivity, dose, time and type of exposure also play a role in the impact of smoke constituents on human fertility.
CONCLUSIONS
All stages of reproductive functions are targets of cigarette smoke toxicants. Further studies are necessary to better understand the deleterious effects of cigarette smoke compounds on the reproductive system in order to improve health care, help to reduce cigarette smoking and provide a better knowledge of the molecular mechanisms involved in reproductive toxicology.
During meiosis, mammalian oocytes undergo two successive cell divisions without an intermediate replicative phase. This brief period, called ‘meiotic maturation’, is crucial for the formation of an egg capable of being fertilized and of generating viable and euploid offspring.
METHODS
We review our current knowledge of the cellular and molecular mechanisms that control asymmetry and appear to be shared between mammalian species, as well as the associated misfunctions that impair the formation of functional female gametes.
RESULTS AND CONCLUSIONS
The two successive divisions that comprise mammalian oogenesis are asymmetric. They lead to the formation of small polar bodies and the large and polarized egg. This asymmetry depends upon the dynamic organization of the oocyte cytoskeleton during both divisions. During meiosis I, microfilaments and associated molecules ensure the targeting of the microtubule spindle at the oocyte periphery. During meiosis II, they anchor the spindle under the plasma membrane. In parallel, the cortex overhanging the spindle is dramatically reorganized. Establishment and maintenance of this cortical domain are crucial for the completion of fertilization. Loss of this differentiated area is characteristic of ageing or low-quality gametes and associated with increased maternal age or post-ovulatory ageing.
Despite increasing contraceptive availability, unintended pregnancy remains a global problem, representing as many as 30% of all known pregnancies. Various strategies have been proposed to reverse this disturbing trend, especially increased use of long-acting reversible contraceptive (LARC) methods. In this review we aim to discuss the role of LARC methods and importance of contraceptive counseling in reducing unintended pregnancy rates.
METHODS
References/resources cited were identified based on searches of medical literature (MEDLINE, 1990–2009), bibliographies of relevant publications and the Internet.
RESULTS AND CONCLUSIONS
LARC methods—copper intrauterine devices (IUDs), progestogen-releasing intrauterine system and injectable and implantable contraceptives—are safe and effective contraceptive options (unintended pregnancy rates with typical versus perfect use: 0.05–3.0 versus 0.05–0.6%) that are appropriate for a wide range of women seeking to limit or space childbearing. Despite their safety and efficacy records, these methods remain underutilized; injectable and implantable methods are used by an estimated 3.4% and intrauterine methods by 15.5% of women worldwide. LARC methods require no daily or coital adherence and avoid the adverse events and health risks of estrogen-containing contraceptives. The copper IUD and progestin-only injections and implants have been shown to be more cost-effective than more commonly used methods, such as condoms and the pill (5-year savings: $13 373–$14 122, LARC; $12 239, condoms; $12 879, pill). Women who are considering use of LARC methods should receive comprehensive contraceptive counseling, as women who receive counseling before use demonstrate higher rates of after-use method satisfaction, continuation and acceptance than those who do not.
Single embryo transfer (SET) is the most effective way of reducing multiple pregnancy rates associated with assisted reproductive technology (ART). Despite published evidence suggesting that the judicious use of elective SET can lead to near-elimination of multiples without compromising cumulative live birth rates, the uptake of this strategy has been variable.
METHODS
Medline, EMBASE and the Cochrane Database of Systematic Reviews (1978–2010) were searched using appropriate MeSH headings. Leading fertility journals along with appropriate cross references were hand searched and information retrieved from national ART registers and websites of national fertility societies in order to determine current rates of SET. We explored social, economic and clinical factors determining the uptake of SET.
RESULTS
It was not possible to distinguish elective from non-elective SET from national ART reports. Data from 31 countries suggest that there has been a gradual increase in SET rates over a 3 year period (2003–2005) but major geographical differences were noted. SET rates are highest in Sweden (69.4%) but are as low as 2.8% in the USA. Access to public funding for ART, availability of good cryopreservation facilities and legislation appear to be the most important reasons favouring the uptake of SET. Personal choice plays a significant role as many subfertile couples have a strong preference for twins. Awareness that double embryo transfer (DET) increases live birth per fresh treatment cycle, inability to accurately identify women at high risk for twins and limitations of existing embryos selection criteria are barriers to a wider acceptance of SET.
CONCLUSIONS
The current variation in the uptake of elective SET is likely to persist until there are major changes in the way ART is viewed, funded and legislated.
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.
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.
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.
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.
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.
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.
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.
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 (ICSIWM) or KSOM medium with amino acids (ICSIKSOMaa) 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 (KSOMaa).
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.
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.
