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Results in Journal Human Reproduction: 22,215

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, , Aurelio Tobias, Bee Tan, Abey Eapen,
Published: 31 October 2017
Human Reproduction, Volume 33, pp 65-80; https://doi.org/10.1093/humrep/dex326

Abstract:
STUDY QUESTIONIs serum vitamin D associated with live birth rates in women undergoing ART?SUMMARY ANSWERWomen undergoing ART who are replete in vitamin D have a higher live birth rate than women who are vitamin D deficient or insufficient.WHAT IS KNOWN ALREADYVitamin D deficiency has been associated with an increased risk of abnormal pregnancy implantation as well as obstetric complications such as pre-eclampsia and fetal growth restriction. However, the effect of vitamin D on conception and early pregnancy outcomes in couples undergoing ART is poorly understood.STUDY DESIGN, SIZE, DURATIONA systematic review and meta-analysis of 11 published cohort studies (including 2700 women) investigating the association between vitamin D and ART outcomes.PARTICIPANTS/MATERIALS, SETTINGS, METHODSLiterature searches were conducted to retrieve studies which reported on the association between vitamin D and ART outcomes. Databases searched included MEDLINE, EMBASE, Cochrane Central Register of Controlled Trials and CINAHL. Eleven studies matched the inclusion criteria.MAIN RESULTS AND THE ROLE OF CHANCELive birth was reported in seven of the included studies (including 2026 patients). Live birth was found to be more likely in women replete in vitamin D when compared to women with deficient or insufficient vitamin D status (OR 1.33 [1.08–1.65]). Five studies (including 1700 patients) found that women replete in vitamin D were more likely to achieve a positive pregnancy test than women deficient or insufficient in vitamin D (OR 1.34 ([1.04–1.73]). All 11 of the included studies (including 2700 patients) reported clinical pregnancy as an outcome. Clinical pregnancy was found to be more likely in women replete in vitamin D (OR 1.46 [1.05–2.02]). Six studies (including 1635 patients) reported miscarriage by vitamin D concentrations. There was no association found between miscarriage and vitamin D concentrations (OR 1.12 [0.81–1.54]. The included studies scored well on the Newcastle-Ottawa quality assessment scale.LIMITATIONS REASONS FOR CAUTIONAlthough strict inclusion criteria were used in the conduct of the systematic review, the included studies are heterogeneous in population characteristics and fertility treatment protocols.WIDER IMPLICATIONS OF THE FINDINGSThe findings of this systematic review show that there is an association between vitamin D status and reproductive treatment outcomes achieved in women undergoing ART. Our results show that vitamin D deficiency and insufficiency could be important conditions to treat in women considering ARTs. A randomized controlled trial to investigate the benefits of vitamin D deficiency treatment should be considered to test this hypothesis.STUDY FUNDING/COMPETING INTERESTSNo external funding was either sought or obtained for this study. The authors have no competing interests to declare.REGISTRATION NUMBERN/A.
P G Artini, C Tatone, S Sperduti, , S Franchi, , R Ciriminna, M Vento, C Di Pietro, L Stuppia, et al.
Published: 26 October 2017
Human Reproduction, Volume 32, pp 2474-2484; https://doi.org/10.1093/humrep/dex320

Abstract:
Is the phosphoinositol 1,3-kinase/protein kinase B (PI3K/AKT) pathway expression profile in cumulus cells (CCs) a potential marker of oocyte competence and predictive of pregnancy outcome? Eleven genes (AKT1, ARHGEF7, BCL2L1, CCND1, E2F1, HRAS, KCNH2, PIK3C2A, SHC1, SOS1 and SPP1) in the PI3K/AKT pathway were significantly down-regulated in CCs from oocytes that went on to produce a pregnancy compared to CCs associated with a negative outcome. The PI3K/AKT pathway plays a pivotal role in the interdependence and continuous feedback between the oocyte and CCs. The expression analysis of 92 transcripts in the PI3K/AKT pathway in CCs from patients with negative or positive pregnancy outcome, after single embryo transfer, was performed. Mouse CCs target gene expression was conducted to associate the expression profile of PI3K/AKT pathway to oocyte developmental profile. Fifty-five good prognosis IVF patients who had been referred to IVF or intracytoplasmic sperm injection treatment for male-factor infertility or tubal disease were enroled. CCs from single cumulus-oocyte complexes (COCs) from 16 patients who underwent a single embryo transfer were analyzed. Twenty-five CD-1 mice were used to assess gene expression in CCs associated with oocytes with different competence in relation to hCG priming. A total 220 human COCs were collected. The RNA extracted from CCs of 16 selected patients was used to analyze PI3K/AKT pathway gene expression employing a 96-well custom TaqMan Array. Expression data of CCs associated to positive IVF outcome were compared to data from negative outcome samples. Mice were sacrificed after 9, 12, 15, 21 and 24 h post-hCG administration to obtain CCs from MII oocytes with different developmental competence. Akt1, Bcl2l2 and Shc1 expression were tested in the collected mouse CCs. In addition, the expression of upstream regulator ESR1, the gene encoding for the oestrogen receptor ERβ, and the downstream effectors of the pathway FOXO1, FOXO3 and FOXO4 was evaluated in human and mouse samples. Transcripts involved in the PI3K Signaling Pathway were selectively modulated according to the IVF/ICSI outcome of the oocyte. Eleven transcripts in this pathway were significantly down-regulated in all samples of CCs from oocytes with positive when compared those with a negative outcome. These outcomes were confirmed in mouse CCs associated with oocytes at different maturation stages. Expression data revealed that the down-regulation of ESR1 could be related to oocyte competence and is likely to be the driver of expression changes highlighted in the PI3K/AKT pathway. Small sample size and retrospective design. The CCs expression profile of PI3K/AKT signaling genes, disclosed a specific CCs gene signature related to oocyte competence. It could be speculated that CCs associated with competent oocytes have completed their role in sustaining oocyte development and are influencing their fate in response to metabolic and hormonal changes by de-activating anti-apoptotic signals. Supported by Merck Serono an affiliate of Merck KGaA, Darmstadt, Germany (research grant for the laboratory session; Merck KGaA reviewed the manuscript for medical accuracy only before journal submission. The authors are fully responsible for the content of this manuscript, and the views and opinions described in the publication reflect solely those of the authors). The authors declare no conflict of interest.
Michelle M Denomme, , Jason C Parks, William B Schoolcraft,
Published: 26 October 2017
Human Reproduction, Volume 32, pp 2443-2455; https://doi.org/10.1093/humrep/dex317

Abstract:
Is there a distinct sperm histone-retained epigenetic signature in unexplained male factor infertility patients resulting in compromised blastocyst development? Using only donor oocyte IVF cycles, sperm DNA methylation patterns and miRNA profiles were significantly altered in normozoospermic patients resulting in poor blastocyst development, reflecting a subset of unexplained male factor infertility. Aberrant sperm DNA methylation has been associated with known male factor infertility, particularly noted in oligozoospermic patients. Unexplained male factor infertility remains a significant proportion of in vitro fertilization failures having unknown underlying physiology. Sperm samples (n = 40) and blastocysts (n = 48) were obtained during fertile donor oocyte IVF cycles with normozoospermic parameters, thereby excluding known female and male infertility factors. Samples were divided into two groups based on blastocyst development (Good Group = ≥20% embryos with D5 grade ‘AA’ blastocysts, and ≥60% embryos of transferable quality on D5 and D6; Poor Group = ≤10% embryos with D5 grade ‘AA’ blastocysts, and ≤40% embryos of transferable quality on D5 and D6). Samples were obtained from patients undergoing IVF treatments with informed consent and institutional review board approval. The Infinium HumanMethylation450 BeadChip microarray was used to identify histone-retained CpG island genes and genomic regions showing differences in sperm DNA methylation between the Good Group and the Poor Group. Pathway and gene network analysis for the significantly altered genes was performed, and targeted DNA methylation validation was completed for 23 genes and two imprinting control regions. Sperm miRNA profiles were assessed using the TaqMan® Human MicroRNA Array Card, with corresponding blastocyst mRNA gene expression examined by qRT-PCR. Our study is the first to investigate unexplained male factor infertility while significantly eliminating confounding female factors from our sample population by using only young fertile donor oocytes. We identified 1634 CpG sites located at retained histone CpG island regions that had significant sperm DNA methylation differentials between the two embryogenesis groups (P< 0.05). A largely hypermethylated profile was evident in the Good Group, with a small but distinct and statistically significant shift (P< 0.05) observed in the Poor Group. Genes involved in embryonic development were highly represented among histone-retained CpG sites with decreased methylation in the Poor Group (P< 0.05). Ten significantly altered sperm miRNAs (P< 0.05), correlated with altered target gene mRNA expression in the blastocysts from the Poor Group (P< 0.05). Taken together, significantly impacted sperm miRNA and target transcript levels in blastocysts from the Poor Group may contribute alongside aberrant sperm DNA methylation to the compromised blastocyst development observed. Our examination of CpG island regions restricted to retained histones represents only a small part of the sperm epigenome. The results observed are descriptive and further studies are needed to elucidate the functional effects of differential sperm DNA methylation on unexplained male factor infertility and blastocyst development. Slight epigenetic changes in sperm may have a cumulative effect on fertility and embryonic developmental competence. Knowledge of sperm epigenetics and inheritance has important implications for future generations, while providing evidence for potential causes of unexplained male factor infertility. No external funding was used for this study. None of the authors have any competing interest.
Derk Kuiper, Annemieke Hoek, , , Douwe J Mulder, Maaike Haadsma, Maas Jan Heineman,
Published: 26 October 2017
Human Reproduction, Volume 32, pp 2540-2548; https://doi.org/10.1093/humrep/dex323

