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Preview: Human Reproduction - current issue

Human Reproduction Current Issue





Published: Fri, 13 Oct 2017 00:00:00 GMT

Last Build Date: Sun, 22 Oct 2017 20:48:22 GMT

 



Placental Nano-vesicles Target to Specific Organs and Modulate Vascular Tone In Vivo

2017-10-13

Abstract
STUDY QUESTION
How do nano-vesicles extruded from normal first trimester human placentae affect maternal vascular function?
SUMMARY ANSWER
Placental nano-vesicles affect the ability of systemic mesenteric arteries to undergo endothelium- and nitric oxide- (NO-) dependent vasodilation in vivo in pregnant mice.
WHAT IS KNOWN ALREADY
Dramatic cardiovascular adaptations occur during human pregnancy, including a substantial decrease in total peripheral resistance in the first trimester. The human placenta constantly extrudes extracellular vesicles that can enter the maternal circulation and these vesicles may play an important role in feto-maternal communication.
STUDY DESIGN, SIZE, DURATION
Human placental nano-vesicles were administered into CD1 mice via a tail vein and their localization and vascular effects at 30 min and 24 h post-injection were investigated.
PARTICIPANTS/MATERIALS, SETTING, METHODS
Nano-vesicles from normal first trimester human placentae were collected and administered into pregnant (D12.5) or non-pregnant female mice. After either 30 min or 24 h of exposure, all major organs were dissected for imaging (n = 7 at each time point) while uterine and mesenteric arteries were dissected for wire myography (n = 6 at each time point). Additional in vitro studies using HMEC-1 endothelial cells were also conducted to investigate the kinetics of interaction between placental nano-vesicles and endothelial cells.
MAIN RESULTS AND THE ROLE OF CHANCE
Nano-vesicles from first trimester human placentae localized to the lungs, liver and kidneys 24 h after injection into pregnant mice (n = 7). Exposure of pregnant mice to placental nano-vesicles for 30 min in vivo increased the vasodilatory response of mesenteric arteries to acetylcholine, while exposure for 24 h had the opposite effect (P < 0.05, n = 6). These responses were prevented by L-NAME, an NO synthase inhibitor. Placental nano-vesicles did not affect the function of uterine arteries or mesenteric arteries from non-pregnant mice. Placental nano-vesicles rapidly interacted with endothelial cells via a combination of phagocytosis, endocytosis and cell surface binding in vitro.
LARGE SCALE DATA
N/A.
LIMITATIONS REASONS FOR CAUTION
As it is not ethical to administer labelled placental nano-vesicles to pregnant women, pregnant CD1 mice were used as a model of pregnancy.
WIDER IMPLICATIONS OF THE FINDINGS
This is the first study to report the localization of placental nano-vesicles and their vascular effects in vivo. This work provides new insight into how the dramatic maternal cardiovascular adaptations to pregnancy may occur and indicates that placental extracellular vesicles may be important mediators of feto-maternal communication in a healthy pregnancy.
STUDY FUNDING/COMPETING INTEREST(S)
This research was supported by the Faculty of Medical and Health Science (FMHS) School of Medicine PBRF research fund to L.W.C. M.T. is a recipient of a University of Auckland Health Research Doctoral Scholarship and the Freemasons Postgraduate Scholarship. No authors have any competing interests to disclose.



Immobilization or mobilization after IUI: an RCT

2017-10-13

Abstract
STUDY QUESTION
Does 15 min of immobilization after IUI improve pregnancy rates?
SUMMARY ANSWER
Immobilization for 15 min after IUI does not improve pregnancy rates.
WHAT IS KNOWN ALREADY
Prior RCTs report a beneficial effect of supine immobilization for 15 min following IUI compared to immediate mobilization, however, these studies can be criticized. Given the importance for the logistics in daily practice and the lack of biological plausibility we planned a replication study prior to potential implementation of this procedure.
STUDY DESIGN, SIZE, DURATION
A single centre RCT, based in an academic setting in the Netherlands, was performed. Participants were randomly assigned for 15 min of supine immobilization following IUI for a maximum of six cycles compared to the standard procedure of immediate mobilization following IUI. Participants and caregivers were not blinded to group assignment. An independent researcher used computer-generated tables to allocate treatments. Stratification occurred to the indication of IUI (unexplained or mild male subfertility). Revelation of allocation took place just before the insemination by the caregiver. The primary outcome was ongoing pregnancy rate per couple.
PARTICIPANTS/MATERIALS, SETTING, METHODS
A total of 498 couples diagnosed with unexplained or mild male subfertility and an indication for treatment with IUI were approached and randomized in the study, of which 244 participants were assigned to 15 min of supine immobilization and 254 participants to immediate mobilization.
MAIN RESULTS AND THE ROLE OF CHANCE
Participant characteristics were comparable between the groups, and 236 participants were analysed in the immobilization group, versus 245 in the mobilization group. The ongoing pregnancy rate per couple was not found to be superior in the immobilization group (one-sided P-value = 0.97) with 76/236 ongoing pregnancies (32.2%) being accomplished in the immobilization and 98/245 ongoing pregnancies (40.0%) in the immediate mobilization group (relative risk 0.81; 95% CI [0.63, 1.02], risk difference: −7.8%, 95% CI [−16.4%, 0.8%]). No difference was found in miscarriage rate, multiple gestation rate, live birth rate and time to pregnancy between the groups.
LIMITATIONS, REASONS FOR CAUTION
Owing to discontinuation of the planned treatment not all participants reached six IUI cycles or an ongoing pregnancy. However, this is as expected in IUI treatment and mirrors clinical practice. These participants were equally distributed across the two groups. Women with tubal pathology and endocrine disorders were excluded for this trial, and this might narrow generalizability.
WIDER IMPLICATIONS OF THE FINDINGS
This study shows no positive effect of 15 min of immobilization following IUI on pregnancy rates. Based on available evidence today, including our study, a possible beneficial effect of supine immobilization after IUI is at least doubtful and straightforward implementation does not seem to be justified.
STUDY FUNDING/COMPETING INTEREST(S)
No funding was received. All authors have nothing to disclose.
TRIAL REGISTRATION NUMBER
Dutch Trial Register NTR 2418.
TRIAL REGISTRATION DATE
20 July 2010.
DATE OF FIRST PATIENT's ENROLMENT
11 August 2010.



Targeted metabolomics reveals reduced levels of polyunsaturated choline plasmalogens and a smaller dimethylarginine/arginine ratio in the follicular fluid of patients with a diminished ovarian reserve

2017-10-13

ABSTRACT
STUDY QUESTION
Does the metabolomic profile of the follicular fluid (FF) of patients with a diminished ovarian reserve (DOR) differ from that of patients with a normal ovarian reserve (NOR)?
SUMMARY ANSWER
The metabolomic signature of the FF reveals a significant decrease in polyunsaturated choline plasmalogens and methyl arginine transferase activity in DOR patients compared to NOR patients.
WHAT IS KNOWN ALREADY
The composition of the FF reflects the exchanges between the oocyte and its microenvironment during its acquisition of gametic competence. Studies of the FF have allowed identification of biomarkers and metabolic pathways involved in various pathologies affecting oocyte quality, but no large metabolomic analysis in the context of ovarian ageing and DOR has been undertaken so far.
STUDY DESIGN, SIZE, DURATION
This was an observational study of the FF retrieved from 57 women undergoing in vitro fertilization at the University Hospital of Angers, France, from November 2015 to September 2016. The women were classified in two groups: one including 28 DOR patients, and the other including 29 NOR patients, serving as controls.
PARTICIPANTS/MATERIALS, SETTING, METHODS
Patients were enrolled in the morning of oocyte retrieval after ovarian stimulation. Once the oocytes were isolated for fertilization and culture, the FF was pooled and centrifuged for analysis. A targeted quantitative metabolomic analysis was performed using high-performance liquid chromatography coupled with tandem mass spectrometry, and the Biocrates Absolute IDQ p180 kit. The FF levels of 188 metabolites and several sums and ratios of metabolic significance were assessed by multivariate and univariate analyses.
MAIN RESULTS AND THE ROLE OF CHANCE
A total of 136 metabolites were accurately quantified and used for calculating 23 sums and ratios. Samples were randomly divided into training and validation sets. The training set, allowed the construction of multivariate statistical models with a projection-supervised method, i.e. orthogonal partial least squares discriminant analysis (OPLS-DA), applied to the full set of metabolites, or the penalized least absolute shrinkage and selection operator with logistic regression (LASSO-LR), applied to the ratios and sums of the metabolites. Both multivariate models showed good predictive performances when applied to the validation set. The final penalized model retained the three most significant variables, i.e. the total dimethylarginine-to-arginine ratio (Total DMA/Arginine), the sum of the polyunsaturated choline plasmalogens (PUFA ae), and the patient's age. The negative coefficients of Total DMA/Arginine and PUFA ae indicated that these FF variables had lower values in DOR patients than in NOR patients.
LARGE SCALE DATA
N/A.
LIMITATIONS REASONS FOR CAUTION
This study presents two limitations. First, with this targeted metabolomics analysis, we have explored only a limited portion of the FF metabolome. Second, although the signature found was highly significant, the mechanism underlying the dysfunction remains undetermined.
WIDER IMPLICATIONS OF THE FINDINGS
The understanding of the mechanisms implied in ovarian ageing is essential for providing an adequate response to affected women desiring pregnancy. Our study proposes an incoming signature that may open new paths towards this goal.
STUDY FUNDING/COMPETING INTEREST(S)
This study was supported by the University Hospital of Angers, the University of Angers, and the French national research centers, INSERM and the CNRS. There were no competing interests.



