Defining the gonadotrophin requirement for the selection of a single dominant follicle in cattle.

The aim was to define the pattern and physiological concentrations of FSH and LH required for the selection of a single dominant follicle in mono-ovulatory species. A series of five experiments was carried out using gonadotrophin-releasing hormone agonist-induced hypogonadal heifers. Animals were infused with different patterns of either FSH and/or LH followed by an ovulatory dose of human chorionic gonadotrophin. Follicular response was monitored by ultrasound scanning and blood samples were collected to measure concentrations of FSH, LH, oestradiol and progesterone. The main findings were: (1) physiological concentrations of FSH given as a continuous infusion and for an adequate duration, in the presence of basal LH, with or without LH pulses, are capable of inducing a superovulatory response, (2) initial exposure to FSH followed by LH pulses alone stimulate the development of multiple preovulatory follicles, confirming that ovarian follicles are capable of transferring dependence on gonadotrophins from FSH to LH, (3) while LH pulses appear not to have a major effect on the pattern of preovulatory follicle development, adequate LH pulsatile support is required for full oestradiol synthesis and (4) the duration of initial exposure to FSH and the ability to transfer the dependence from FSH to LH are critical for the selection of a single dominant follicle. In conclusion, this experimental series confirms that the duration of initial exposure to FSH and the ability of the selected follicle to transfer its gonadotrophic dependence from FSH to LH are critical for the selection of a single dominant follicle in cattle.

Random Allocation of Blastomere Descendants to the Trophectoderm and ICM of the Bovine Blastocyst.

The first lineage specification during mammalian embryo development can be visually distinguished at the blastocyst stage. Two cell lineages are observed on the embryonic-abembryonic axis of the blastocyst: the inner cell mass and the trophectoderm. The timing and mechanisms driving this process are still not fully understood. In mouse embryos, cells seem prepatterned to become certain cell lineage because the first cleavage plane has been related with further embryonic-abembryonic axis at the blastocyst stage. Nevertheless, this possibility has been very debatable. Our objective was to determine whether this would be the case in another mammalian species, the bovine. To achieve this, cells of in vitro produced bovine embryos were traced from the 2-cell stage to the blastocyst stage. Blastocysts were then classified according to the allocation of the labeled cells in the embryonic and/or abembryonic part of the blastocyst. Surprisingly, we found that there is a significant percentage of the embryos (∼60%) with labeled and nonlabeled cells randomly distributed and intermingled. Using time-lapse microscopy, we have identified the emergence of this random pattern at the third to fourth cell cycle, when cells started to intermingle. Even though no differences were found on morphokinetics among different embryos, these random blastocysts and those with labeled cells separated by the embryonic-abembryonic axis (deviant pattern) are significantly bigger; moreover deviant embryos have a significantly higher number of cells. Interestingly, we observed that daughter cells allocation at the blastocyst stage is not affected by biopsies performed at an earlier stage.

Cell density-mediated pericellular hypoxia and the local dynamic regulation of VEGF-a splice variants in ovine ovarian granulosa cells.

The primary aims of this study were to utilize a specialized culture system to further elucidate the functional significance of pericellular hypoxia within the granulosa cell (GC) compartment of growing follicles, and to clarify its effects on the production of vascular endothelial growth factor (VEGF)-A isoforms and steroid hormones. Multilaminar clusters formed rapidly in ovine GCs seeded at high density (HD), and Hypoxyprobe-1 protein adducts appeared markedly more abundant and HIF-1 activation significantly (P < 0.001) greater than in cells seeded at low density (LD). Four proangiogenic VEGF mRNA transcript variants were identified in cultured GCs. Most abundant were VEGF120 and VEGF164, but VEGF182 and VEGF188 were also detected. Total VEGF mRNA was shown to be up-regulated transiently in the HD cells (P < 0.001) and VEGF164 mRNA appeared to contribute most to this. The hypoxia mimetic cobalt chloride also induced marked increases in HIF-1 activation (P < 0.01) and total VEGF mRNA (P < 0.01) production. HD cells increased levels of HIF-1alpha (P < 0.001) and VEGF receptor type 1 (P < 0.05), but not VEGF receptor type 2 mRNA, compared to LD cells or cells grown under chemically induced hypoxia. Both 17beta-estradiol (E2) and progesterone (P4) were markedly lower (P < 0.001) in the HD, cells but though cobalt chloride treatment accompanied significantly reduced P4 production (P < 0.05), E2 levels remained similar to those in untreated cells. These outcomes suggest that pericellular hypoxia may be an important mediator of VEGF production in the GCs of growing follicles, but that local regulation is complex and may involve multiple mechanisms such as mediation by steroid hormones and differential variant mRNA production.