Abstract:
Are the in vitro procedure, ovarian hyperstimulation or a combination of these two associated with blood pressure (BP) of 9-year-old IVF children born to subfertile couples? Our study demonstrates that ovarian hyperstimulation and the in vitro procedure are not associated with BP values in 9-year-old children born to subfertile couples. Possible long-term effects of IVF on child health and development have been studied relatively little. This is surprising, as it is known that environmental conditions may influence embryonic and foetal development which may result in health related problems in later life. Some studies suggested that IVF is associated with higher BP at pre-school age. Yet, it is unclear whether this may be also true for older children and if so, which component of IVF, i.e. the ovarian hyperstimulation, the embryo culture or a combination of these, attributes to this potentially less favourable BP. The Groningen Assisted Reproductive Technology cohort-study is a prospective assessor-blinded study of children followed from before birth onwards. In total, 170 children were assessed at the age of 9 years. The attrition rate up until the 9-year-old assessment was 21%. We evaluated cardiovascular health, focusing on BP (in mmHg and the internationally recognized percentiles of the US National High BP Education Program), heart rate and anthropometrics of 57 children born following controlled ovarian hyperstimulation-IVF/ICSI (COH-IVF/ICSI); 47 children born after modified natural cycle-IVF/ICSI (MNC-IVF/ICSI); and 66 children who were conceived naturally by subfertile couples (Sub-NC). Cardiovascular parameters were measured multiple times on one day. In addition, anthropometric data, including BMI and skinfold thickness, were collected. Systolic BP in mmHg did not differ between the COH-IVF/ICSI (mean 106.9, SD 6.7), MNC-IVF/ICSI (mean 104.8, SD 5.9) and Sub-NC (mean 106.3, SD 5.3) groups. In addition, systolic BP percentiles did not differ between the groups: COH-IVF/ICSI (mean 62.4, SD 20.2); MNC-IVF/ICSI (mean 56.3, SD 19.3); and Sub-NC (mean 62.3, SD17.8). Also, after adjustment for confounders BP in the three groups was similar. Heart rate and anthropometric values in the three groups did not differ. For instance, BMI values in the COH-IVF/ICSI-children were 16.3 (median value, range 13.0–24.7), in MNC-IVF/ICSI-children 16.1 (range 12.7–22.5) and in Sub-NC children 16.3 (range 12.7–24.0). The size of our study groups does not allow for pertinent conclusions on the effect of ovarian hyperstimulation and the in vitro procedure. The lack of a fertile control group may be regarded as another limitation. Our study suggests that ovarian hyperstimulation and in vitro procedures are not associated with cardiovascular health in 9-year-old. Yet, BP percentiles of the three groups were higher than the expected 50th percentile. This might indicate that children of subfertile couples have a higher BP than naturally conceived children. The study was financially supported by the University Medical Center Groningen (UMCG), the two graduate schools of the UMCG, BCN, SHARE and the Cornelia Stichting. The sponsors of the study had no role in study design, data collection, data analysis, data interpretation or writing of the report. The authors have no conflicts of interest to declare.
, G Mariani, N Holtmann, P Celada, J Remohí, E Bosch
Published: 13 October 2017
Human Reproduction, Volume 32, pp 2437-2442; https://doi.org/10.1093/humrep/dex316

Abstract:
Is there a relationship between serum progesterone (P) and endometrial volume on the day of embryo transfer (ET) with ongoing pregnancy rate (OPR) in artificial endometrium preparation cycles? Patients with serum P< 9.2 ng/ml on the day of ET had a significantly lower OPR but endometrial volume was not related with OPR. A window of optimal serum P levels during the embryo implantation period has been described in artificial endometrium preparation cycles. A very low endometrial volume is related to poor reproductive outcome. Prospective cohort study with 244 patients who underwent ET in an oocyte donation cycle after an artificial endometrial preparation cycle with estradiol valerate and vaginal micronized progesterone (400 mg/12 h). The study period went from 22 February 2016 to 25 October 2016 (8 months). Sample size was calculated to detect a 20% difference in OPR (35–55%) between two groups according to serum P levels in a two-sided test (80% statistical power, 95% confidence interval (CI)). Patients undergoing their first/second oocyte donation cycle, aged <50, BMI < 30 kg/m2, triple layer endometrium >6.5 mm and 1–2 good quality transferred blastocysts. A private infertility centre. Serum P determination and 3D ultrasound of uterine cavity were performed on the day of ET. Endometrial volume measurements were taken using a virtual organ computer-aided analysis (VOCAL™) system. The primary endpoint was OPR beyond pregnancy week 12. About 211 of the 244 recruited patients fulfilled all the inclusion/exclusion criteria. Mean serum P on the day of embryo transfer was 12.7 ± 5.4 ng/ml (Centiles 25, 9.2; 50, 11.8; 75,15.8). OPRs according to serum P quartiles were: Q1: 32.7%; Q2: 49.1%; Q3: 58.5%; Q4: 50.9%. The OPR of Q1 was significantly lower than Q2–Q4: 32.7% versus 52.8%; P = 0.016; RR (95% CI): 0.62 (0.41–0.94). The mean endometrial volume was 3.4 ± 1.9 ml. Serum P on the day of ET did not correlate with endometrial volume. A logistic regression analysis, adjusted for all the potential confounders, showed that OPR significantly lowered between women with serum P< 9.2 ng/ml versus ≥9.2 ng/ml (OR: 0.297; 95%CI: 0.113–0.779); P = 0.013. The ROC curve showed a significant predictive value of serum P levels on the day of ET for OPR, with an AUC (95%CI) = 0.59 (0.51–0.67). Only the women with normal uterine cavity, appropriate endometrial thickness and good quality blastocysts transfer were included. Extrapolation to an unselected population or to other routes and/or doses of administering P needs to be validated. The role of endometrial volume could not be fully defined as very few patients presented a very low volume. The present study suggests a minimum threshold of serum P values on the day of ET that needs to be reached in artificial endometrial preparation cycles to optimize outcome. No upper threshold could be defined. None. NCT02696694
, Latiefa Vinoos,
Published: 21 October 2017
Human Reproduction, Volume 32, pp 2431-2436; https://doi.org/10.1093/humrep/dex315

Abstract:
How do households recover financially from direct out-of-pocket payment for government subsidized ART? After a mean of 3.8 years, there was poor recovery from initiated financial coping strategies with the poorest households being disproportionatley affected. Out-of-pocket payment for health services can create financial burdens for households and inequities in access to care. A previous study conducted at a public-academic institution in South Africa documented that patient co-payment for one cycle of ART resulted in catastrophic expenditure for one in five households, and more frequently among the poorest, requiring diverse financial coping strategies to offset costs. An observational follow-up study was conducted ~4 years later to assess financial recovery among the 135 couples who had participated in this previous study. Data were collected over 12 months from 73 informants. The study was conducted at a level three referral hospital in the public-academic health sector of South Africa. At this institution ART is subsidized but requires patient co-payments. A purpose-built questionnaire capturing socio-economic information and recovery from financial coping strategies which had been activated was administered to all informants. Financial recovery was defined as the resolution of strategies initiated for the specific purpose of covering the original ART cycle. Results were analysed by strategy and household with the latter including analysis by tertiles based on socio-economic status at the time of the original expenditure. In addition to descriptive statistics, the Pearson Chi squared test was used to determine differences between socioeconomic tertiles and associations between recovery and other variables. The participation rate in this follow-up study was 54.1% with equal representation from the three socio-economic tertiles. The average duration of follow-up was 46.1 months (±9.78 SD) and respondents’ mean age was 42 years (range 31–52). The recovery rate was below 50% for four of five strategies evaluated: 23.1% of households had re-purchased a sold asset; 23.5% had normalized a previous reduction in household spending, 33.8% had regained their savings, and 48.7% were no longer bolstering income through additional work. Two-thirds of households (60.0%) had repaid all loans and debts. The poorest households showed lower rates of recovery when compared to households in the richest tertile. Complete recovery from all strategies initiated was reported by only 10 households (13.7%): 1 of 19 in the lowest tertile, 3 of 30 in the middle and by 6 of 24 households in the richest tertile (P > 0.05). No association was found between the degree of financial recovery and additional cost burdens incurred, including related to babies born; or between the degree of recovery and ongoing pursuit of ART. The sample size was limited. The participation rate was just over 50%. Results were dependent on participants’ narrative and recall. The willingness of patients to pay for ART does not necessarily imply the ability to pay. As a result, the lack of comprehensive third-party funding for ART can create immediate and long-term financial hardship which is more pronounced among poorer households. While more data on the impact of out-of-pocket payment for ART are needed to illustrate the problem in other low resource settings, the results from South Africa provide useful information for similar developing countries. The current absence of more extensive data should therefore not be a barrier to the promotion of financial risk protection for infertile couples, especially the poorest, in need of ART. The study was supported by a Masters Student Grant from the Faculty of Health Sciences, University of Cape Town. The authors had no competing interests. Not applicable.
X Y Jin, L J Zhao, D H Luo, L Liu, Y D Dai, X X Hu, Y Y Wang, X Lin, F Hong, , et al.
Published: 13 October 2017
Human Reproduction, Volume 32, pp 2394-2403; https://doi.org/10.1093/humrep/dex312