High gonadotropin dosage does not affect euploidy and pregnancy rates in IVF PGS cycles with single embryo transfer

2017-10-12

Abstract
STUDY QUESTION
Does high gonadotropin dosage affect euploidy and pregnancy rates in PGS cycles with single embryo transfer?
SUMMARY ANSWER
High gonadotropin dosage does NOT affect euploidy and pregnancy rates in PGS cycles with single embryo transfer.
WHAT IS KNOWN ALREADY
PGS has been proven to be the most effective and reliable method for embryo selection in IVF cycles. Euploidy and blastulation rates decrease significantly with advancing maternal age. In order to recruit an adequate number of follicles, the average dosage of gonadotropins administered during controlled ovarian stimulation in IVF cycles often increases significantly with advancing maternal age.
STUDY DESIGN, SIZE, AND DURATION
A retrospective study of SNP (Single Nucleotide Polymorphism) PGS outcome data from blastocysts biopsied on day 5 or day 6 was conducted to identify differences in euploidy and clinical pregnancy rates. Seven hundred and ninety four cycles of IVF treatment with PGS between January 2013 and January 2017 followed by 651 frozen embryo transfers were included in the study (506 patients, maternal age (y.o.) – 37.2 ± 4.31).
PARTICIPANTS/MATERIALS, SETTING, METHODS
A total of 4034 embryos were analyzed (5.1 ± 3.76 per case) for euploidy status. All embryos were vitrified after biopsy, and selected embryos were subsequently thawed for a hormone replacement frozen embryo transfer cycle. All cycles were analyzed by total gonadotropin dosage (<3000 IU, 3000–5000 IU and >5000 IU), by number of eggs retrieved (1–5, 5–10, 10–15 and >15 eggs) and patient's age (<35, 35–37, 38–40 and ≥41 y.o.). Clinical pregnancy rate was defined by the presence of a fetal heartbeat at 6–7 weeks of gestation.
MAIN RESULTS AND THE ROLE OF CHANCE
Euploidy rates within the same age group were not statistically different regardless of the total dosage of gonadotropins used or the number of eggs retrieved. In the youngest group of patients (<35 y.o. – 187 IVF cycles) euploidy rates ranged from 62.3% (<3000 IU were used in the IVF cycle) to 67.5% (>5000 IU were used in the IVF cycle) (OR = 0.862, 95% CI 0.687–1.082, P = 0.2) and from 69.5% (1–5 eggs retrieved) to 60.0% (>15 eggs retrieved) (OR = 0.658, 95% CI 0.405–1.071, P = 0.09). Similar data were obtained in the oldest group of patients (≥41 y.o. – 189 IVF cycles): euploidy rates ranged from 30.7 to 26.4% (OR = 0.811, 95% CI 0.452–1.454, P = 0.481) when analyzed by total dosage of gonadotropins used in the IVF cycle and from 40.0 to 30.7% (OR = 0.531, 95% CI 0.204–1.384, P = 0.19), when assessed by the total number of eggs retrieved. Ongoing pregnancy rates were similar, not only within particular age groups, but also between different age groups regardless of the total dosage of gonadotropins used: ranging from to 63.6% (<3000 IU, < 35 y.o.) to 54.8% (>5000 IU, ≥41 y.o) (OR = 0.696, 95% CI 0.310–1.565, P = 0.38).
LIMITATIONS, REASONS FOR CAUTION
Retrospective study and heterogeneity of patients included.
WIDER IMPLICATIONS OF THE FINDINGS
These data are reassuring for the common practice of increasing gonadotropin dosages in PGS cycles, particularly in older woman.
STUDY FUNDING/COMPETING INTEREST(S)
No formal funding has been received for this study.
TRIAL REGISTRATION NUMBER
N/A.



Taste receptor polymorphisms and male infertility

2017-10-12

Abstract
STUDY QUESTION
Are polymorphisms of taste receptor genes associated with male infertility?
SUMMARY ANSWER
This study has showed the associations between three single nucleotide polymorphisms (SNPs) in taste receptors genes (TASR) and male infertility.
WHAT IS KNOWN ALREADY
Recent studies showed the expression of taste receptors in the testis and in spermatozoa, suggesting their possible role in infertility. The vast genetic variability in taste genes results in a large degree of diversity in various human phenotypes.
STUDY DESIGN, SIZE, DURATION
In this study, we genotyped 19 SNPs in 12 taste related genes in a total of 494 Caucasian male patients undergoing semen evaluation at the Centre of Couple Sterility of the Siena University Hospital. Consecutive patients were enrolled during infertility investigations from October 2014 to February 2016.
PARTICIPANTS/MATERIALS, SETTING, METHODS
Median age of the patients was 36 years (18–58) and 141 were smokers. Genotyping was performed using the allele-specific PCR. The statistical analysis was carried out using generalized linear model (GLM) to explore the association between age, smoking, the genetic polymorphisms and sperm parameters.
MAIN RESULTS AND THE ROLE OF CHANCE
We observed that the homozygous carriers of the (G) allele of the TAS2R14-rs3741843 polymorphism showed a decreased sperm progressive motility compared to heterozygotes and (A) homozygotes (P = 0.003). Moreover, the homozygous carriers of the (T) allele of the TAS2R3-rs11763979 SNP showed fewer normal acrosome compared with the heterozygous and the homozygous carriers of the (G) allele (P = 0.002). Multiple comparisons correction was applied and the Bonferroni-corrected critical P-value was = 0.003.
LIMITATIONS, REASONS FOR CAUTION
The analysis is restricted to SNPs within genes and to men of Caucasian ancestry.
WIDER IMPLICATIONS OF THE FINDINGS:
In silico analyses strongly point towards a functional effect of the two SNPs: TAS2R14-rs3741843 regulates TAS2R43 expression, a gene that is involved in cilia motility and therefore could influences sperm mobility; the (T) allele of TAS2R3-rs11763979 increases the expression of the WEE2 antisense RNA one gene (WEE2-AS1). According to Genotype-Tissue Expression (GTEx) project the WEE2 gene is expressed in the testes where presumably it has the role of down regulating meiotic cell division. It is plausible to hypothesize that the WEE2-AS1 increased expression may down regulate WEE2 which in turn can alter the natural timing of sperm maturation increasing the number of abnormal sperm cells.
STUDY FUNDING/COMPETING INTEREST(S)
None



Constructing the crystal ball: how to get reliable prognostic information for the management of subfertile couples

2017-10-12

Abstract
Couples in whom the results of an initial fertility workup fail to identify the presence of any obvious barriers to conception are diagnosed with unexplained subfertility. Couples who have tried to conceive for a relatively short time have a good chance of natural conception and thus may not benefit from immediate access to ART. As fertility decreases over time, the main dilemma that clinicians and couples face is when to abandon an expectant approach in favour of active treatment. Several prognostic or predictive models have been used to try to discriminate between couples with high and low chances of conception but have been unable to compare individualized chances of conception associated with ART relative to chances of natural conception at various time points. These models are also unable to recalculate the chances of pregnancy at subsequent time points in those who return after a period of unsuccessful expectant management. In this paper, we discuss currently available models. We conclude that in order to provide accurate, individualized and dynamic fertility prognoses associated with and without treatment at different points in time, we need to develop, validate and update clinical prediction models which are fit for purpose. We suggest several steps to move the field forwards.