What exactly do we mean by 'recurrent implantation failure'? A systematic review and opinion.

Recurrent implantation failure (RIF) is an iatrogenic condition, being the result of repetitive unsuccessful cycles of IVF or intracytoplasmic sperm injection (ICSI) treatment. The aim of this review was to assess the definitions of RIF used in literature as well as suggest a uniform definition of this condition. A systematic search of MEDLINE, Embase and Cochrane Library was conducted. The most commonly stated definitions described RIF as 'three or more failed treatment cycles' or 'two or more failed cycles'. Other identified definitions were based solely on the number of embryos transferred in previous cycles or combined the number of previously failed cycles with the number of transferred embryos. Several other definitions were also identified. This review highlights the lack of uniformity of the definition of RIF. Based on the available literature and the expert opinion of the authors, RIF should be defined as the absence of implantation after two consecutive cycles of IVF, ICSI or frozen embryo replacement cycles where the cumulative number of transferred embryos was no less than four for cleavage-stage embryos and no less than two for blastocysts, with all embryos being of good quality and of appropriate developmental stage.

Assessment of endometrial and subendometrial blood flow in women undergoing frozen embryo transfer cycles.

This study evaluated whether 3D power Doppler (3DPD) indices from endometrium and subendometrium can identify increases in endometrial volume/vascularity induced by exogenous oestradiol and subsequent introduction of progestogens in women undergoing frozen-thawed embryo transfer (FET). Oral oestradiol was administered at increasing doses after down-regulation to prepare the endometrium and progestogens were used for luteal support. 3DPD data sets were acquired at down-regulation, on days 5, 10 and 15 of oestradiol administration and at the time of FET. Endometrial thickness was measured using the multiplanar method and endometrial volume and blood flow from the endometrium and subendometrium were estimated using virtual organ computer-aided analysis and shell-imaging. This study evaluated 45 women at least once: 19 achieved clinical pregnancy (CP); 21 were evaluated at down-regulation (eight CP), 26 at day 5 (10 CP), 31 at day 10 (12 CP), 31 at day 15 (13 CP) and 16 at FET (seven CP). Changes were observed in all parameters between the examinations; however, differences between women who achieved CP and those who did not were not significant. 3DPD angiography is not a sufficiently sensitive tool to predict the outcome of FET. We evaluate whether 3D ultrasound using power Doppler (3DPD) indices from endometrium and subendometrium can identify predictable increases in endometrial volume and vascularity induced by serial increments in exogenous oestradiol and the subsequent introduction of progestogens in women undergoing frozen-thawed embryo transfer (FET) using hormone replacement therapy to prepare the endometrium. Oral oestradiol was administered at increasing doses after down-regulation to prepare the endometrium and progestogens were used for luteal support. 3DPD data sets of the uterus were acquired at down-regulation, on days 5, 10, and 15 of oestradiol administration, and at the time of FET. Endometrial thickness was measured. Endometrial volume and blood flow from the endometrium and subendometrium were measured using virtual organ computer-aided analysis (VOCAL) and shell imaging. This study evaluated 45 women at least once: 19 achieved clinical pregnancy (CP); 21 were evaluated at down-regulation (eight CP), 26 at day 5 (10 CP), 31 at day 10 (12 CP), 31 at day 15 (13 CP) and 16 at FET (seven CP). Changes were observed in all the parameters between the examinations; however, differences between women who achieved CP and those who did not were not significant, suggesting that quantitative 3D power Doppler angiography is not a sufficiently sensitive tool to predict the outcome of FET treatment.