Abstract:
Is pinopode measurement of any prognostic value? Pinopode expression was significantly associated with the occurrence of pregnancy after frozen embryo transfer. Pinopodes are expressed in the endometrium during the implantation period. Pinopode measurement has been proposed as a marker of endometrial receptivity. A prospective cohort study was conducted at the Center of Reproductive Medicine, Sir Run Run Shaw Hospital, between 2014 and 2016, recruiting 172 women with infertility and undergoing frozen embryo transfer following IVF treatment. Among 172 participants, 46 women took part in the first study to quantify the daily changing pattern of pinopodes 3–7 days after the initiation of progesterone therapy in the hormone replacement cycles and the remaining 126 women with infertility participated in a study to examine the relationship between pinopode count and pregnancy outcome following frozen embryo transfer in hormone replacement cycles. The mean age of participants was 29 years old. All participants received an artificial hormone replacement protocol capable of supporting successful implantation. Endometrial biopsies from 46 women were obtained 3, 4, 5, 6 and 7 days after the initiation of progesterone therapy (P + 3, n = 6; P + 4, n = 6; P + 5, n = 11; P + 6, n = 13; P + 7, n = 10, respectively). Another 126 endometrial biopsies were obtained precisely 6 days after the initiation of progesterone. Scanning electron microscopy was used to capture the pinopode images, followed by use of the image J program to quantify the count and subtype of the pinopodes. We found that at least 60 microscopic fields were necessary to achieve a reproducible result. An intra-observer variability study showed good agreement between two measurements regarding the developing pinopode (DP) subtype (r = 0.95) and the fully developed pinopode (FDP) subtype (r = 0.86) but not for the regressing (RP) pinopode subtype (r = 0.39). The proportion of DP/total pinopodes (TP) declined rapidly form day P + 4 to a minimum on day P + 6. The percentage of FDP/TP increased rapidly from day P + 4 to reach a peak on day P + 6. On the other hand, the percentage of RP/TP reached a peak on day P + 7. Participants who conceived had a significantly (P = 0.011) higher percentage of FDP/TP on day P + 6 and significantly (P = 0.005) lower percentage of DP/TP on the same day compared with participants who did not become pregnant. Using a scoring system incorporating the percentages of DP and FDP, it was found that the pregnancy rate and the embryo implantation rate of women with a high pinopode score (82.3%; 63.0%) was significantly (P = 0.001; P = 0.046) higher than that of women with a low pinopode score (53.3%; 46.7%), respectively. There remains a possibility that the observations could have arisen due to chance. This study examined pinopode count and subtype in the HRT cycles, and it is uncertain whether the same observations apply to in natural cycles. Pinopodes have been questioned as a potential marker of endometrial receptivity for many years. Our results suggested that pinopode measurement may be of value in predicting pregnancy. The study was supported by the grants from the general project of medicine and health in Zhejiang Province of China (2015KYA142; 2018KY106), the Key Research and Development Program of Zhejiang Province (2017C03022) and the National Natural Science Foundation of China (81701514).The funders had no role in the study design, data collection and analysis, decision to publish or preparation of the manuscript. We have no competing interests to declare. ISRCTN26300668
, , R Hersmus, J Kaprová, S L S Drop, H Stoop, A J M Gillis, J W Oosterhuis, E M F Costa, S Domenice, et al.
Published: 7 November 2017
Human Reproduction, Volume 32, pp 2561-2573; https://doi.org/10.1093/humrep/dex300

Abstract:
What is the prevalence of malignant testicular germ cell tumors (TGCT) and its precursors, (pre-) germ cell neoplasia in situ (GCNIS), in late teenagers and adults who have androgen insensitivity syndrome (AIS) and the impact of an individual's genetic susceptibility to development of TGCT? No GCNIS or TGCT was diagnosed, but pre-GCNIS was identified in 14 and 10% of complete and partial AIS patients, respectively, and was associated with a higher genetic susceptibility score (GSS), with special attention for KITLG (rs995030) and ATFZIP (rs2900333). Many adult women with AIS decline prophylactic gonadectomy, while data regarding the incidence, pathophysiology and outcomes of TGCT in postpubertal individuals with AIS are lacking. The relevance of genetic factors, such as single nucleotide polymorphisms (SNPs), in predisposing AIS individuals to TGCT is unknown. This multicenter collaborative study on prophylactically removed gonadal tissue was conducted in a pathology lab specialized in germ cell tumor biology. Material from 52 postpubertal individuals with molecularly confirmed AIS (97 gonadal samples) was included; the median age at surgery was 17.5 (14–54) years. Immunohistochemical studies and high-throughput profiling of 14 TGCT-associated SNPs were performed. The main outcome measures were the prevalence of pre-GCNIS, GCNIS and TGCT, and its correlation with a GSS, developed based on the results of recent genome-wide association studies. The earliest recognizable change preceding GCNIS, referred to as pre-GCNIS, was present in 14% of individuals with complete and 10% of those with partial AIS at a median age of 16 years. No GCNIS or invasive TGCT were found. The median GSS was significantly greater for those with, compared to those without, pre-GCNIS (P = 0.01), with an overlap between groups. Our data suggest important roles for risk alleles G at KITLG (rs995030) and C at ATFZIP (rs2900333), among the 14 studied TGCT-associated SNPs. N/A. A limited number of cases were included. Our data suggest that the prevalence of pre-GCNIS in individuals with AIS beyond puberty is around 15%. Genetic susceptibility likely contributes to pre-GCNIS development in AIS but factors related to malignant progression remain unclear. Although data in older patients remain scarce, malignant progression appears to be a rare event, although the natural history of the premalignant lesion remains unknown. Therefore, the practice of routine prophylactic gonadectomy in adults with AIS appears questionable and the patient's preference, after having been fully informed, should be decisive in this matter. This study was supported by research grants from the Research Foundation Flanders (FWO) (to M.C.), the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq G0D6713N) (to B.B.M. and M.C.) and the European Society for Pediatric Endocrinology (ESPE), granted by Novo Nordisk AB (to J.K.). There are no competing interests.
, M R Caanen, V Mijatovic, C G Vergouw, P M Van De Ven, , R Schats
Published: 13 October 2017
Human Reproduction, Volume 32, pp 2218-2224; https://doi.org/10.1093/humrep/dex302

Abstract:
Does 15 min of immobilization after IUI improve pregnancy rates? Immobilization for 15 min after IUI does not improve pregnancy rates. 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. 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. 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. 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. 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. 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. No funding was received. All authors have nothing to disclose. Dutch Trial Register NTR 2418. 20 July 2010. 11 August 2010.
M Uijldert, A Meißner, , , , A A De Melker, , M D Van De Wetering,
Published: 12 October 2017
Human Reproduction, Volume 32, pp 2366-2372; https://doi.org/10.1093/humrep/dex306

Abstract:
Is testicular growth affected by a testicular biopsy intended for fertility preservation in pre-pubertal boys with cancer?
, , , H Tournaye, C M A Van Ravenswaaij-Arts, J A Land
Published: 13 October 2017
Human Reproduction, Volume 32, pp 2574-2580; https://doi.org/10.1093/humrep/dex307