Fertility-related knowledge and reproductive goals in childhood cancer survivors: short communication

2017-09-29

Abstract
STUDY QUESTION
Do young adult survivors of childhood cancer know their fertility status, in the context of their parenthood goals and screening for gonadal functioning?
SUMMARY ANSWER
While 80% of survivors (who were without children) wanted children in the future, most did not know their fertility status, and screening for gonadal functioning was underutilized.
WHAT IS KNOWN ALREADY
Survivors of childhood cancer are at risk for infertility, but fertility counseling and assessment are underutilized. Separate studies indicated that survivors’ fertility-related knowledge is poor and that they often wanted to have children. Yet, studies have not investigated the intersection of both issues, as well as potential distress if parenthood goals are not met.
STUDY DESIGN, SIZE, DURATION
Young adult male and female survivors of childhood cancer (N = 149) completed cross-sectional surveys, and data for those without children (n = 105, 70.5%) are presented here.
PARTICIPANTS/MATERIALS, SETTING, METHODS
Participants were 20–40 years old (M = 26.5), diagnosed 5–33 years prior to study participation, and completed questionnaires online. Knowledge of fertility status, parenthood goals, and potential distress if survivors were unable to have children were assessed. Medical records were reviewed for hormone levels as indicators of screening for gonadal functioning.
MAIN RESULTS AND THE ROLE OF CHANCE
Most survivors (n = 81; 77.1%) did not know their fertility status, while over 80% (n = 89) wanted children (neither aspect varied by socio-demographic/cancer-specific factors). Two-thirds of survivors indicated they would be distressed if parenthood goals remained unfulfilled; especially female (versus male, t = 2.64; P = 0.01) or partnered (versus single, t = −3.45; P < 0.001) survivors. Forty survivors (38.1%) had documented assessments of gonadal functioning, of which 33 (82.5%) reported not knowing their fertility status.
LIMITATIONS, REASONS FOR CAUTION
Relevant risk factors may have not been identified owing to limited sample size and missing treatment information. The underutilization of screening for gonadal functioning needs further exploration in other pediatric centers.
WIDER IMPLICATIONS OF THE FINDINGS
Most adult childhood cancer survivors want to become parents, but do not know their fertility status, which could cause significant psychological distress. Healthcare providers should continuously address fertility among survivors, but more research is needed on how to implement routine fertility counseling and/or testing.
STUDY FUNDING/COMPETING INTEREST(S)
This study was funded by the Research Institute at Nationwide Children's Hospital (V.L.) and Dutch Cancer Society (RUG2009-4442, M.A.T.). All authors have no conflict of interest to declare.



Genomic fragmentation and extrachromosomal telomeric repeats impact assessment of telomere length in human spermatozoa: quantitative experiments and systematic review

2017-09-29

ABSTRACT
STUDY QUESTION
Can differences in DNA isolation alter assessment of sperm telomere length (spTL) and do they account for conflicting results in the literature on spTL and male fertility?
SUMMARY ANSWER
DNA isolation methods preferentially include or exclude short, extrachromosomal (EC) telomere-specific sequences that alter spTL measurements, and are responsible for a proportion of the disparity observed between investigations.
WHAT IS KNOWN ALREADY
The relationship between spTL and male fertility has become an active area of research. The results across investigations, however, have been discordant, generating a need to critically evaluate the existing body of knowledge to guide future investigations.
STUDY DESIGN, SIZE, DURATION
Quantitative experiments determined the effect of DNA isolation on the integrity of sperm DNA and measures of spTL, while a systematic analysis of the current literature evaluated the effect of DNA isolation and study design on experimental outcomes.
PARTICIPANTS/MATERIALS, SETTING, METHODS
Two DNA isolation methods were compared: Genomic Tips which isolate ‘High Molecular Weight’ (HMW) DNA exclusively, and QIAamp® DNA Mini which isolates ‘Total’ genomic DNA irrespective of size. DNA quality was assessed via field inversion gel electrophoresis (FIGE) and spTL was measured via terminal restriction fragment analysis. In addition, major databases in medicine, health and the life sciences were subject to a targeted search, and results were independently screened according to defined exclusion/inclusion criterion. Findings from primary articles were analyzed for concordance and study designs were compared across six moderator variables (sample size, participant age, fertility status, semen fraction, telomere population and type of analysis).
MAIN RESULTS AND THE ROLE OF CHANCE
HMW DNA spTL was significantly longer than spTL measured from total DNA (P < 0.01), indicating that Total DNA contained short, EC telomeric repeats that shifted downstream assessment towards shorter spTL. HMW DNA spTL reflected the length of intact, chromosomal telomeres. Major findings on spTL showed the greatest concordance amongst studies that implemented HMW DNA isolation prior to spTL assessment. Studies that utilized Total DNA varied in concordance, but outcomes were similar if (i) a comparative analysis was applied or (ii) a sample size threshold of 81 was achieved for correlative analysis.
LIMITATIONS, REASONS FOR CAUTION
Chromosomal and EC telomeric DNA were distinguished based on outcomes of HMW DNA isolation and size. Further experiments are required to determine the nature and function of these two types of telomeric sequences.
WIDER IMPLICATIONS OF THE FINDINGS
This study reveals a dramatic impact of upstream DNA processing and study design on measurements of spTL, which accounts for conflicting results in the literature. Future assessments of spTL should incorporate independent detection of chromosomal and EC telomeric DNA and specific experimental planning.
STUDY FUNDING/COMPETING INTERESTS
This study was funded by CReATe Fertility Centre, Toronto, Ontario, Canada. The authors have declared no conflict of interest.
REGISTRATION NUMBER
N/A.



Endometriosis fertility index predicts live births following surgical resection of moderate and severe endometriosis

2017-09-29

AbstractSTUDY QUESTIONCan live birth be accurately predicted following surgical resection of moderate-severe (Stage III–IV) endometriosis?SUMMARY ANSWERLive births can accurately be predicted with the endometriosis fertility index (EFI), with adnexal function being the most important factor to predict non-assisted reproductive technology (non-ART) fertility or the requirement for ART (www.endometriosisefi.com).WHAT IS KNOWN ALREADYFertility prognosis is important to many women with severe endometriosis. Controversy persists regarding optimal post-operative management to achieve pregnancy and the counselling of patients regarding duration of conventional treatments before undergoing ART. The EFI is reported to correlate with expectant management pregnancy rate, although external validation has been performed without specifically addressing fertility in women with moderate and severe endometriosis.STUDY DESIGN, SIZE, DURATIONRetrospective cohort study of 279 women from September 2001 to June 2016.PARTICIPANTS/MATERIALS, SETTINGS, METHODSWe included women undergoing laparoscopic resection of Stage III–IV endometriosis who attempted pregnancy post-operatively. The EFI was calculated based on detailed operative reports and surgical images. Fertility outcomes were obtained by direct patient contact. Kaplan–Meier model, log rank test and Cox regression were used for analyses.MAIN RESULTS AND THE ROLE OF CHANCEThe follow-up rate was 84% with a mean duration of 4.1 years. A total of 147 women (63%) had a live birth following surgery, 94 of them (64%) without ART. The EFI was highly associated with live births (P < 0.001): for women with an EFI of 0–2 the estimated cumulative non-ART live birth rate at five years was 0% and steadily increased up to 91% with an EFI of 9–10, while the proportion of women who attempted ART and had a live birth, steadily increased from 38 to 71% among the same EFI strata (P = 0.1). A low least function score was the most significant predictor of failure (P = 0.003), followed by having had a previous resection (P = 0.019) or incomplete resection (P = 0.028), being older than 40 compared to <35 years of age (P = 0.027), and having leiomyomas (P = 0.037).LIMITATIONS REASONS FOR CAUTIONThe main limitation of this study is its retrospective design. Imprecision was higher with low EFI due to smaller sample size in this subgroup. Finally, the EFI is somewhat subjective and could be prone to intra- and inter-observer variations.WIDER IMPLICATIONS OF THE FINDINGSWomen with a high EFI score have excellent fertility prognosis and may be advised to try to become pregnant with timed intercourse compared to women with a low score, for which prompt referral to ART seems more reasonable. Other prognostic factors can be used to guide the management of women with an intermediate EFI score. These data follow women over many years post-resection and represent longitudinal fertility data rarely demonstrated in such a cohort. The location and impact of lesions on the ability of the adnexa to function seems crucial for the fertility prognosis and should be further investigated.STUDY FUNDING/COMPETING INTEREST(S)This study was funded by the GRACE Research funds. S.M.-L. is the recipient of a Training Award from the Fonds de Recherche Quebec-Sante. D.A. is the primary author of the Endometriosis Fertility Index. All authors have no conflicts of interest to declare.TRIAL REGISTRATION NUMBERN/A.[...]