The activity and copy number of mitochondrial DNA in ovine oocytes throughout oogenesis in vivo and during oocyte maturation in vitro.

Mitochondria are responsible for the production of ATP, which drives cellular metabolic and biosynthetic processes. This is the first study to quantify the mtDNA copy number across all stages of oogenesis in a large monovulatory species, it includes assessment of the activity of mitochondria in germinal vesicle (GV) and metaphase II (MII) oocytes through JC1 staining. Primordial to early antral follicles (n = 249) were isolated from the sheep ovarian cortex following digestion at 37°C for 1 h and all oocytes were disaggregated from their somatic cells. Germinal vesicle oocytes (n = 133) were aspirated from 3- to 5-mm diameter antral follicles, and mature MII oocytes (n = 71) were generated following in vitro maturation (IVM). The mtDNA copy number in each oocyte was quantified using real-time PCR and showed a progressive, but variable increase in the amount of mtDNA in oocytes from primordial follicles (605 ± 205, n = 8) to mature MII oocytes (744 633 ± 115 799, n = 13; P < 0.05). Mitochondrial activity (P > 0.05) was not altered during meiotic progression from GV to MII during IVM. The observed increase in the mtDNA copy number across oogenesis reflects the changing ATP demands needed to orchestrate cytoskeletal and cytoplasmic reorganization during oocyte growth and maturation and the need to fuel the resumption of meiosis in mature oocytes following the pre-ovulatory gonadotrophin surge.

Efficacy of dehydroepiandrosterone to improve ovarian response in women with diminished ovarian reserve: a meta-analysis.

Women with diminished ovarian reserve often respond poorly to controlled ovarian stimulation resulting in retrieval of fewer oocytes and reduced pregnancy rates. It has been proposed that pre-IVF Dehydroepiandrosterone (DHEA) adjuvant therapy may improve ovarian response and pregnancy rates in women with diminished ovarian reserve. This meta-analysis aims to investigate efficacy of DHEA as an adjuvant to improve ovarian response and IVF outcome in women with diminished ovarian reserve. Electronic databases were searched under the following terms: (DHEA) and (diminished ovarian reserve) and/or (poor response). Studies were included if they reported at least one of the following outcomes; clinical pregnancy rate, number of oocytes retrieved, miscarriage rate. We identified 22 publications determining effects of DHEA in clinical trials. Only 3 controlled studies were eligible for meta-analysis. There was no significant difference in the clinical pregnancy rate and miscarriage rates between women pre-treated with DHEA compared to those without DHEA pre-treatment (RR 1.87, 95% CI 0.96-3.64; and RR 0.59, 95% CI 0.21-1.65, respectively). The number of oocytes retrieved (WMD -1.88, 95% CI -2.08, 1.67; P < 0.001) was significantly lower in the DHEA group. In conclusion, based on the limited available evidence from a total of approximately 200 IVF cycles, there are insufficient data to support a beneficial role of DHEA as an adjuvant to controlled ovarian stimulation in IVF cycle. Well-designed, randomised controlled trials as well as more exact knowledge about DHEA mechanisms of action are needed to support use of DHEA in standard practice for poor-responders.

Development of an acute ovine model of polycystic ovaries to assess the effect of ovarian denervation.