Abstract:
What is the prevalence of chromosomal abnormalities in azoospermic men and what are the clinical consequences in terms of increased risk for absent spermatogenesis, miscarriages and offspring with congenital malformations? The prevalence of chromosomal abnormalities in azoospermia was 14.4%, and the number of azoospermic men needed to be screened (NNS) to identify one man with a chromosomal abnormality with increased risk for absence of spermatogenesis was 72, to prevent one miscarriage 370–739 and to prevent one child with congenital malformations 4751–23 757. Infertility guidelines worldwide advise screening of non-iatrogenic azoospermic men for chromosomal abnormalities, but only few data are available on the clinical consequences of this screening strategy. This retrospective multicentre cross-sectional study of non-iatrogenic azoospermic men was performed at the University Hospital Brussels, Belgium, and the University Medical Centre Groningen, The Netherlands, between January 2000 and July 2016. Analysis of clinical registries retrospectively identified 1663 non-iatrogenic azoospermic men with available results of karyotyping and FSH serum levels. Iatrogenic azoospermia was an exclusion criterion, defined as azoospermia after spermatotoxic medical treatment, exogenous androgen suppletion or vasectomy and/or vasovasostomy. Also, men with a clinical diagnosis of anejaculation or hypogonadotropic hypo-androgenism and/or FSH values <1.0 U/l were excluded. Chromosomal abnormalities were categorized according to their (theoretical) impact on clinical consequences for the patient (i.e. an increased risk for absence of spermatogenesis) and adverse pregnancy outcomes (i.e. miscarriage or offspring with congenital malformations), in both normogonadotropic (FSH < 10 U/l) and hypergonadotropic (FSH ≥ 10 U/l) azoospermia. We estimated the NNS for chromosomal abnormalities to identify one man with absence of spermatogenesis and to prevent one miscarriage or one child with congenital malformations, and calculated the surgical sperm retrieval rates per chromosomal abnormality. The overall prevalence of chromosomal abnormalities in azoospermia was 14.4% (95% CI 12.7–16.1%), its prevalence being higher in hypergonadotropic azoospermia (20.2%, 95% CI 17.8–22.7%) compared to normogonadotropic azoospermia (4.9%, 95% CI 3.2–6.6%, P< 0.001). Klinefelter syndrome accounted for 83% (95% CI 77–87%) of abnormalities in hypergonadotropic azoospermia. The NNS to identify one man with increased risk for absence of spermatogenesis was 72, to prevent one miscarriage 370–739, and to prevent one child with congenital malformations 4751–23 757. There was no clinically significant difference in NNS between men with normogonadotropic and hypergonadotropic azoospermia. The surgical sperm retrieval rate was significantly higher in azoospermic men with a normal karyotype (60%, 95% CI 57.7–63.1%) compared to men with a chromosomal abnormality (32%, 95% CI 25.9–39.0%, P< 0.001). The sperm retrieval rate in Klinefelter syndrome was 28% (95% CI 20.7–35.0%). The absolute number of chromosomal abnormalities associated with clinical consequences and adverse pregnancy outcomes in our study was limited, thereby increasing the role of chance. Further, as there are currently no large series on outcomes of pregnancies in men with chromosomal abnormalities, our conclusions are partly based on assumptions derived from the literature. The NNS found can be used in future cost-effectiveness studies and the evaluation of current guidelines on karyotyping in non-iatrogenic azoospermia. None to declare.
, B W Whitcomb, , M E Boutot, J E Manson, S E Hankinson, B A Rosner, E R Bertone-Johnson
Published: 25 October 2017
Human Reproduction, Volume 32, pp 2522-2531; https://doi.org/10.1093/humrep/dex304

Abstract:
Is adult adiposity associated with early menopause? Overall and abdominal adiposity were non-linearly associated with odds for early natural menopause with elevated odds observed among women who were underweight in early or mid-adulthood compared to lean-normal weight women. High and low adiposity have been associated with reproductive function and may potentially impact timing of menopause. It is unclear whether various aspects of adiposity are associated with risk of early menopause. Prospective cohort study that examined data from 78 759 premenopausal women from the Nurses’ Health Study II who were followed from 1989 to 2011 for incidence of early natural menopause. Participants were aged 25–42 years and premenopausal at baseline in 1989, when information on menopausal status, height and weight was reported via questionnaire. Information on menopausal status, type of menopause (natural, surgical, radiation/chemotherapy), hormone therapy use and weight was updated every two years along with information on smoking, physical activity and other behavioral and health-related factors. Multivariable logistic regression was used to estimate odds ratios for early menopause, defined as natural menopause before age 45 years, by aspects of adiposity. Early natural menopause was reported by 2804 participants. Body mass index (BMI) was non-linearly associated with risk for early menopause. Compared to women with BMI = 18.5–22.4 kg/m2, those with BMI < 18.5 kg/m2 had a significant 30% higher odds of early menopause (95% confidence interval (CI) = 1.08, 1.57), while women with BMIs between 25.0–29.9 kg/m2 had significant 21–30% lower odds. Odds were not higher in women with BMI ≥ 35.0 kg/m2 in fully adjusted analysis. Non-linear associations with higher odds in underweight women were also observed for age 18 and age 35 BMI, though lower odds for overweight women was only observed for age 35 BMI. Odds were highest among women with age 18 BMI < 18.5 kg/m2 reporting severe weight cycling. Though weight and early menopause status were self-reported, validation studies conducted among Nurses’ Health Study participants suggest that self-reported weight is highly correlated with directly measured weight, and prospective self-reported menopausal status is highly reproducible. It is possible that underweight women may have been misclassified with an earlier age at menopause if being underweight led to amenorrhea. In one of the few studies to prospectively examine a variety of adiposity measures and risk for early menopause, our findings that women who were underweight in early or mid-adulthood had elevated risk for early menopause can assist in efforts to better understand the etiology of early menopause. Additional prospective research is needed to understand how low adiposity may physiologically impact timing of menopause. This study was conducted with funding from NIH UM1CA176726 and R01HD078517. The authors declare no conflicts of interest. Not applicable.
, Maaike Catteeuw, , , , Urban Besenfelder, Vitezslav Havlicek, Katrien Smits, , Andres Salumets, et al.
Published: 23 September 2017
Human Reproduction, Volume 32, pp 2348-2357; https://doi.org/10.1093/humrep/dex286

Abstract:
Is the rate and nature of chromosome instability (CIN) similar between bovine in vivo-derived and in vitro-cultured cleavage-stage embryos? There 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. CIN 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. Because 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). Single 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. The 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). Genotyping data obtained in this study has been submitted to NCBI Gene Expression Omnibus (GEO; accession number GSE95358) There 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. Although 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. 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 polymorphic variant allelic frequencies’), licensed to Cartagenia (Agilent Technologies).
Rasa Pelanis, , , , , Juha S Tapanainen, , Johanna Puurunen, Angelica Lindén Hirschberg, Peter Fedorcsak, et al.
Published: 29 September 2017
Human Reproduction, Volume 32, pp 2279-2286; https://doi.org/10.1093/humrep/dex294

Abstract:
Is oral glucose tolerance test (OGTT) needed in all women with polycystic ovary syndrome (PCOS)? OGTT is not routinely needed in women with PCOS and BMI < 25 kg/m2. 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. A Nordic cross-sectional study including 876 women. 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. 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. 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. Routine OGTT may not be indicated in normal-weight women with PCOS. None. N/A.
Qian Ma, YuChi Li, Manling Luo, Huan Guo, Shouren Lin, Jianbo Chen, Y Du, Zhimao Jiang,
Published: 12 September 2017
Human Reproduction, Volume 32, pp 2178-2187; https://doi.org/10.1093/humrep/dex290

Abstract:
What are the features of FAM71D (Family with sequence similarity 71, member D) expression and is there an association between FAM71D expression and sperm motility? FAM71D, a novel protein exclusively expressed in the testis, is located in sperm flagella and is functionally involved in sperm motility. Some 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. FAM71D 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. Testes 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. Our 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). N/A. The 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. This 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. This 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.
, M Kirkman, C Stern, R I McLachlan, G Clarke, F Agresta, , L Rombauts, B Vollenhoven, J R W Fisher
Published: 17 October 2017
Human Reproduction, Volume 32, pp 2423-2430; https://doi.org/10.1093/humrep/dex314

Abstract:
What are the reproductive experiences and outcomes of people who store reproductive material before cancer treatment? Of respondents who had tried to achieve pregnancy since completing cancer treatment almost all had succeeded, in most cases through natural conception. People of reproductive age who are diagnosed with cancer can cryopreserve reproductive material to guard against the adverse effects on fertility of gonadotoxic treatment. Little is known about the reproductive outcomes of people who undergo fertility preservation before cancer treatment. Cross-sectional survey. Women and men who had stored reproductive material before cancer treatment at two private and one public fertility clinics up to June 2014 and were at least 18 years old at the time were identified from medical records and invited to complete an anonymous questionnaire about their reproductive experiences. Of the 870 potential respondents 302 (171 female and 131 male) returned completed questionnaires yielding a response rate of 34.5% (39.5% and 29.7% for female and male respondents, respectively). Current age was similar for women and men (37.2 years) but men had been diagnosed with cancer significantly earlier in life than women (28.2 versus 30.3 years, P = 0.03). Almost two-thirds of respondents wished to have a child or another child in the future, some of whom knew that they were unable to. One in ten respondents was a parent before the cancer diagnosis and around one-third had had a child since diagnosis or was pregnant (or a partner in pregnancy) at the time of the survey. Of those who had tried to conceive since completing cancer treatment (N = 119) 84% (79% of women and 90% of men) had had a child or were pregnant (or a partner in pregnancy). Most of the pregnancies since the diagnosis of cancer occurred after natural conception (58/100, 58%). Of the 22 women (13% of all women) and 35 men (27% of all men) who had used their stored reproductive material four women (18%) and 28 men (80%) had had a child or were pregnant or a partner in pregnancy at the time of completing the survey. The most commonly stated reason for not using the stored material was not being ready to try for a baby. The relatively low response rate, particularly among men, means that participation bias may have influenced the findings. As type of cancer was self-reported and we did not ask questions about respondents’ cancer treatments, it is not possible to link reproductive outcomes to type of cancer or cancer treatment. Also, there is no way of comparing the sample with the populations they were drawn from as data on reproductive outcomes of people who store reproductive material before cancer treatment are not collected routinely. This might have led to over- or underestimates of the reproductive experiences and outcomes reported in this paper. The findings add to the limited evidence about the reproductive outcomes of this growing group of people and can be used to inform the advice given to those contemplating fertility preservation in the context of cancer. The study was funded by the National Health and Medical Research Council (APP1042347). The authors have no conflicts of interest to declare. Not applicable.
Yifan Liu, Ming Han, Xiaoshuang Li, Hui Wang, Minyue Ma, Shihui Zhang, Yifan Guo, Shuling Wang, Yuanfen Wang, Na Duan, et al.
Published: 17 October 2017
Human Reproduction, Volume 32, pp 2465-2473; https://doi.org/10.1093/humrep/dex309