The prevalence of Type 2 diabetes is not increased in normal-weight women with PCOS

2017-09-29

Abstract
STUDY QUESTION
Is oral glucose tolerance test (OGTT) needed in all women with polycystic ovary syndrome (PCOS)?
SUMMARY QNSWER
OGTT is not routinely needed in women with PCOS and BMI < 25 kg/m2.
WHAT IS KNOWN ALREADY
PCOS is associated with insulin resistance and increased prevalence of prediabetes and Type 2 diabetes (T2D) which is closely linked to obesity and possibly age, ethnicity and PCOS phenotype. Several guidelines recommend OGTT upon diagnosis of PCOS and during follow-up.
STUDY DESIGN, SIZE, DURATION
A Nordic cross-sectional study including 876 women.
PARTICIPANTS/MATERIALS, SETTING, METHODS
The 876 Nordic women with PCOS, aged 14–57 years, were examined for T2D and prediabetes (impaired glucose tolerance [IGT] or impaired fasting glucose (IFG) by OGTT.
MAIN RESULT AND THE ROLE OF CHANCE
Of all study subjects 3% (23/876) had T2D, 23% (204/876) prediabetes and 74% (649/876) had normal glucose tolerance (NGT). Increased BMI and waist circumference were significantly (P < 0.001) associated with prevalence of prediabetes and T2D. No normal-weight woman (BMI < 25 kg/m2) was diagnosed with T2D. The prevalence of BMI ≥ 25 kg/m2 was 66% (578/ 876). 91% of women (21/23) with T2D had BMI ≥ 30 kg/m2. Testosterone levels and PCOS phenotype did not predict 2-h glucose levels during OGTT after adjustment for BMI and age.
LIMITATIONS, REASONS FOR CAUTION
The present study included cross-sectional data and prospective studies are needed to confirm our results. These results may not apply to populations of other ethnic origin.
WIDER IMPLICATIONS OF THE FINDINGS
Routine OGTT may not be indicated in normal-weight women with PCOS.
STUDY FUNDING/COMPETING INTEREST(S)
None.
TRIAL REGISTRATION NUMBER
N/A.



Cultural determinants influence assisted reproduction usage in Europe more than economic and demographic factors

2017-09-29

Abstract
STUDY QUESTION
To what extent do financial, demographic and cultural determinants explain the vast cross-national differences in ART treatments in Europe?
SUMMARY ANSWER
The normative cultural acceptance of ART is a major driver of ART treatments in Europe, above and beyond differences in country wealth, demographic aspects and religious composition.
WHAT IS KNOWN ALREADY
There are vast differences in the number of ART treatments across European countries, which are to some extent related to country affluence, regulation, and insurance coverage and costs. The role and impact of cultural and normative factors has not been explored in a larger cross-national comparison.
STUDY DESIGN, SIZE, DURATION
A descriptive and comparative cross-national analysis of ART treatment prevalence in over 30 European countries in 2010, with the outcome defined as the total number of ART cycles per million women of reproductive age (15–44 years). Data is drawn from multiple sources (ICMART, US Census Bureau Library, World Bank, Barro–Lee Educational Attainment Dataset, IFFS Surveillance reports, European Values Study and World Religion Database).
PARTICIPANTS/MATERIALS, SETTING, METHODS
Our sample includes data from 35 European countries, where we describe the associations between demographic and cultural factors and the prevalence of ART treatments. Bivariate correlation and ordinary least squares multiple regression analysis serves to establish the relationships between predictor variables and the number of ART treatments per million women aged 15–44 years in a country.
MAIN RESULTS AND THE ROLE OF CHANCE
A one-percent increase in national GDP is associated with 382 (95% CI: 177–587) additional ART procedures per million women of reproductive age, yet this effect is reduced to 99 (−92 to 290) treatments once cultural values and demographic factors are accounted for. In our fully adjusted model, normative cultural values measuring the acceptability of ART are the strongest predictor of ART usage, with a one-point increase of average approval in a country associated with 276 (167–385) additional ART treatments per million women of reproductive age.
LIMITATIONS, REASONS FOR CAUTION
Findings are based on a cross-sectional, cross-national analysis, making formal tests of causality impossible and prohibiting inferences to the individual level.
WIDER IMPLICATIONS OF THE FINDINGS
Results indicate that reproductive health policy should openly acknowledge the importance of cultural norms in informally shaping and regulating the wider availability of ART treatment.
STUDY FUNDING/COMPETING INTEREST(S)
Funding for this project was provided by the European Union's Seventh Framework Program (FP7 2007–2013) (No. 320116 Families and Societies), European Research Council for the SOCIOGENOME Consolidator Grant (ERC-2013-CoG-615603) and the Wellcome Trust Institutional Strategic Support Fund (all to M.C.M.). The authors have no conflicts of interest to declare.
TRIAL REGISTRATION NUMBER
N/A.



N-cadherin identifies human endometrial epithelial progenitor cells by in vitro stem cell assays

2017-09-27

AbstractSTUDY QUESTIONIs there a specific surface marker that identifies human endometrial epithelial progenitor cells with adult stem cell activity using in vitro assays?SUMMARY ANSWERN-cadherin isolates clonogenic, self-renewing human endometrial epithelial progenitor cells with high proliferative potential that differentiate into cytokeratin+ gland-like structures in vitro and identifies their location in some cells of gland profiles predominantly in basalis endometrium adjacent to the myometrium.WHAT IS KNOWN ALREADYHuman endometrium contains a small population of clonogenic, self-renewing epithelial cells with high proliferative potential that differentiate into large gland-like structures, but their identity and location is unknown. Stage-specific embryonic antigen-1 (SSEA-1) distinguishes the epithelium of basalis from functionalis and is a marker of human post-menopausal (Post-M) endometrial epithelium.STUDY DESIGN, SIZE, DURATIONProspective observational study of endometrial epithelial cells obtained from hysterectomy samples taken from 50 pre-menopausal (Pre-M) and 24 Post-M women, of which 4 were from women who had taken daily estradiol valerate 2 mg/day for 8 weeks prior.PARTICIPANTS/MATERIALS, SETTING, METHODSGene profiling was used to identify differentially expressed surface markers between fresh EpCAM (Epithelial Cell Adhesion Molecule)-magnetic bead-selected basalis-like epithelial cells from Post-M endometrium compared with predominantly functionalis epithelial cells from Pre-M endometrium and validated by qRT-PCR. In vitro clonogenicity and self-renewal assays were used to assess the stem/progenitor cell properties of magnetic bead-sorted N-cadherin+ and N-cadherin− epithelial cells. The cellular identity, location and phenotype of N-cadherin+ cells was assessed by dual colour immunofluorescence and confocal microscopy for cytokeratin, proliferative status (Ki-67), ERα, SSEA-1, SOX9 and epithelial mesenchymal transition (EMT) markers on full thickness human endometrium.MAIN RESULTS AND THE ROLE OF CHANCECDH2 (N-cadherin gene) was one of 11 surface molecules highly expressed in Post-M compared to Pre-M endometrial epithelial cells. N-cadherin+ cells comprise a median 16.7% (n = 8) and 20.2% (n = 5) of Pre-M endometrial epithelial cells by flow cytometry and magnetic bead sorting, respectively. N-cadherin+ epithelial cells from Pre-M endometrium were more clonogenic than N-cadherin− cells (n = 12, P = 0.003), underwent more population doublings (n = 7), showed greater capacity for serial cloning (n = 7) and differentiated into cytokeratin+ gland-like organoids. N-cadherin immunolocalised to the lateral and apical membrane of epithelial cells in the bases of glands in the basalis of Pre-M endometrium and Post-M gland profiles, co-expressing cytokeratin, ERα but not SSEA-1 or SOX9, which localized on gland profiles proximal to N-cadherin+ cells. N-cadherin+ cells were quiescent (Ki-67−) in the basalis and in Post-M endometrial glands and co-localized with EMT markers vimentin and E-cadherin.LARGE SCALE DATAThe raw and processed data files from the gene microarray have been deposited in the National Center for Biotechnology Information Gene Expression Omnibus data set with accession number GSE35221.LIMITATIONS, REASONS FOR CAUTIONThis is a descriptive study in human endometrium only using in vitro stem cell assays. The differential ability of N-cadherin+ and N-cadherin−cells to generate endometrial glands in vivo was not determined. A small number of uterine tissues analysed contained adenomyosis for which N-cadherin has been implicated in epithelial-EMT.WIDER IMPLICATIONS OF THE FINDINGSA new marker enriching for human endometrial epithelial progenitor cells identifies a different and potentially more[...]