Introduction: Polycystic ovary syndrome (PCOS) seems to be associated with increased ovarian sympathetic nerve activity and in rodent models of PCOS reducing the sympathetic drive to the ovary, through denervation or neuromodulation, improves ovulation rate. We hypothesised that sympathetic nerves work with gonadotropins to promote development and survival of small antral follicles to develop a polycystic ovary phenotype. Methods: Using a clinically realistic ovine model we showed a rich sympathetic innervation to the normal ovary and reinnervation after ovarian transplantation. Using needlepoint diathermy to the nerve plexus in the ovarian vascular pedicle we were able to denervate the ovary resulting in reduced intraovarian noradrenaline and tyrosine hydroxylase immunostained sympathetic nerves. We developed an acute polycystic ovary (PCO) model using gonadotrophin releasing hormone (GnRH) agonist followed infusion of follicle stimulating hormone (FSH) with increased pulsatile luteinising hormone (LH). This resulted in increased numbers of smaller antral follicles in the ovary when compared to FSH infusion suggesting a polycystic ovary. Results: Denervation had no effect of the survival or numbers of follicles in the acute PCO model and did not impact on ovulation, follicular and luteal hormone profiles in a normal cycle. Discussion: Although the ovary is richly inervated we did not find evidence for a role of sympathetic nerves in ovarian function or small follicle growth and survival.

Steroids and miRNAs in assessment of ovarian tissue damage following cryopreservation.

Ovarian cortical tissue cryopreservation is a relatively novel approach to preserving fertility in women diagnosed with cancer. However, the effects of freezing-thawing are not fully understood, mainly due to the lack of suitable methods to assess tissue's survival after thawing. Disparities in steroid production have been associated with ovarian failure by disrupting folliculogenesis, ovulation and oocyte apoptosis. Moreover, specific microRNAs, identified in human ovarian follicles, are thought to play a fundamental role in folliculogenesis. In this study, we investigated the possible interplay between the ovarian steroidal production and microRNA expression patterns in spent culture media, as potential non-invasive markers for ovarian tissue damage after cryopreservation. Cryopreservation of ovarian cortical tissue decreased (P<0.05) both steroid production (oestradiol and progesterone) and expression of microRNA-193b and 320A in spent culture media over 5 days, however, expression of microRNA-24 increased (P<0.05). The number of primordial follicles were also reduced (P<0.05) in fresh-cultured and cryopreserved-cultured cortical tissues when compared with fresh tissues. Downregulation of microRNA-193b and microRNA-320A together with upregulation of microRNA-24 could have a synergistic role in cell apoptosis, and consequently leading to reduced oestradiol and progesterone production. Thus, there appears to be an interplay between these microRNAs, ovarian steroid production and cell damage, which can be further explored as novel non-invasive markers of cell damage following cryopreservation.

In vivo and in vitro studies of MUC1 regulation in sheep endometrium.

In this study, we investigated the expression of mucin 1 (MUC1) mRNA and protein in sheep endometrium at different time points during follicular and luteal phases of estrous cycle, and also determined the effect of steroid hormone treatments and interferon tau (IFNτ) on MUC1 mRNA expression in endometrial cell culture in vitro. In experiment one, 15 Welsh mountain ewes were synchronized to a common estrus and killed at precise stages of estrous cycle corresponding to (1) pre-LH peak, (2) LH peak, (3) post-LH peak, (4) early luteal, and (5) mid-luteal. Reproductive tracts were harvested and mRNA was extracted from the endometrial tissues. Parts of the uterine horns were fixed for immunohistochemistry. In experiment two, mixed populations of ovine endometrial cells (from slaughterhouse material collected at the postovulatory stage of the estrous cycle) were cultured to 70% confluence before treatment with (1) progesterone (P4, 10 ng/mL, for 48 hours), (2) estradiol (E2, 100 pg/mL, for 48 hours), or with (3) E2 priming for 12 hours (100 pg/mL) followed by P4 (10 ng/mL) for 36 hours. These were compared with: (4) IFNτ (10 ng/mL, for 48 hours), and (5) basic medium (Dulbecco Modified Eagle Medium /F12) as control. The results showed that MUC1 mRNA and protein expression in sheep endometrium were highest during the midluteal stage and very low during the post-LH period compared with the other stages (P < 0.05). MUC1 immunostaining in the luminal epithelium was apically restricted and was not significantly different across all stages of estrous cycle except at the post-LH peak where it was significantly low. In cell culture, MUC1 mRNA expression was significantly upregulated by both steroids either singly or in combination (P < 0.05), and downregulated in the presence of IFNτ. In conclusion, endometrial MUC1 expression is cyclically regulated by both E2 and P4in vivo and in vitro, and directly downregulated by IFNτ treatment in vitro.