Abstract:
What changes in the mitochondria of human mural granulosa cells (mGCs) with maternal aging? The mitochondrial membrane potential (MMP) and the ability of oxidative phosphorylation (OXPHOS) of mGCs declines with reproductive aging, accompanied with more abnormal mitochondria. Mitochondria play an important role in the dialogue between the mGCs and oocytes. However, the underlying mechanism of mitochondrial dysfunction in mGCs in aging is still poorly understood. In total, 149 infertile women underwent IVF in the ART Centre of the Chinese PLA General Hospital, China from September 2016 to May 2017. Two age groups were investigated: the young group (<38 years old) and the old group (≥38 years old). The mitochondrial ultrastructure of mGCs was observed by transmission electron microscopy, and real-time quantitative polymerase chain reaction was applied to quantify the mitochondrial DNA (mtDNA) copy number, 4977-bp deleted DNA and mRNA expression of mitochondrial ATP synthases ATP5A1 and ATP5I. MMP was detected by flow cytometry and fluorescence microscopy, respectively. Reactive oxygen species (ROS) was tested by flow cytometry. A luminometer was used to measure the ATP levels and western blot to analyse the OXPHOS complex. In the young group, mitochondria were mostly round or oval, with a few intact parallel tubular–vesicular cristae and homogenous matrix density, while elongated mitochondria were mainly observed in the old group, which had numerous cristae and more high-density matrix particles. Abnormal mitochondria were more common in aging women (P = 0.012). mtDNA relative copy number was positively correlated with maternal age (r = 0.294, P = 0.009) and we found no one with 4977-bp deleted mitochondria. JC-1 (dye used as an indicator of MMP) ratio in the old group was significantly lower than the young group (3.01 ± 0.21 vs 3.85 ± 0.27, P = 0.033). Intracellular ROS levels between the groups did not differ significantly (P = 0.191). The intracellular ATP level in the young group was 1.75-fold higher than that of the advanced-age group (7.17 ± 1.16 vs 4.15 ± 0.60, P = 0.025). The protein expression of ATP5A1, as one of five proteins of OXPHOS, decreased with aging (P< 0.001). ATP5A1 mRNA expression was negatively correlated with aging (r = −0.341, P = 0.012). The quantity of mGCs from some individual patient, especially an advanced-age individual, was small, which cannot meet the demands of all the detections. mGCs dysfunction with aging is mainly linked to impaired mitochondrial function, especially OXPHOS function. Improving the OXPHOS ability in mGCs should be the focus in resolving infertility among advanced age women and making mGCs the proper mitochondria donor cells in the autologous mitochondria transplantation to oocytes. This work was supported by the grants of the National High Technology Research and Development Program of China, 863 Program No. SS2015AA020402, and the Key Projects of Military Medical Research, No. BWS11J058. There were no competing interests.
, , Z. Bochdanovits, A. Takizawa, J. Peter, , , J.C.M. Meijers, H. Weiler, , et al.
Published: 12 September 2017
Human Reproduction, Volume 32, pp 2332-2339; https://doi.org/10.1093/humrep/dex287

Abstract:
Is the thrombophilia mutation factor V Leiden (FVL) associated with an increased total sperm count? Carriers 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. FVL 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. We 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 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. Human 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. The 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. A 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. 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 Health and the Danish Environmental Protection Agency. Not applicable.
Erratum
, Sezcan Mumusoglu, Kiper Aslan, Ayse Seyhan, Isil Kasapoglu, Berrin Avci, Bulent Urman, Gurkan Bozdag, , Isıl Kasapoglu, et al.
Human Reproduction, Volume 32, pp 1539-1539; https://doi.org/10.1093/humrep/dex225

Abstract:
Hum Reprod 2017;32: doi:10.1093/humrep/dex099
, S Santos-Ribeiro, A Van De Vijver, , , H Tournaye,
Published: 8 September 2017
Human Reproduction, Volume 32, pp 2234-2242; https://doi.org/10.1093/humrep/dex285

Abstract:
STUDY QUESTIONWhat is the optimal endometrial preparation protocol for a frozen embryo transfer (FET)?SUMMARY ANSWERAlthough 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 ALREADYAmid 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, DURATIONThis is a review of the current literature on FET preparation methods, with special attention to the timing of the embryo transfer.PARTICIPANTS/MATERIALS, SETTING, METHODSLiterature on the topic was retrieved in PubMed and references from relevant articles were investigated until June 2017.MAIN RESULTS AND THE ROLE OF CHANCEThe 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 CAUTIONAs 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 FINDINGSCaution 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 NUMBERNot applicable.
J M Reyes, E Silva, J L Chitwood, W B Schoolcraft, R L Krisher,
Published: 15 September 2017
Human Reproduction, Volume 32, pp 2199-2208; https://doi.org/10.1093/humrep/dex284

Abstract:
What effect does maternal age have on the human oocyte's molecular response to in vitro oocyte maturation? Although 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. Microarray 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. The 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. Patients 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. A 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. Raw data from this study can be accessed through GSE95477. The human oocytes used in the current study were obtained from patients with varying causes of infertility (e.g. decreased oocyte quality and oocyte quality-independent factors), possibly affecting oocyte gene expression. Oocytes in this study were retrieved at the GV stage following hCG administration and the MII oocytes were derived by IVM of patient oocytes. Although the approach has the benefit of identifying intrinsic differences between samples, it may not be completely representative of in vivo matured oocytes. Transcriptome profiles of YNG and AMA oocytes, particularly at the MII stage, suggest that aberrant transcript abundance may contribute to the age-associated decline in fertility. J.M.R. was supported by an Austin Eugene Lyons Fellowship awarded by the University of California, Davis. The Eunice Kennedy Shriver National Institute of Child Health and Human Development of the National Institutes of Health (awarded to P.J.R.; R01HD070044) and the Fertility Laboratories of Colorado partly supported the research presented in this manuscript.
, G David Adamson, Silke Dyer, Catherine Racowsky, Jacques de Mouzon, Rebecca Sokol, Laura Rienzi, Arne Sunde, , Ian D Cooke, et al.
Human Reproduction, Volume 32, pp 1786-1801; https://doi.org/10.1093/humrep/dex234

Abstract:
Can a consensus and evidence-driven set of terms and definitions be generated to be used globally in order to ensure consistency when reporting on infertility issues and fertility care interventions, as well as to harmonize communication among the medical and scientific communities, policy-makers, and lay public including individuals and couples experiencing fertility problems? A set of 283 consensus-based and evidence-driven terminologies used in infertility and fertility care has been generated through an inclusive consensus-based process with multiple stakeholders. In 2006 the International Committee for Monitoring Assisted Reproductive Technologies (ICMART) published a first glossary of 53 terms and definitions. In 2009 ICMART together with WHO published a revised version expanded to 87 terms, which defined infertility as a disease of the reproductive system, and increased standardization of fertility treatment terminology. Since 2009, limitations were identified in several areas and enhancements were suggested for the glossary, especially concerning male factor, demography, epidemiology and public health issues. Twenty-five professionals, from all parts of the world and representing their expertise in a variety of sub-specialties, were organized into five working groups: clinical definitions; outcome measurements; embryology laboratory; clinical and laboratory andrology; and epidemiology and public health. Assessment for revisions, as well as expansion on topics not covered by the previous glossary, were undertaken. A larger group of independent experts and representatives from collaborating organizations further discussed and assisted in refining all terms and definitions. Members of the working groups and glossary co-ordinators interacted through electronic mail and face-to-face in international/regional conferences. Two formal meetings were held in Geneva, Switzerland, with a final consensus meeting including independent experts as well as observers and representatives of international/regional scientific and patient organizations. A consensus-based and evidence-driven set of 283 terminologies used in infertility and fertility care was generated to harmonize communication among health professionals and scientists as well as the lay public, patients and policy makers. Definitions such as ‘fertility care’ and ‘fertility awareness’ together with terminologies used in embryology and andrology have been introduced in the glossary for the first time. Furthermore, the definition of ‘infertility’ has been expanded in order to cover a wider spectrum of conditions affecting the capacity of individuals and couples to reproduce. The definition of infertility remains as a disease characterized by the failure to establish a clinical pregnancy; however, it also acknowledges that the failure to become pregnant does not always result from a disease, and therefore introduces the concept of an impairment of function which can lead to a disability. Additionally, subfertility is now redundant, being replaced by the term infertility so as to standardize the definition and avoid confusion. All stakeholders agreed to the vast majority of terminologies included in this glossary. In cases where disagreements were not resolved, the final decision was reached after a vote, defined before the meeting as consensus if passed with 75%. Over the following months, an external expert group, which included representatives from non-governmental organizations, reviewed and provided final feedback on the glossary. Some terminologies have different definitions, depending on the area of medicine, for example demographic or clinical as well as geographic differences. These differences were taken into account and this glossary represents a multinational effort to harmonize terminologies that should be used worldwide. None. N/A.
Maria C Magnus, Sara Ghaderi, Nils-Halvdan Morken, Per Magnus, Liv Bente Romundstad, Rolv Skjærven, , Siri Eldevik Håberg
Human Reproduction, Volume 32, pp 2298-2304; https://doi.org/10.1093/humrep/dex277