Corifollitropin alfa followed by highly purified HMG versus recombinant FSH in young poor ovarian responders: a multicentre randomized controlled clinical trial

2017-09-23

AbstractSTUDY QUESTIONDoes administration of corifollitropin alfa followed by highly purified (hp) HMG result in higher ongoing pregnancy rates compared with daily recombinant FSH (rFSH) in young poor responders?SUMMARY ANSWERCorifollitropin alfa followed by hp-HMG does not increase ongoing pregnancy rates compared with rFSH in young poor responders, although more supernumerary cryopreserved embryos were obtained with corifollitropin alfa and hp-HMG.WHAT IS KNOWN ALREADYPoor ovarian response remains one of the main therapeutic challenges in women undergoing ovarian stimulation, given that very low live birth rates of 6% have been reported in this particular group of infertile patients. Nevertheless, concerns have been raised that a degree of heterogeneity remains, as the prognostic effect of individual factors is still unclear, particularly for the young poor responder group. The rationale for conducting the current randomized trial was based on the results of a previous pilot study demonstrating promising results with the administration of hp-HMG following corifollitropin alpha in women younger than 40 years of age, fulfilling the ‘Bologna’ criteria.STUDY DESIGN, SIZE, DURATIONA multicenter, phase III, superiority, randomized trial was conducted using a parallel two-arm design. The study included 152 patients younger than 40 years old and fulfilling the ‘Bologna’ criteria for poor ovarian response, from one tertiary referral centre in Europe and one tertiary referral centre in Asia. Enrolment was performed from March 2013 to May 2016.PARTICIPANTS/MATERIALS, SETTING, METHODSEligible patients were randomized to either administration of 150 μg corifollitropin alfa followed by 300 IU hp-HMG (Group A) or to 300 IU of daily recombinant FSH (Group B) in a fixed GnRH antagonist protocol. The randomization sequence was created using a computer generated randomization list stratified by centre, using 1:1 allocation. The primary outcome was ongoing pregnancy rate (defined as the presence of an intrauterine gestational sac with an embryonic pole demonstrating cardiac activity at 9–10 weeks of gestation). Secondary outcomes included embryo cryopreservation rates, clinical and biochemical pregnancy rates and number of oocytes retrieved.MAIN RESULTS AND THE ROLE OF CHANCEOverall, 152 poor ovarian responders defined by the ‘Bologna’ criteria were included in the study. Using an intention-to treat analysis, the ongoing pregnancy rates did not differ significantly between Group A 11/77 (14.3%) and Group B 11/70 (15.7%), absolute difference: −0.4 (−11.5 to 10.8), OR = 0.9 (0.4–2.4). Biochemical and clinical pregnancy rates, live birth rates and the number of oocytes retrieved were also comparable between the two groups. Nevertheless, more patients in the corifollitropin alfa group had cryopreserved embryos compared to the rFSH group [22 (28.6%) versus 10 (14.3%), OR = 2.4 (1.01–5.5)]. Incidentally, Asian patients had significantly lower cancellation rates compared to European poor responders [2/64 (3.1%) versus 17/83 (20.4%), OR = 0.12 (0.03–0.5)]. This discrepancy could be explained by the fact that Asian women were better prognosis patients than European patients, with significantly lower FSH [9.8 (5.3) versus 11.5 (5.4), P = 0.017] and significantly higher AMH [1.1 (0.9) versus 0.4 (0.3), P-value <0.001] levels.LIMITATIONS, REASONS FOR CAUTIONOngoing pregnancy rates close to 14% for both treatment groups differ significantly from the hypothesized primary outcome rates used in the power calculation. Therefore, our randomized trial might have been underpowered to detect smaller differences. The use of multiple secondary outcomes and multiple comparisons could have increased a Type 1 error[...]



Clinical implications of mitochondrial DNA quantification on pregnancy outcomes: a blinded prospective non-selection study

2017-09-23

AbstractSTUDY QUESTIONCan quantification of mitochondrial DNA (mtDNA) in trophectoderm (TE) biopsy samples provide information concerning the viability of a blastocyst, potentially enhancing embryo selection and improving IVF treatment outcomes?SUMMARY ANSWERThis study demonstrated that euploid blastocysts of good morphology, but with high mtDNA levels had a greatly reduced implantation potential.WHAT IS KNOWN ALREADYBetter methods of embryo selection leading to IVF outcome improvement are necessary, as the transfer of chromosomally normal embryos of high morphological grade cannot guarantee the establishment of an ongoing pregnancy. The quantity of mtDNA in embryonic cells has been proposed as a new biomarker of viability—higher levels of mtDNA associated with reduced implantation potential.STUDY DESIGN, SIZE, DURATIONmtDNA was quantified in 199 blastocysts, previously biopsied and shown to be chromosomally normal using preimplantation genetic testing for aneuploidy (PGT-A). These were generated by 174 couples (average female age 37.06 years). All patients underwent IVF in a single clinic. The study took place in a blinded, non-selection manner—i.e. mtDNA quantity was not known at the time of single embryo transfer. The fate of the embryos transferred was subsequently compared to the mtDNA levels measured.PARTICIPANTS/MATERIALS, SETTING, METHODSEmbryos were biopsied at the blastocyst stage. The TE samples obtained were subjected to whole genome amplification followed by comprehensive chromosome analysis via next generation sequencing. The same biopsy specimens were also tested using quantitative PCR, allowing highly accurate mtDNA quantification. After blastocyst transfer, the code used for blinding was broken and analysis undertaken to reveal whether the amount of mtDNA had any association with embryo implantation.MAIN RESULTS AND THE ROLE OF CHANCEmtDNA analysis of the 199 blastocysts revealed that 9 (5%) contained unusually high levels of mtDNA. All embryo transfers involved a single chromosomally normal blastocyst of good morphology. Of these, 121 (60%) led to ongoing pregnancies, 11(6%) led to biochemical pregnancies, and 10 (5%) spontaneously miscarried. All (100%) of these blastocysts had mtDNA levels considered to be normal/low. The remaining 57 (29%) blastocysts failed to implant. Among these non-viable embryos there were 9 (16%) with unusually high levels of mtDNA. This meant that the ongoing pregnancy rate for morphologically good, euploid blastocysts, with normal/low levels of mtDNA was 64% (121/190). In contrast, the ongoing pregnancy rate for the same type of embryos, but with elevated mtDNA levels, was 0/9 (0%). This difference was highly statistically significant (P < 0.0001).LIMITATIONS REASONS FOR CAUTIONTo determine the true extent of any clinical benefits a randomized clinical trial will be necessary. Research is needed to improve understanding of the biology of mtDNA expansion.WIDER IMPLICATIONS OF THE FINDINGSThis is the first investigation to evaluate the clinical impact of increased mtDNA in a prospective blinded manner. Results confirm that embryos with elevated mtDNA rarely implant, supporting its use as a viability biomarker. A total of 64% of euploid blastocysts with normal/low mtDNA implanted versus 60% for the cohort as a whole.STUDY FUNDING/COMPETING INTEREST(S)This study was supported by institutional funding (Reprogenetics UK and Reprogenetics). DW is supported by the National Institute for Health Research (NIHR) Oxford Biomedical Research Centre Programme. None of the authors have any competing interests.[...]



Genome stability of bovine in vivo -conceived cleavage-stage embryos is higher compared to in vitro -produced embryos

2017-09-23

AbstractSTUDY QUESTIONIs the rate and nature of chromosome instability (CIN) similar between bovine in vivo-derived and in vitro-cultured cleavage-stage embryos?SUMMARY ANSWERThere is a major difference regarding chromosome stability of in vivo-derived and in vitro-cultured embryos, as CIN is significantly lower in in vivo-derived cleavage-stage embryos compared to in vitro-cultured embryos.WHAT IS KNOWN ALREADYCIN is common during in vitro embryogenesis and is associated with early embryonic loss in humans, but the stability of in vivo-conceived cleavage-stage embryos remains largely unknown.STUDY DESIGN, SIZE, DURATIONBecause human in vivo preimplantation embryos are not accessible, bovine (Bos taurus) embryos were used to study CIN in vivo. Five young, healthy, cycling Holstein Friesian heifers were used to analyze single blastomeres of in vivo embryos, in vitro embryos produced by ovum pick up with ovarian stimulation (OPU-IVF), and in vitro embryos produced from in vitro matured oocytes retrieved without ovarian stimulation (IVM-IVF).PARTICIPANTS/MATERIALS, SETTING, METHODSSingle blastomeres were isolated from embryos, whole-genome amplified and hybridized on Illumina BovineHD BeadChip arrays together with the bulk DNA from the donor cows (mothers) and the bull (father). DNA was also obtained from the parents of the bull and from the parents of the cows (paternal and maternal grandparents, respectively). Subsequently, genome-wide haplotyping and copy-number profiling was applied to investigate the genomic architecture of 171 single bovine blastomeres of 16 in vivo, 13 OPU-IVF and 13 IVM-IVF embryos.MAIN RESULTS AND THE ROLE OF CHANCEThe genomic stability of single blastomeres in both of the in vitro-cultured embryo cohorts was severely compromised (P < 0.0001), and the frequency of whole chromosome or segmental aberrations was higher in embryos produced in vitro than in embryos derived in vivo. Only 18.8% of in vivo-derived embryos contained at least one blastomere with chromosomal anomalies, compared to 69.2% of OPU-IVF embryos (P < 0.01) and 84.6% of IVM-IVF embryos (P < 0.001).LARGE SCALE DATAGenotyping data obtained in this study has been submitted to NCBI Gene Expression Omnibus (GEO; accession number GSE95358)LIMITATIONS REASONS FOR CAUTIONThere were two main limitations of the study. First, animal models may not always reflect the nature of human embryogenesis, although the use of an animal model to investigate CIN was unavoidable in our study. Second, a limited number of embryos were obtained, therefore more studies are warranted to corroborate the findings.WIDER IMPLICATIONS OF THE FINDINGSAlthough CIN is also present in in vivo-developed embryos, in vitro procedures exacerbate chromosomal abnormalities during early embryo development. Hence, the present study highlights that IVF treatment compromises embryo viability and should be applied with care. Additionally, our results encourage to refine and improve in vitro culture conditions and assisted reproduction technologies.STUDY FUNDING/COMPETING INTEREST(S)The study was funded by the Agency for Innovation by Science and Technology (IWT) (TBM-090878 to J.R.V. and T.V.), the Research Foundation Flanders (FWO; G.A093.11 N to T.V. and J.R.V. and G.0392.14 N to A.V.S. and J.R.V.), the European Union's FP7 Marie Curie Industry-Academia Partnerships and Pathways (IAPP, SARM, EU324509 to J.R.V., T.V., O.T, A.D., A.S. and A.K.) and Horizon 2020 innovation programme (WIDENLIFE, 692065 to J.R.V., O.T., T.V., A.K. and A.S.). M.Z.E., J.R.V. and T.V. are co-inventors on a patent application ZL913096-PCT/EP2014/068315-WO/2015/028576 (‘Haplotyping and copy-number typing using polymorph[...]