Relationship between hair and salivary cortisol and pregnancy in women undergoing IVF.

Evidence for an association between cortisol and clinical pregnancy in women undergoing In Vitro Fertilisation (IVF) is mixed with previous studies relying exclusively on short term measures of cortisol in blood, saliva, urine, and/or follicular fluid. Hair sampling allows analysis of systemic levels of cortisol over the preceding 3-6 months. The present study sought to explore the relationship between cortisol and clinical pregnancy outcome in women undergoing IVF utilising multiple indices of cortisol derived from both saliva and hair measured prior to commencing gonadotrophin treatment. A total of 135 women (mean age 34.5 SD+/-4.8) were recruited from an English fertility clinic (December 2012-April 2014) 60% of whom became pregnant (n=81). Salivary cortisol data were obtained over two days: upon awakening, 30min post awakening, and at 22:00. A subsample (n=88) of the women providing salivary samples were approached consecutively to provide hair samples for the measurement of cortisol. Independent Logistic regression analyses revealed that salivary cortisol measures including cortisol awakening response (CAR) (p=0.485), area under the curve with respect to ground (AUCg) (p=0.527), area under the curve with respect to increase (AUCi) (p=0.731) and diurnal slope (p=0.889) did not predict clinical pregnancy. In contrast, hair cortisol concentrations significantly predicted clinical pregnancy (p=0.017). Associations between hair cortisol and clinical pregnancy remained when controlling for accumulations of salivary cortisol (p=0.034) accounting for 26.7% of the variance in pregnancy outcome. These findings provide preliminary evidence that longer term systemic cortisol may influence reproductive outcomes; and in turn suggests that interventions to reduce cortisol prior to commencing IVF could improve treatment outcomes.

Personality and gene expression: Do individual differences exist in the leukocyte transcriptome?

BACKGROUND: The temporal and situational stability of personality has led generations of researchers to hypothesize that personality may have enduring effects on health, but the biological mechanisms of such relationships remain poorly understood. In the present study, we utilized a functional genomics approach to examine the relationship between the 5 major dimensions of personality and patterns of gene expression as predicted by 'behavioural immune response' theory. We specifically focussed on two sets of genes previously linked to stress, threat, and adverse socio-environmental conditions: pro-inflammatory genes and genes involved in Type I interferon and antibody responses. METHODS: An opportunity sample of 121 healthy individuals was recruited (86 females; mean age 24 years). Individuals completed a validated measure of personality; questions relating to current health behaviours; and provided a 5ml sample of peripheral blood for gene expression analysis. RESULTS: Extraversion was associated with increased expression of pro-inflammatory genes and Conscientiousness was associated with reduced expression of pro-inflammatory genes. Both associations were independent of health behaviours, negative affect, and leukocyte subset distributions. Antiviral and antibody-related gene expression was not associated with any personality dimension. CONCLUSIONS: The present data shed new light on the long-observed epidemiological associations between personality, physical health, and human longevity. Further research is required to elucidate the biological mechanisms underlying these associations.

4-D Assessment of Endometrial Vascularity Using Spatiotemporal Image Correlation: A Study Comparing Spherical Sampling and Whole-Tissue Analysis.