Abstract:
Among babies born by ART, do singleton survivors of a vanishing twin have lower birth weight than other singletons? Vanishing 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. Previous 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. This was a Norwegian population-based registry study, including 17 368 mothers with 20 410 ART singleton deliveries between January 1984 and December 2013. The 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. ART 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. We 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. The 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. 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. N/A.
Carolina Vaz-De-Macedo,
Human Reproduction, Volume 32, pp 1951-1956; https://doi.org/10.1093/humrep/dex272

Abstract:
Conventionally, the search for carrier status was based on ethnicity and/or family history and targeted to a restricted number of genetic conditions and mutations. This is now being replaced by extended panels testing for hundreds of genetic disorders with a broad range of phenotypes, in what is called ‘expanded carrier screening’. While the ultimate aim of these panels is to increase the reproductive autonomy of the individuals and couples by providing preconception knowledge that could lead to the broadest range of available options, including PGD, we argue that: (i) Given the number and heterogeneity of the conditions included in panels, it cannot be guaranteed that a couple who tests positive for one of those conditions will be eligible for PGD; patients should be informed of this potential limitation before undertaking screening. (ii) Family history is typically lacking in couples identified through panels as being at high-risk for certain disorders. This should promote a reflection on the inclusion of personal experience with a condition as a consideration for PGD in disorders with incomplete penetrance or for which treatment options are available. (iii) With the advent of next-generation sequencing panels, cases of couples in which one member carries a disease-causing variant and the other has a variant of uncertain significance found in the same gene are likely to become more common and need to be discussed from the PGD perspective. (iv) With comprehensive panels where healthy individuals are likely to be identified as carriers for several conditions, testing of carrier status for embryos and prioritisation of the embryos to transfer needs reassessing. We believe that these points should be included in the discussion on expanded carrier screening and that all stakeholders, patients included, must be aware of the challenges and limitations that may come with a positive result.
, Molly S Estill, Alexander Shershebnev, , Stephen A Krawetz, Brian W Whitcomb, Holly Dinnie, Tayyab Rahil, Cynthia K Sites,
Published: 12 September 2017
Human Reproduction, Volume 32, pp 2159-2169; https://doi.org/10.1093/humrep/dex283

Abstract:
Are preconception phthalate and phthalate replacements associated with sperm differentially methylated regions (DMRs) among men undergoing IVF? Ten phthalate metabolites were associated with 131 sperm DMRs that were enriched in genes related to growth and development, cell movement and cytoskeleton structure. 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. An exploratory cross-sectional study with 48 male participants from the Sperm Environmental Epigenetics and Development Study (SEEDS). 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). 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. 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. 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. This work was supported by grant K22-ES023085 from the National Institute of Environmental Health Sciences. The authors declare no competing interests.
Yash S. Khandwala, Chiyuan A. Zhang, ,
Human Reproduction, Volume 32, pp 2110-2116; https://doi.org/10.1093/humrep/dex267

Abstract:
STUDY QUESTIONHow has the mean paternal age in the USA changed over the past 4 decades?SUMMARY ANSWERThe age at which men are fathering children in the USA has been increasing over time, although it varies by race, geographic region and paternal education level.WHAT IS KNOWN ALREADYWhile the rise in mean maternal age and its implications for fertility, birth outcomes and public health have been well documented, little is known about paternal characteristics of births within the USA.STUDY DESIGN, SIZE, DURATIONA retrospective data analysis of paternal age and reporting patterns for 168 867 480 live births within the USA since 1972 was conducted.PARTICIPANTS/MATERIALS, SETTING, METHODSAll live births within the USA collected through the National Vital Statistics System (NVSS) of the Centers for Disease Control and Prevention (CDC) were evaluated. Inverse probability weighting (IPW) was used to reduce bias due to missing paternal records.MAIN RESULTS AND THE ROLE OF CHANCEMean paternal age has increased over the past 44 years from 27.4 to 30.9 years. College education and Northeastern birth states were associated with higher paternal age. Racial/ethnic differences were also identified, whereby Asian fathers were the oldest and Black fathers were the youngest. The parental age difference (paternal age minus maternal age) has decreased over the past 44 years. Births to Black and Native American mothers were most often lacking paternal data, implying low paternal reporting. Paternal reporting was higher for older and more educated women.LIMITATIONS, REASONS FOR CAUTIONAlthough we utilized IPW to reduce the impact of paternal reporting bias, our estimates may still be influenced by the missing data in the NVSS.WIDER IMPLICATIONS OF THE FINDINGSPaternal age is rising within the USA among all regions, races and education levels. Given the implications for offspring health and demographic patterns, further research on this trend is warranted.STUDY FUNDING/COMPETING INTEREST(S)No funding was received for this study and there are no competing interests.TRIAL REGISTRATION NUMBERN/A.
J. Aizpurua, L. Medrano, M. Enciso, J. Sarasa, , M.A. Fernández, M.J. Gómez-Torres
Human Reproduction, Volume 32, pp 2007-2015; https://doi.org/10.1093/humrep/dex281

Abstract:
Is permeable cryoprotectant-free vitrification of native sperm samples a good alternative to conventional slow freezing? The permeable cryoprotectant-free sperm vitrification protocol tested in this study renders considerably better recovery rates of good quality sperm compared to slow freezing. Slow freezing is currently the most commonly used technique for sperm cryopreservation, though this method has been repeatedly shown to have negative effects on both structural and functional sperm features. New alternative methods such as vitrification have been established as a successful alternative in other reproductive cell types, but vitrification of spermatozoa is still a rather unexplored methodology, with limited studies showing its efficacy in male gametes. This study included 18 normozoospermic sperm samples from patients seeking ART treatment between 2014 and 2015. The effects of a new vitrification protocol on functional and structural sperm quality parameters in comparison to fresh and slow-frozen samples were assessed. All samples were divided into three aliquots: fresh (F), slow freezing–thawing (S) and vitrification-warming (V). Sperm concentration, motility, morphology, vitality, DNA fragmentation, cytoskeleton integrity and spontaneous acrosome reaction were assessed and compared between the groups. Results showed improved preservation of sperm features after vitrification compared to conventional freezing. Permeable cryoprotectant-free vitrification presented a significantly higher percentage of live spermatozoa, than slow freezing, better preservation of acrosomes was achieved in vitrified samples and DNA fragmentation was reduced approximately one-third on average compared to slow freezing. Regarding tubulin assay, three different labelling patterns were observed. The frequency of these labelling patterns was similar in F and V groups but this was not the case of the S group. The multivariate analysis of all sperm quality parameters studied revealed that the V group presented features that are closer to the F group than the S group, indicating that samples are better preserved through vitrification than slow freezing. This validation has been undertaken only on normozoospermic sperm samples. It would be necessary to compare these results in pathological samples and also to evaluate the influence of the application of this methodology on clinical outcomes. The sperm vitrification protocol here described warrants better maintenance of sperm quality parameters than traditional freezing methods and may be a good alternative to preserve sperm samples from patients seeking IVF treatment. This study was funded by IVF-Spain Foundation. The authors have no conflicts of interest to declare.
Comment
Dagan Wells, Krithika Ravichandran, Caroline McCaffrey, , Arlene Morales, Mark Perloe, Santiago Munne,
Human Reproduction, Volume 32, pp 2150-2151; https://doi.org/10.1093/humrep/dex279

, , Ying Zhu, Wei Zhang, Xi Wang, Beili Chen, Tengyan Li, Hong Pan, Jing Wang, , et al.
Human Reproduction, Volume 32, pp 2138-2146; https://doi.org/10.1093/humrep/dex263

Abstract:
Does a novel heterozygous KHDRBS1 variant, identified using whole-exome sequencing (WES) in two patients with primary ovarian insufficiency (POI) in a pedigree, cause defects in mRNA alternative splicing? The heterozygous variant of KHDRBS1 was confirmed to cause defects in alternative splicing of many genes involved in DNA replication and repair. Studies in mice revealed that Khdrbs1 deficient females are subfertile, which manifests as delayed sexual maturity and significantly reduced numbers of secondary and pre-antral follicles. No mutation of KHDRBS1, however, has been reported in patients with POI. This genetic and functional study used WES to find putative mutations in a POI pedigree. Altogether, 215 idiopathic POI patients and 400 healthy controls were screened for KHDRBS1 mutations. Two POI patients were subjected to WES to identify sequence variants. Mutational analysis of the KHDRBS1 gene in 215 idiopathic POI patients and 400 healthy controls were performed. RNA-sequencing was carried out to find the mis-regulation of gene expression due to KHDRBS1 mutation. Bioinformatics was used to analyze the change in alternative splicing events. We identified a heterozygous mutation (c.460A >G, p.M154V) in KHDRBS1 in two patients. Further mutational analysis of 215 idiopathic POI patients with the KHDRBS1 gene found one heterozygous mutation (c.263C >T, p.P88L). We failed to find these two mutations in 400 healthy control women. Using RNA-sequencing, we found that the KGN cells expressing the M154V KHDRBS1 mutant had different expression of 66 genes compared with wild-type (WT) cells. Furthermore, 145 genes were alternatively spliced in M154V cells, and these genes were enriched for DNA replication and repair function, revealing a potential underlying mechanism of the pathology that leads to POI. Although the in vitro assays demonstrated the effect of the KHDRBS1 variant on alternative splicing, further studies are needed to validate the in vivo effects on germ cell and follicle development. This finding provides researchers and clinicians a better understanding of the etiology and molecular mechanism of POI. This study was supported by the Ministry of Science and Technology of China (2012CB944704; 2012CB966702), National Research Institute for Family Planning (2017GJZ05), the National Natural Science Foundation of China (31171429) and Beijing Advanced Innovation Center for Structural Biology. The authors declare no conflict of interest.
L. Abadia, Y.-H. Chiu, P.L. Williams, T.L. Toth, I. Souter, R. Hauser, , , for the EARTH Study Team
Human Reproduction, Volume 32, pp 1846-1854; https://doi.org/10.1093/humrep/dex237