Cumulative live birth rates following miscarriage in an initial complete cycle of IVF: a retrospective cohort study of 112 549 women

2017-09-20

AbstractSTUDY QUESTIONIn women undergoing IVF/ICSI who miscarry in their first complete cycle, what is the chance of a live birth in subsequent complete cycles, and how does this compare with those whose first complete cycle ends with live birth or without a pregnancy?SUMMARY ANSWERAfter two further complete cycles of IVF/ICSI, women who had miscarried or had a live birth in their first complete cycle had a higher chance of live birth (40.9 and 49.0%, respectively) than those who had no pregnancies (30.1%).WHAT IS KNOWN ALREADYCumulative live birth rates (CLBRs) after one or more complete cycles of IVF have been reported previously, as have some of the risk factors associated with miscarriage, both in general populations and in those undergoing IVF. Chances of cumulative live birth after a number of complete IVF cycles involving replacement of fresh followed by frozen embryos after an initial miscarriage in a population undergoing IVF treatment have not been reported previously.STUDY DESIGN, SIZE, DURATIONNational population-based cohort study of 112 549 women who started their first IVF treatment between 1999 and 2008.PARTICIPANTS/MATERIALS, SETTING, METHODSData from the UK Human Fertilisation and Embryology Authority (HFEA) register on IVF/ICSI treatments, using autologous gametes were analysed. CLBRs were estimated in women who (i) had miscarriage (and no live birth), (ii) at least one live birth or (iii) no pregnancy in their first complete cycle of IVF/ICSI (including fresh and frozen embryo transfers following a single oocyte retrieval episode). A multivariable analysis was performed to assess the effect of first complete cycle outcome on subsequent CLBRs after adjusting for confounding factors such as female age, duration of infertility and cause of infertility.MAIN RESULTS AND THE ROLE OF CHANCEIn their first complete cycle, 9321 (8.3%) women had at least one miscarriage (and no live birth); 33 152 (29.5%) had at least one live birth and 70 076 (62.3%) had no pregnancies. After two further complete cycles, conservative CLBRs (which assume that women who discontinued treatment subsequently never had a live birth) were 40.9, 49.0 and 30.1%, while optimal CLBRs (which assume that women who discontinue have the same chance of live birth as those treated) were 49.5, 57.9 and 38.4% in the miscarriage, live birth and no pregnancy groups respectively. Odds of cumulative live birth for women who miscarried in their first complete cycle were 42% higher than those who had no pregnancy [odds ratio (95% CI) = 1.42 (1.34, 1.50)], and twice as high for live birth versus no pregnancy [2.04 (1.89, 2.20)]. Negative predictors for live birth in all women included tubal infertility [0.88 (0.82, 0.94)] and increasing age [18–40 years = 0.94 (0.94, 0.95); >40 years = 0.63 (0.59, 0.66) per year].LIMITATIONS AND REASON FOR CAUTIONCLBRs could not be estimated for treatments occurring after September 2008 due to potentially incomplete data following regulatory changes regarding consent for data use in research. Additionally, covariates not included in the HFEA database (including BMI, smoking, previous history of miscarriage and gestational age at miscarriage) could not be adjusted for in our analysis.WIDER IMPLICATIONS OF THE FINDINGSMiscarriage following IVF can be devastating for couples who are uncertain about their ultimate prognosis. Our findings will provide reassurance to these couples as they consider their options for continuing treatment.STUDY FUNDING/COMPETING INTEREST(S)N.J.C. received an Aberdeen Summer Research Scholarship funded by the Institute of Applied Health Sciences (University of Aberdeen), through the Aberde[...]



Differing molecular response of young and advanced maternal age human oocytes to IVM

2017-09-15

AbstractSTUDY QUESTIONWhat effect does maternal age have on the human oocyte's molecular response to in vitro oocyte maturation?SUMMARY ANSWERAlthough polyadenylated transcript abundance is similar between young and advanced maternal age (AMA) germinal vesicle (GV) oocytes, metaphase II (MII) oocytes exhibit a divergent transcriptome resulting from a differential response to in vitro oocyte maturation.WHAT IS KNOWN ALREADYMicroarray studies considering maternal age or maturation stage have shown that either of these factors will affect oocyte polyadenylated transcript abundance in human oocytes. However, studies considering both human oocyte age and multiple stages simultaneously are limited to a single study that examined transcript levels for two genes by qPCR. Thus, polyadenylated RNA sequencing (RNA-Seq) could provide novel insight into age-associated aberrations in gene expression in GV and MII oocytes.STUDY DESIGN, SIZE, DURATIONThe effect of maternal age (longitudinal analysis) on polyadenylated transcript abundance at different stages was analyzed by examining single GV and single in vitro matured MII oocytes derived from five young (YNG; < 30 years; average age 26.8; range 20–29) and five advanced maternal age (AMA; ≥40 years; average age 41.6 years; range 40–43 years) patients. Thus, a total of 10 YNG (5 GV and 5 MII) and 10 AMA (5 GV and 5 MII) oocytes were individually processed for RNA-Seq analysis.PARTICIPANTS/MATERIALS, SETTINGS, METHODSPatients undergoing infertility treatment at the Colorado Center for Reproductive Medicine (Lone Tree, CO, USA) underwent ovarian stimulation with FSH and received hCG for final follicular maturation prior to ultrasound guided oocyte retrieval. Unused GV oocytes obtained at retrieval were donated for transcriptome analysis. Single oocytes were stored (at −80°C in PicoPure RNA Extraction Buffer; Thermo Fisher Scientific, USA) immediately upon verification of immaturity or after undergoing in vitro oocyte maturation (24 h incubation), representing GV and MII samples, respectively. After isolating RNA and generating single oocyte RNA-Seq libraries (SMARTer Ultra Low Input RNA HV kit; Clontech, USA), Illumina sequencing (100 bp paired-end reads on HiSeq 2500) and bioinformatics analysis (CLC Genomics Workbench, DESeq2, weighted gene correlation network analysis (WGCNA), Ingenuity Pathway Analysis) were performed.MAIN RESULTS AND THE ROLE OF CHANCEA total of 12 770 genes were determined to be expressed in human oocytes (reads per kilobase per million mapped reads (RPKM) > 0.4 in at least three of five replicates for a minimum of one sample type). Differential gene expression analysis between YNG and AMA oocytes (within stage) identified 1 and 255 genes that significantly differed (adjusted P < 0.1 and log2 fold change >1) in polyadenylated transcript abundance for GV and MII oocytes, respectively. These genes included CDK1, NLRP5 and PRDX1, which have been reported to affect oocyte developmental potential. Despite the similarity in transcript abundance between GV oocytes irrespective of age, divergent expression patterns emerged during oocyte maturation. These age-specific differentially expressed genes were enriched (FDR < 0.05) for functions and pathways associated with mitochondria, cell cycle and cytoskeleton. Gene modules generated by WGCNA (based on gene expression) and patient traits related to oocyte quality (e.g. age and blastocyst development) were correlated (P < 0.05) and enriched (FDR < 0.05) for functions and pathways associated with oocyte maturation.LARGE SCALE DATARaw data from this study can be accessed[...]