Our aim in the study described here was to assess the feasibility of spatiotemporal image correlation power Doppler quantification of the endometrium with two techniques: spherical samples and whole tissue. We scanned 51 women in the midluteal phase of the menstrual cycle: STIC assessment of the whole endometrium was not possible in 10% of cases, whereas spherical analysis was possible in all. The time taken for data set analysis was much longer for the whole endometrium compared with spherical analysis (1478.9 ± 291 s vs. 266.8 ± 39.3 s, p < 0.05). Intra-class correlation coefficients for the vascularization flow index (VFI) were similar for both methods. Volumetric vascularity indices were higher when spherical sampling was conducted. Significant cycle-to-cycle variability in the vascularity indices was present, with coefficients of variation exceeding 20% for both techniques. We found that STIC power Doppler quantification of the whole endometrium is possible in the majority of cases, however, it is time consuming and limited by significant cycle-to-cycle variability.

Evidence of a role for follicle-stimulating hormone in controlling the rate of preantral follicle development in sheep.

Autografting ovarian cortex results in the loss of growing follicles and elevated gonadotropins. This paradigm was employed to examine the effect of gonadotropins on preantral follicle development in sheep. Ovarian tissue was recovered at 1, 2, 3, and 4 months after grafting from ewes that were either hyper- (n = 12; untreated) or hypogonadotropic (n = 12; GnRH-agonist and estradiol implants). Compared with the Hypo group, Hyper ewes had higher (P < 0.001) gonadotropins, had greatly enlarged grafts, had reestablished a normal follicular hierarchy 2 months earlier (P < 0.05), had higher (P < 0.05) levels of proliferating cell nuclear antigen expression in tertiary, preantral, and antral follicles, and had higher (P < 0.01) concentrations of inhibin A and estradiol. Compared with time zero controls, increases in the number of primary follicles and the rate of proliferation in primary and secondary follicles in both groups of autografts (P < 0.05) were also observed. In conclusion, the results of this experiment provide the first evidence that gonadotropins can affect the rate of development of preantral follicles in vivo in a large monovulatory species. Furthermore data are presented to support the existence of a gonadotropin-independent intraovarian feedback loop regulating both the rate of primordial follicle initiation and primary and secondary follicle development.

Effect of ageing on hormone secretion and follicular dynamics in sheep with and without the Booroola gene.

It has been suggested that ewes carrying the Booroola gene (Fec(B)) consistently ovulate more follicles because they recruit more primordial follicles and/or have a lower rate of atresia. If the former is correct, the pool of follicles would be depleted sooner in Fec(B) animals. We have studied follicular dynamics and endocrine function during follicular and early luteal phases of the estrous cycle of older ewes with or without the fecundity gene and compared this data with data obtained 6 yr previously in the same animals. Older sheep carrying the Booroola gene maintained a significantly higher ovulation rate than noncarrier ewes [4.2 +/- 0.8 vs. 2.2 +/- 0.6 corpora lutea (CL), respectively; P < 0.05], and in keeping with data from young animals, both ovulatory follicles and CL (4.7 +/- 0.3 vs. 6.9 +/- 0.7 mm and 12.8 +/- 0.5 vs. 16.7 +/- 0.8 mm, respectively) were smaller than those of noncarrier ewes (P < 0.05). The interval from luteolysis to the onset of the LH surge increased with age in all the animals (from 52.0 +/- 8.0 to 67.0 +/- 7.5 h in gene carrier sheep and from 56.0 +/- 2.0 to 79.5 +/- 9.6 h in noncarrier sheep, P < 0.05). The concentration of estradiol and inhibin A in the early luteal phase was lower in older noncarrier ewes (P = 0.08 and P < 0.05, respectively), and the level of inhibin A was inversely related to the level of FSH in aged sheep of both genotypes (P < 0.0001). In contrast, the number of developing follicles in older ewes of both genotypes was similar to the number found in younger ewes, suggesting that increased ovulation rate in sheep carrying the Fec(B) mutation is related to a reduced rate of atresia.