Abstract:
Is pre-treatment alcohol and caffeine intake associated with infertility treatment outcomes among women undergoing ART? Low to moderate alcohol and caffeine intakes in the year prior to infertility treatment were not related to ART outcomes. Alcohol and caffeine intake have been found to be associated with infertility in some studies. Nevertheless, data on their relation with outcomes of infertility treatments are scarce and inconsistent. We included 300 women (493 ART cycles) from the Environment and Reproductive Health Study, an ongoing cohort study (2006–2016). Pre-treatment intakes of alcohol and caffeine were assessed retrospectively using a validated food frequency questionnaire. Intermediate and clinical endpoints of ART were abstracted from electronic medical records. Generalized linear mixed models with random intercepts to account for multiple ART cycles per woman were used to evaluate the association with ART outcomes adjusting for age, BMI, smoking status, infertility diagnosis, protocol type, race, dietary patterns, and calories, vitamin B12 and folate intake. Median (range) pre-treatment alcohol and caffeine intakes were 5.6 (0.0–85.8) g/day and 124.9 (0.3–642.2) mg/day, respectively. The adjusted percentage of initiated cycles resulting in live birth (95% CI) for women in increasing categories of pre-treatment alcohol intake was 34% (20, 52%) for non-consumers, 46% (36, 57%) for 0.1–6 g/day, 41% (29, 53%) for 6.1–12 g/day, 42% (31, 55%) for 12.1–24 g/day, and 41% (22, 63%) for >24 g/day (P, trend = 0.87). The adjusted percentage of cycles resulting in live birth (95% CI) for women in increasing categories of caffeine intake was 46% (36–57%) for 300 mg/day (P, trend = 0.34). When specific types of alcoholic and caffeinated beverages were evaluated, no relations with ART treatment outcomes were observed. Residual confounding by other diet and lifestyle factors cannot be ruled out owing to the observational nature of this study. It is also unclear how generalizable these results are to women who are conceiving without the assistance of ART. Our results provide reassurance that low to moderate intakes of alcohol (e.g. ≤12 g/day) and caffeine (e.g. <200 mg/day) in the year prior to infertility treatment initiation do not have an adverse effect on intermediate or clinical outcomes of ART. The authors are supported by National Institutes of Health (NIH) grants ES022955, R01ES009718, R01ES000002, P30DK46200 and L50-HD085359. No conflicts of interest to declare. NCT00011713.
, I. Scholten, F. Mol, , B.W. Mol, F. Van Der Veen
Human Reproduction, Volume 32, pp 2117-2122; https://doi.org/10.1093/humrep/dex273

Abstract:
STUDY QUESTIONAre randomized controlled trials (RCTs) on IVF and ICSI subject to selective outcome reporting and is this related to sponsorship?SUMMARY ANSWERThere are inconsistencies, independent from sponsorship, in the reporting of primary outcome measures in the majority of IVF and ICSI trials, indicating selective outcome reporting.WHAT IS KNOWN ALREADYRCTs are subject to bias at various levels. Of these biases, selective outcome reporting is particularly relevant to IVF and ICSI trials since there is a wide variety of outcome measures to choose from. An established cause of reporting bias is sponsorship. It is, at present, unknown whether RCTs in IVF/ICSI are subject to selective outcome reporting and whether this is related with sponsorship.STUDY DESIGN, SIZE, DURATIONWe systematically searched RCTs on IVF and ICSI published between January 2009 and March 2016 in MEDLINE, EMBASE, the Cochrane Central Register of Controlled Trials and the publisher subset of PubMed. We analysed 415 RCTs.PARTICIPANTS/MATERIALS, SETTING, METHODSPer included RCT, we extracted data on impact factor of the journal, sample size, power calculation, and trial registry and thereafter data on primary outcome measure, the direction of trial results and sponsorship.MAIN RESULTS AND THE ROLE OF CHANCEOf the 415 identified RCTs, 235 were excluded for our primary analysis, because the sponsorship was not reported. Of the 180 RCTs included in our analysis, 7 trials did not report on any primary outcome measure and 107 of the remaining 173 trials (62%) reported on surrogate primary outcome measures. Of the 114 registered trials, 21 trials (18%) provided primary outcomes in their manuscript that were different from those in the trial registry. This indicates selective outcome reporting. We found no association between selective outcome reporting and sponsorship. We ran additional analyses to include the trials that had not reported sponsorship and found no outcomes that differed from our primary analysis.LIMITATIONS, REASONS FOR CAUTIONSince the majority of the trials did not report on sponsorship, there is a risk on sampling bias.WIDER IMPLICATIONS OF THE FINDINGSIVF and ICSI trials are subject, to a large extent, to selective outcome reporting. Readers should be aware of this to avoid implementation of false or misleading results in clinical practice.STUDY FUNDING/COMPETING INTERESTSNo funding received and there are no conflicts of interest.TRIAL REGISTRATION NUMBERN/A
, Nikita Sinha, Kaitlyn Wald, Eve Harris, Molly Quinn, Tal Imbar, Evelyn Mok-Lin, A. Jo Chien, Mitchell Rosen
Published: 9 September 2017
Human Reproduction, Volume 32, pp 2123-2129; https://doi.org/10.1093/humrep/dex276

Abstract:
Is random start ovarian stimulation associated with delays in initiation of neoadjuvant chemotherapy for breast cancer? Among women who complete fertility preservation (FP) consultation, random start ovarian stimulation is unlikely to delay time to initiation of neoadjuvant chemotherapy start. Neoadjuvant chemotherapy is now a widely accepted treatment modality for operable breast cancer and random start ovarian stimulation is an increasingly-utilized modality for FP. While conventional ovarian stimulation does not appear to delay starting adjuvant chemotherapy, the relationship between random start ovarian stimulation and neoadjuvant chemotherapy start is not well-understood. Cross-sectional study of all women seen between from January 2011 to April 2017 for FP consultation prior to starting neoadjuvant chemotherapy for breast cancer. A chart-review was performed. Study inclusion criteria were female sex; age 18–45; non-metastatic breast cancer diagnosis; underwent FP consultation; underwent neoadjuvant chemotherapy. Referrals for FP evaluation came from a regional referral base of oncology clinics. Various time-points related to cancer diagnosis, FP or chemotherapy were obtained from medical record review. We compared time-points between those who underwent ovarian stimulation for FP versus those who did not using T-tests and linear modeling. A total of 89 women who had FP consultation prior to neoadjuvant chemotherapy were identified. Sixty-seven percent underwent ovarian stimulation prior to cancer treatment and 33% did not. Women who underwent ovarian stimulation were similar in parity and clinical cancer stage to those who did not. Overall, the average time from cancer diagnosis to chemotherapy start was similar between the group that did undergo ovarian stimulation and those who did not (38.1 ± 11.3 versus 39.4 ± 18.5 days, P = 0.672). Those that underwent ovarian stimulation were referred 9.4 ± 6.8 days after diagnosis versus 17.9 ± 15.3 days for those who did not undergo ovarian stimulation (P< 0.001). Retrospective study with potential for selection bias among those who underwent ovarian stimulation versus those who did not. Reasons for caution include the possibility of unmeasured differences among those who did and did not undergo ovarian stimulation, including: patients’ and providers’ perceptions of the urgency to start chemotherapy, ongoing oncology work-up and treatment planning, FP decision-making, and the pursuit of second and third opinions. The difference in time from referral to FP consultation may have also influenced patients’ decisions about whether to undergo ovarian stimulation. In this study, FP with random start ovarian stimulation was not associated with a delay cancer treatment in the neoadjuvant setting, so long as there was a prompt FP referral. Patients undergoing neoadjuvant chemotherapy should be informed of these findings to avoid unnecessary anxiety due to concern for delays. This study was supported by departmental research funding within the University of California, San Francisco Department of Obstetrics, Gynecology and Reproductive Sciences. There are no conflicts of interest to declare.
Human Reproduction, Volume 32, pp 2069-2075; https://doi.org/10.1093/humrep/dex275