Preconception urinary phthalate concentrations and sperm DNA methylation profiles among men undergoing IVF treatment: a cross-sectional study

2017-09-12

Abstract
STUDY QUESTION
Are preconception phthalate and phthalate replacements associated with sperm differentially methylated regions (DMRs) among men undergoing IVF?
SUMMARY ANSWER
Ten phthalate metabolites were associated with 131 sperm DMRs that were enriched in genes related to growth and development, cell movement and cytoskeleton structure.
WHAT IS KNOWN ALREADY
Several phthalate compounds and their metabolites are known endocrine disrupting compounds and are pervasive environmental contaminants. Rodent studies report that prenatal phthalate exposures induce sperm DMRs, but the influence of preconception phthalate exposure on sperm DNA methylation in humans is unknown.
STUDY DESIGN, SIZE, DURATION
An exploratory cross-sectional study with 48 male participants from the Sperm Environmental Epigenetics and Development Study (SEEDS).
PARTICIPANTS/MATERIALS, SETTING, METHODS
The first 48 couples provided a spot urine sample on the same day as semen sample procurement. Sperm DNA methylation was assessed with the HumanMethylation 450 K array. Seventeen urinary phthalate and 1,2-Cyclohexane dicarboxylic acid diisononyl ester (DINCH) metabolite concentrations were measured from spot urine samples. The A-clust algorithm was employed to identify co-regulated regions. DMRs associated with urinary metabolite concentrations were identified via linear models, corrected for false discovery rate (FDR).
MAIN RESULTS AND ROLE OF CHANCE
Adjusting for age, BMI, and current smoking, 131 DMRs were associated with at least one urinary metabolite. Most sperm DMRs were associated with anti-androgenic metabolites, including mono(2-ethylhexyl) phthalate (MEHP, n = 83), mono(2-ethyl-5-oxohexyl) phthalate (MEOHP, n = 16), mono-n-butyl phthalate (MBP, n = 22) and cyclohexane-1,2-dicarboxylic acid-monocarboxy isooctyl (MCOCH, n = 7). The DMRs were enriched in lincRNAs as well as in regions near coding regions. Functional analyses of DMRs revealed enrichment of genes related to growth and development as well as cellular function and maintenance. Finally, 13% of sperm DMRs were inversely associated with high quality blastocyst-stage embryos after IVF.
LIMITATIONS, REASONS FOR CAUTION
Our modest sample size only included 48 males and additional larger studies are necessary to confirm our observed results. Non-differential misclassification of exposure is also a concern given the single spot urine collection.
WIDER IMPLICATIONS OF THE FINDINGS
To our knowledge, this is the first study to report that preconception urinary phthalate metabolite concentrations are associated with sperm DNA methylation in humans. These results suggest that paternal adult environmental conditions may influence epigenetic reprogramming during spermatogenesis, and in turn, influence early-life development.
STUDY FUNDING/COMPETING INTEREST(S)
This work was supported by grant K22-ES023085 from the National Institute of Environmental Health Sciences. The authors declare no competing interests.



The expression characteristics of FAM71D and its association with sperm motility

2017-09-12

AbstractSTUDY QUESTIONWhat are the features of FAM71D (Family with sequence similarity 71, member D) expression and is there an association between FAM71D expression and sperm motility?SUMMARY ANSWERFAM71D, a novel protein exclusively expressed in the testis, is located in sperm flagella and is functionally involved in sperm motility.WHAT IS KNOWN ALREADYSome testis-specific proteins have been reported as potential diagnostic biomarkers to evaluate the spermatogenesis process and sperm quality. We have identified a novel testis-specific protein, FAM71D, through microarray data analysis, yet little is known about its expression and function.STUDY DESIGN, SIZE, DURATIONFAM71D mRNA and protein expression was quantified during mouse testis development. Its localization in germ cells was detected by dual-labeled immunostaining in testis sections and sperm smears. The clinical significance was assessed by comparing FAM71D expression in spermatozoa from normozoospermic controls and asthenozoospermic patients.PARTICIPANTS/MATERIALS, SETTING, METHODSTestes were dissected from C57BL/6 J male mice at postnatal ages of 1, 2, 3, 4, 6, 8 weeks and 6 months, and sperm was collected from cauda epididymides of adult mice by the swim-up method. Human spermatozoa were isolated from 100 human semen samples by density gradient Percoll centrifugation. RT-qPCR and western blot were performed to semi-quantify the expression of FAM71D in mouse testis, and in the ejaculated spermatozoa of normozoospermic controls and asthenozoospermic patients. Immunofluorescence staining was used to detect the localization of FAM71D. Co-immunoprecipitation assay was performed to evaluate the interaction between FAM71D and calmodulin. An antibody blocking assay was employed to assess the role of FAM71D in sperm motility.MAIN RESULTS AND THE ROLE OF CHANCEOur results showed that FAM71D was exclusively expressed in the testis in an age-dependent manner. FAM71D expression exhibited dynamic change in the cytoplasm of spermatids during spermiogenesis and was finally retained in sperm flagella. FAM71D could interact with calmodulin. Use of anti-FAM71D antibody on sperm significantly decreased sperm motility. Expression level of FAM71D was markedly reduced in the ejaculated spermataozoa of asthenozoospermic patients (P < 0.05), and this was correlated with sperm progressive motility (r = 0.7435, P < 0.0001).LARGE SCALE DATAN/A.LIMITATIONS, REASONS FOR CAUTIONThe sample size was limited and it is necessary to verify the correlation of FAM71D expression with sperm motility in larger cohorts. Furthermore, our results were descriptive and follow-up studies would be needed to elucidate the detailed role of FAM71D in sperm motility.WIDER IMPLICATIONS OF THE FINDINGSThis is the first systematic study to document the expression of endogenous FAM71D and a function for FAM71D in sperm motility. It provides new insights into our understanding of sperm motility regulation and causes of male infertility.STUDY FUNDING/COMPETING INTERESTSThis study was funded by the National Natural Science Foundation of China, Guangdong Natural Science Foundation and the Shenzhen Project of Science and Technology. The authors have no competing interests.[...]



Factor V Leiden is associated with increased sperm count

2017-09-12

AbstractSTUDY QUESTIONIs the thrombophilia mutation factor V Leiden (FVL) associated with an increased total sperm count?SUMMARY ANSWERCarriers of FVL have a higher total sperm count than non-FVL-carriers, which could not be explained by genetic linkage or by observations in a FVL-mouse model.WHAT IS KNOWN ALREADYFVL has a high prevalence in Caucasians despite detrimental health effects. Carriers have been shown to have higher fecundity, which might partly explain this evolutionary paradox.STUDY DESIGN, SIZE, DURATIONWe determined FVL status in two cohorts (Dutch, n = 627; Danish, n = 854) of consecutively included men without known causes for spermatogenic failure, and performed an individual patient data meta-analysis of these two cohorts together with one previously published (Dutch, n = 908) cohort. We explored possible biological underpinnings for the relation between sperm count and FVL, by use of a FVL-mouse model and investigations of genetic linkage.PARTICIPANTS/MATERIALS, SETTING, METHODSParticipants were male partners of subfertile couples (two Dutch cohorts) and young men from the general population (Danish cohort): FVL carrier rate was 4.0%, 4.6% and 7.3%, respectively. There were differences in smoking, abstinence time and age between the cohorts. We corrected for these in the primary analysis, which consisted of a mixed linear effects model, also incorporating unobjectified population differences. In public haplotype data from subjects of European descent, we explored linkage disequilibrium of FVL with all known single nucleotide polymorphisms in a 1.5 MB region around the F5 gene with an R2 cutoff of 0.8. We sequenced exons of four candidate genes hypothesized to be linked to FVL in a subgroup of FVL carriers with extreme sperm count values. The animal studies consisted of never mated 15–18-week-old C57BL/J6 mice heterozygous and homozygous for FVL and wild-type mice. We compared spermatogenesis parameters (normalized internal genitalia weights, epididymis sperm content and sperm motility) between FVL and wild-type mice.MAIN RESULTS AND THE ROLE OF CHANCEHuman FVL carriers have a higher total sperm count than non-carriers, with an adjusted mean difference of 31 × 106 (95%CI 0.2–61.7; P = 0.048). Mice with the FVL mutation do not have increased spermatogenesis as compared to wildtype mice. None of the studied polymorphisms was in linkage disequilibrium, either in the public databases or in a subgroup of FVL carriers with extremely high sperm counts.LIMITATIONS, REASONS FOR CAUTIONThe difference in total sperm count would benefit from confirmation in other cohorts. The finding of higher count in carriers was consistent however, with no heterogeneity between the cohorts. The lack of effect of murine FVL might suggest there is no direct causality. The exploratory efforts on genetic linkage do not rule out that the association is a reflection of FVL co-inheritance with a non-studied causative polymorphism.WIDER IMPLICATIONS OF THE FINDINGSA high sperm count in FVL-carrying males contributes to understanding the high prevalence of this otherwise disadvantageous mutation. The findings might provide directions for future research on male fertility.STUDY FUNDING/COMPETING INTEREST(S)No conflicts of interest. Research was conducted with funding from the Netherlands Organisation for Scientific Research (NWO, VIDI innovative research grant 016.126.364 awarded to S. Middeldorp). The Danish cohort was supported by the Innovation Fund Denmark (InnovationsFonden, grant no. 14-2013-4), The Danish Ministry of[...]