Multiple factors affecting the efficiency of multiple ovulation and embryo transfer in sheep and goats.

This review offers an overview of the basic characteristics of in vivo embryo technologies, their current status, the main findings and the advances gained in recent years, and the outstanding subjects for increasing their efficiency. The use of superovulation and embryo transfer procedures remains affected by a high variability in the ovulatory response to hormonal treatment and by a low and variable number of transferable embryos and offspring obtained. This variability has been classically identified with both extrinsic (source, purity of gonadotrophins and protocol of administration) and intrinsic factors (breed, age, nutrition and reproductive status), which are reviewed in this paper. However, emerging data indicate that the main causes of variability are related to endocrine and ovarian factors, and so the number of studies and procedures addressing a better understanding and control of these factors may be increased in the future. The accomplishment of this objective, the improvement of procedures for embryo conservation and for the selection and management of recipient females, will allow further development and application of this technology.

Efficacy of dehydroepiandrosterone to overcome the effect of ovarian ageing (DITTO): a proof of principle randomised controlled trial protocol.

INTRODUCTION: Dehydroepiandrosterone (DHEA) has been proposed to improve pregnancy rates in women with diminished ovarian reserve undergoing in vitro fertilisation (IVF) treatment. However, evidence regarding its efficacy is supported by a limited number of randomised controlled trials (RCTs). This double-blinded RCT aims to measure the effect of DHEA supplementation prior to and during controlled ovarian hyperstimulation on ovarian response prior to IVF treatment in women predicted to have poor ovarian reserve. METHODS AND ANALYSIS: Sixty women with ovarian antral follicle count ≤10 and serum anti-Mullerian hormone ≤5 pmol/L undergoing IVF/intracytoplasmic sperm injection (ICSI) treatment at the Nurture fertility clinic, Nottingham will be recruited. They will be randomised to either receive DHEA capsule 75 mg/day or placebo for at least 12 weeks before egg collection. All participants will undergo standard long down regulation protocol using human menopausal gonadotropin 300 IU/day. Serum samples and follicular fluids at the time of egg collection will be collected for hormonal immunoassays. For ICSI participants, cumulus cells stripped from oocyte will be collected for cumulus gene expression analyses regarding oocyte competence. Microdrops of oocyte culture media before the time of ICSI will be assessed for glucose, pyruvate and lactate utilisation. Embryo transfer will be performed on day 2, 3 or 5 based on the number and quality of the embryos available. Pregnancy will be defined as urine pregnancy test positive (biochemical pregnancy) and 6-8 weeks ultrasound scan with fetal heart beat (clinical pregnancy) and live birth. It is planned to perform the molecular and nutritional fingerprint analyses in batches after finishing the clinical phase of the study. ETHICS AND DISSEMINATION: The approval of the study was granted by the NHS Research Ethics Committee (Ref number NRES 12/EM/0002), the Medicines and Healthcare products Regulatory Agency (MHRA), and the Nottingham University Hospitals Trust Research and Development department. All participants shall provide written informed consent before being randomised into allocated treatment groups. TRIAL REGISTRATION NUMBER: Protocol V.2.0; EudraCT number: 2011-002425-21; http://www.clinicaltrials.gov; NCT01572025; CTA reference: 03057/0053/001-0002.

Effects of assisted reproductive technologies on human sex ratio at birth.