Frozen embryo transfer: a review on the optimal endometrial preparation and timing

2017-09-08

ABSTRACT
STUDY QUESTION
What is the optimal endometrial preparation protocol for a frozen embryo transfer (FET)?
SUMMARY ANSWER
Although the optimal endometrial preparation protocol for FET needs further research and is yet to be determined, we propose a standardized timing strategy based on the current available evidence which could assist in the harmonization and comparability of clinic practice and future trials.
WHAT IS KNOWN ALREADY
Amid a continuous increase in the number of FET cycles, determining the optimal endometrial preparation protocol has become paramount to maximize ART success. In current daily practice, different FET preparation methods and timing strategies are used.
STUDY DESIGN, SIZE, DURATION
This is a review of the current literature on FET preparation methods, with special attention to the timing of the embryo transfer.
PARTICIPANTS/MATERIALS, SETTING, METHODS
Literature on the topic was retrieved in PubMed and references from relevant articles were investigated until June 2017.
MAIN RESULTS AND THE ROLE OF CHANCE
The number of high quality randomized controlled trials (RCTs) is scarce and, hence, the evidence for the best protocol for FET is poor. Future research should compare both the pregnancy and neonatal outcomes between HRT and true natural cycle (NC) FET. In terms of embryo transfer timing, we propose to start progesterone intake on the theoretical day of oocyte retrieval in HRT and to perform blastocyst transfer at hCG + 7 or LH + 6 in modified or true NC, respectively.
LIMITATIONS REASONS FOR CAUTION
As only a few high quality RCTs on the optimal preparation for FET are available in the existing literature, no definitive conclusion for benefit of one protocol over the other can be drawn so far.
WIDER IMPLICATIONS OF THE FINDINGS
Caution when using HRT for FET is warranted since the rate of early pregnancy loss is alarmingly high in some reports.
STUDY FUNDING/COMPETING INTEREST(S)
S.M. is funded by the Research Fund of Flanders (FWO). H.T. and C.B. report grants from Merck, Goodlife, Besins and Abbott during the conduct of the study.
TRIAL REGISTRATION NUMBER
Not applicable.



Vanishing twin syndrome among ART singletons and pregnancy outcomes

2017-08-31

AbstractSTUDY QUESTIONAmong babies born by ART, do singleton survivors of a vanishing twin have lower birth weight than other singletons?SUMMARY ANSWERVanishing twin syndrome (VTS) was associated with lower birth weight among ART singletons; a sibship analysis indicated that the association was not confounded by maternal characteristics that remain stable between deliveries.WHAT IS KNOWN ALREADYPrevious studies indicate that ART singletons with VTS have increased risk of adverse pregnancy outcomes, compared with other ART singletons. The potential contribution of unmeasured maternal background characteristics has been unclear.STUDY DESIGN, SIZE AND DURATIONThis was a Norwegian population-based registry study, including 17 368 mothers with 20 410 ART singleton deliveries between January 1984 and December 2013.PARTICIPANTS/MATERIALS, SETTING, METHODSThe study population included 17 291 ART singletons without VTS, 638 ART singletons with VTS and 2418 ART singletons with uncertain vanishing twin status. We estimated differences in birth weight and gestational age comparing ART singletons with VTS first to all ART singletons without VTS, and subsequently to their ART siblings without VTS, using random- and fixed-effects linear regression, respectively. The corresponding comparisons for the associations with preterm birth and small for gestational age (SGA) were conducted using random-and fixed-effects logistic regression. The sibling analysis of preterm birth included 587 discordant siblings, while the sibling analysis of SGA included 674 discordant siblings.MAIN RESULTS AND THE ROLE OF CHANCEART singletons with VTS had lower birth weight when compared to all ART singletons without VTS, with an adjusted mean difference (95% CI) of −116 g (−165, −67). When we compared ART singletons with VTS to their ART singletons sibling without VTS, the adjusted mean difference was −112 g (−209, −15). ART singletons with VTS also had increased risk of being born SGA, with an adjusted odds ratio (OR) (95% CI) of 1.48 (1.07, 2.03) compared to all ART singletons without VTS, and 2.79 (1.12, 6.91) in the sibship analyses. ART singletons with VTS were also more likely to be born preterm, although this difference did not reach statistical significance.LIMITATIONS REASONS FOR CAUTIONWe did not have information on maternal socio-economic status, but this factor is accounted for in the sibship analyses. We also had no information on whether fresh or frozen embryos were replaced.WIDER IMPLICATIONS OF THE FINDINGSThe reduction in birth weight and increased risk of SGA in ART singletons with VTS may suggest the presence of harmful intrauterine factors with long-term health impact. While vanishing twins are not routinely observed in naturally conceived pregnancies, loss of a twin is potentially a risk factor for the surviving foetus in any pregnancy. This could be further explored in large samples of naturally conceived pregnancies with the necessary information.STUDY FUNDING/COMPETING INTEREST(S)The authors of this study are supported in part by the UK Medical Research Council, US National Institute of Environmental Health Sciences and the Norwegian Research Council. The authors have no conflicts of interest.TRIAL REGISTRATION NUMBERN/A.[...]



Presence of polycystic ovary syndrome is associated with longer anogenital distance in adult Mediterranean women

2017-08-30

AbstractSTUDY QUESTIONIs polycystic ovary syndrome (PCOS) associated with anogenital distance (AGD), a biomarker of fetal androgen exposure, in adult Mediterranean women?SUMMARY ANSWERLonger AGD is associated with PCOS in adult Mediterranean women.WHAT IS KNOWN ALREADYAGD is a biomarker of prenatal androgen milieu. Human observational studies have reported that associations between AGD and reproductive parameters in both sexes. Exposure of the female fetus to intrauterine androgens may be a risk factor for PCOS in adulthood.STUDY DESIGN, SIZE, DURATIONThis was a case–control study of 126 women with PCOS and 159 controls between September 2014 and May 2016.PARTICIPANTS/MATERIALS, SETTING, METHODSCases were attending the gynecology unit of the ‘Virgen de la Arrixaca’ University Clinical Hospital (Murcia, Spain), and were diagnosed following the Rotterdam criteria. Phenotypic subtypes of PCOS were also assessed. Both prevalent and incident (newly diagnosed) cases were included. Controls were women without PCOS attending the gynecological outpatient clinic for routine gynecological exams. All women completed health questionnaires, and underwent physical and gynecological examinations, including transvaginal ultrasound and blood draw. We obtained measures from the anterior clitoral surface to the upper verge of the anus (AGDAC), and from the posterior fourchette to the upper verge of the anus (AGDAF). Gynecologists performing the AGD measures were blind to the status of the patients. We used unconditional multiple logistic regression to evaluate the association between AGD measurements and PCOS while accounting for relevant covariates and confounders, such as BMI, age and episiotomy.MAIN RESULTS AND THE ROLE OF CHANCECases showed significantly longer AGDAF and AGDAC compared to controls in bivariate analyses (P-values < 0.05). In the final adjusted models, AGDAC, but not AGDAF, was associated with the presence of PCOS (P-values = 0.002–0.008). Women with AGDAC in the upper compared to the lowest tertile were 2.9-times (95% CI 1.4–5.9; P-trend = 0.008) more likely to have PCOS. AGDAC measures were also significantly associated with all of the different phenotypic subtypes of PCOS (ORs = 3.1–5.1; P-values < 0.05).LIMITATIONS REASONS FOR CAUTIONWe took into account known and suspected covariates and confounders, but the possibility of chance findings or residual confounding should be noted. As with all observational studies, causal inference is limited, and study selection and information bias should not be ruled out.WIDER IMPLICATIONS OF THE FINDINGSOur results support the hypothesis that PCOS has an intrauterine origin, and that the hormonal environment in which the fetus develops may be highly relevant.STUDY FUNDING/COMPETING INTERESTThis work was supported by the Ministry of Economy and Competitiveness, Instituto de Salud Carlos III (ISCIII) (AES, Acción Estratégica en Salud), grant No. PI13/01237, and The Seneca Foundation, Murcia Regional Agency of Science and Technology, grant No. 19443/PI/14. There are no competing interests.TRIAL REGISTRATION NUMBERNot applicable.[...]