OBJECTIVE: To investigate the effect of assisted reproductive technology (ART) treatments on the sex ratio of babies born. DESIGN: Assessment of direct effects of assisted conception through retrospective data analysis on the progeny sex ratio of treated women in the United Kingdom. SETTING: The study uses the anonymized register of the Human Fertilisation and Embryology Authority. PATIENT(S): A total of 106,066 babies of known gender born to 76,994 treated mothers and 85,511 treatment cycles between 2000 and 2010 in the United Kingdom. INTERVENTION(S): Intrauterine insemination, IVF, or intracytoplasmic sperm injection (ICSI). MAIN OUTCOME MEASURE(S): Sex ratio of babies born. RESULT(S): Intrauterine insemination, IVF, and ICSI lead to different sex ratios, highest after IVF (proportion male = mean 0.521 ± confidence interval 0.0056) and lowest under ICSI embryo transfer (0.493 ± 0.0031). In addition, for both ICSI and IVF, transferring embryos at a later stage (blastocyst) results in approximately 6% more males than after early cleavage-stage ET. CONCLUSION(S): Because the cumulative number of IVF babies born is increasing significantly in Britain and elsewhere, more research is needed into the causes of gender bias after ART and into the public health impact of such gender bias of offspring born observed on the rest of the population.

The role of anti-Müllerian hormone (AMH) during follicle development in a monovulatory species (sheep).

Knockout studies in mice have suggested that anti-Müllerian hormone (AMH) modulates primordial follicle recruitment and the response of growing follicles to FSH. Little is known of the physiology of AMH in monovular species, despite intense clinical interest in this factor. Using sheep as a model, we sought to investigate the functional role of AMH in modulating follicle development in monovular species. In contrast to the rodent, the results indicate that AMH does not affect the rate of primordial follicle recruitment but appears to regulate the rate at which follicles progress through the gonadotropin-responsive phase, during which it is maximally expressed. Thus, knockdown of AMH bioactivity by active immunization lead to a decline in the population of gonadotropin-responsive preantral and small antral follicles (P < 0.01) and increases in both the number of gonadotropin-dependent antral follicles (P < 0.01) and ovulation rate (P < 0.05). These in vivo findings were consistent with the results of other studies examining the pattern of expression of AMH, which was negatively correlated with aromatase (P < 0.001), and in vitro supplementation experiments, which supported an inhibitory role for AMH in modulating the response of both theca and granulosa cells to LH and FSH, respectively. The elucidation of a functional relationship between AMH and LH-stimulated thecal androgen production may be significant in terms of the etiology of common forms of anovulatory infertility in women. Furthermore, the observed increase in both the number of recruitable antral follicles and ovulatory quota in response to AMH knockdown may have therapeutic value in women who respond poorly to ovarian stimulation.

The interovarian variation in three-dimensional ultrasound markers of ovarian reserve in women undergoing baseline investigation for subfertility.

OBJECTIVE: To evaluate differences in the three-dimensional (3D) ultrasound markers of ovarian reserve between the ovaries within an individual undergoing investigation for subfertility. DESIGN: Prospective observational study. SETTING: University-based assisted conception unit. PATIENT(S): Two hundred seventy women undergoing baseline early follicular phase ultrasound as an investigation for subfertility. INTERVENTION(S): Three-dimensional ultrasound scan in early follicular phase between days 2 and 5 of the menstrual cycle. MAIN OUTCOME MEASURE(S): Variations in 3D ultrasound markers of ovarian reserve between the two ovaries within same individual. RESULT(S): Two hundred fifteen subjects were analyzed for ovarian volume and antral follicle count, and 205 subjects for 3D power Doppler indices. Significant differences were noted (median, range) in the number of antral follicles measuring >6.0 mm and ovarian volume. Significant correlation was noted between the two ovaries in antral follicles measuring 6.0 mm or less, ovarian volume, and 3D power Doppler indices. On stratifying the antral follicles according to size using sonography-based automated volume calculation with postprocessing, maximum variation was seen in follicles measuring more than 6.0 mm as measured using limits of agreement. CONCLUSION(S): There are significant differences in the antral follicles measuring >6.0 mm and ovarian volume, as measured using 3D ultrasound, that require consideration when comparing the two ovaries within an individual.