Abstract:
Do women who place high importance on career success have different perceptions of pregnancy planning, delayed reproduction, and the ethical acceptability of ART than women with less emphasis on their career? Career-focused women place more importance on pregnancy planning, have greater confidence in delayed childbearing, and are more ethically accepting of donor gamete ART than women who do not place as much importance on career success. Women in high-professional careers are more likely to delay childbearing while simultaneously possessing a stronger desire for motherhood. The underlying values which enable these competing desires have not been elucidated. This cross-sectional study utilized data from the National Survey of Fertility Barriers (NSFB), a nationally representative telephone survey of US women aged 25–45. Funded by the Eunice Kennedy Shriver National Institute of Child Health and Human Development, the NSFB surveyed 4712 women from 2004 to 2007. In addition to demographic data, the NSFB obtained information about the reproductive history and personal values of participants. Weighted multivariate regression analysis was used to assess reproductive values in career-focused women. In total, 48.8% of women considered success in work very important, while 17.3% considered it somewhat or not important. Women who placed less value on career success were less likely to consider pregnancy planning important and were less optimistic about the success of delayed childbearing than their work-centric counterparts. Women less focused on their careers were also more likely to have serious ethical concerns about donor gametes, but less likely to have ethical concerns about IUI or IVF, when compared to career-focused women. Intention to bear children could not be evaluated in the setting of career intentions due to a lack of data on when the participant intended on pursuing motherhood. Political preferences on reproductive health were also not evaluated. The validity of the career priority questions has not been assessed. Additionally, respondents’ value statements were not matched to subsequent actions, so it remains possible that these values do not directly impact reproductive behaviors. Our results suggest that reproductive counseling for career-focused women should focus on effective contraception when attempting to delay pregnancy, improved knowledge about age-related fertility decline, and the scope and limitations of current reproductive technologies. In addition, the unique reproductive views of career-focused women suggest that they may benefit from increased employer/insurer support for strategies to enable delayed childbearing, such as fertility preservation and third-party reproduction. None.
Suresh Ramaswamy, William H. Walker, Paula Aliberti, Rahil Sethi, Gary R. Marshall, Alyxzandria Smith, Seyedmehdi Nourashrafeddin, Alicia Belgorosky, Uma R. Chandran, Mark P. Hedger, et al.
Human Reproduction, Volume 32, pp 2088-2100; https://doi.org/10.1093/humrep/dex270

, , Ana I Hernández-Peñalver, Shiana Corbalán-Biyang, Ana Carmona-Barnosi, , Aníbal Nieto,
Human Reproduction, Volume 32, pp 2315-2323; https://doi.org/10.1093/humrep/dex274

Abstract:
Is polycystic ovary syndrome (PCOS) associated with anogenital distance (AGD), a biomarker of fetal androgen exposure, in adult Mediterranean women? Longer AGD is associated with PCOS in adult Mediterranean women. AGD 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. This was a case–control study of 126 women with PCOS and 159 controls between September 2014 and May 2016. Cases 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. Cases 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). We 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. Our results support the hypothesis that PCOS has an intrauterine origin, and that the hormonal environment in which the fetus develops may be highly relevant. This 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. Not applicable.
The European IVF-Monitoring Consortium (EIM), , , M S Kupka, J De Mouzon, , , T Motrenko, , , et al.
Human Reproduction, Volume 32, pp 1957-1973; https://doi.org/10.1093/humrep/dex264

Abstract:
Are there any changes in the treatments involving ART and IUI initiated in Europe during 2013 compared with previous years? An increase in the overall number of ART cycles resulting from a higher number of countries reporting data was evident, the pregnancy rates (PRs) in 2013 remained stable compared with those reported in 2012, the number of transfers with multiple embryos (3+) was lower than ever before yet the multiple delivery rates (DRs) remained unchanged, and IUI activity and success rates were similar to those of last years. Since 1997, ART data in Europe have been collected and reported in 16 manuscripts, published in Human Reproduction. Retrospective data collection of European ART data by the European IVF-monitoring Consortium for ESHRE. Data for cycles between 1 January and 31 December 2013 were collected from National Registers, when existing, or on a voluntary basis by personal information. From 38 countries (+4 compared with 2012), 1169 clinics reported 686 271 treatment cycles including 144 299 of IVF, 330 367 of ICSI, 154 712 of frozen embryo replacement (FER), 40 244 of egg donation (ED), 247 of IVM, 9791 of PGD/PGS and 6611 of frozen oocyte replacements. European data on intrauterine insemination using husband/partner's semen (IUI-H) and donor semen (IUI-D) were reported from 1095 IUI labs in 22 countries. A total of 175 467 IUI-H and 43 785 IUI-D cycles were included. In 17 countries where all clinics reported to their ART register, a total of 374 177 ART cycles were performed in a population of around 310 million inhabitants, corresponding to 1175 cycles per million inhabitants (range, 235–2703 cycles per million inhabitants). For all IVF cycles, the clinical PRs per aspiration and per transfer were stable with 29.6% (29.4% in 2012) and 34.5% (33.8% in 2012), respectively. For ICSI, the corresponding rates also were stable with 27.8% (27.8% in 2012) and 32.9% (32.3% in 2012). In FER-cycles, the PR per thawing/warming increased to 27.0% (23.1% in 2012). In ED cycles, the PR per fresh transfer increased to 49.8% (48.4% in 2012), to 38.5% (35.9% in 2012) per thawed transfer, and to 46.4% for transfers after FOR (45.1% in 2012). The DRs after IUI remained stable at 8.6% (8.5% in 2012) after IUI-H and was slightly lower after IUI-D (11.1% versus 12.0% in 2012). In IVF and ICSI cycles, 1, 2, 3 and 4+ embryos were transferred in 31.4, 56.3, 11.5, and 1.0% of the cycles, respectively (corresponding numbers were 30.2, 55.4, 13.3 and 1.1% in 2012). The proportions of singleton, twin and triplet deliveries after IVF and ICSI (added together) were 82., 17.5 and 0.5%, respectively, resulting in a total multiple DR of 18.0% compared to 17.9% in 2012. In FER-cycles, the multiple DR was 12.8% (12.5% twins and 0.3% triplets), nearly the same as in 2012 (12.5, 12.2 and 0.3% respectively). Twin and triplet DRs associated with IUI cycles were 9.5%/0.6% and 7.5%/0.3%, following treatment with husband/donor semen, respectively. The method of reporting varies among countries, and registers from a number of countries have been unable to provide some of the relevant data such as initiated cycles and deliveries. As long as data are incomplete and generated through different methods of collection, the results should be interpreted with caution. The 17th ESHRE report on ART shows a continuing expansion of the number of treatment cycles in Europe, with more than 685 000 cycles reported in 2013 and an increasing contribution to birth rate in many countries. However, the need to improve and standardize the national registries, and to establish validation methodologies, remains manifest. The study has no external funding; all costs are covered by ESHRE. There are no competing interests.
, V Goossens, G Kokkali, M Meijer-Hoogeveen, E Coonen, C Moutou
Human Reproduction, Volume 32, pp 1974-1994; https://doi.org/10.1093/humrep/dex265

Abstract:
STUDY QUESTIONHow does the data collection XIV–XV of the European Society of Human Reproduction and Embryology (ESHRE) PGD Consortium compare with the cumulative data for data collections I–XIII?SUMMARY ANSWERThe 14th and 15th retrospective collection represents valuable data on PGD/PGS cycles, pregnancies and children: the main trend observed is the increased application of array technology at the cost of FISH testing in PGS cycles and in PGD cycles for chromosomal abnormalities.WHAT IS KNOWN ALREADYSince 1999, the PGD Consortium has collected, analysed and published 13 previous data sets and an overview of the first 10 years of data collections.STUDY DESIGN, SIZE, DURATIONData were collected from each participating centre using a FileMaker Pro database (versions 5–12). Separate predesigned FileMaker Pro files were used for the cycles, pregnancies and baby records. The study documented cycles performed during the calendar years 2011 and 2012 and follow-up of the pregnancies and babies born which resulted from these cycles (until October 2013).PARTICIPANTS/MATERIALS, SETTINGS, METHODData were submitted by 71 centres (full PGD Consortium members). Records with incomplete or inconsistent data were excluded from the calculations. Corrections, calculations and tables were made by expert co-authors.MAIN RESULTS AND THE ROLE OF CHANCEFor data collection XIV–XV, 71 centres reported data for 11 637 cycles with oocyte retrieval (OR), along with details of the follow-up on 2147 pregnancies and 1755 babies born. A total of 1953 cycles to OR were reported for chromosomal abnormalities, 144 cycles to OR for sexing for X-linked diseases, 3445 cycles to OR for monogenic diseases, 6095 cycles to OR for PGS and 38 cycles to OR for social sexing. From 2010 until 2012, the use of arrays for genetic testing increased from 4% to 20% in PGS and from 6% to 13% in PGD cycles for chromosomal abnormalities; the uptake of biopsy at the blastocyst stage (from <1% up to 7%) was only observed in cycles for structural chromosomal abnormalities, alongside the application of array comparative genomic hybridization.LIMITATIONS, REASONS FOR CAUTIONThe findings apply to the 71 participating centres and may not represent worldwide trends in PGD.WIDER IMPLICATIONS OF THE FINDINGSThe annual data collections provide an important resource for data mining and for following trends in PGD/PGS practice.STUDY FUNDING/COMPETING INTEREST(S)None.
, , N.P. Dickson, , W.R. Gillett
Human Reproduction, Volume 32, pp 2042-2048; https://doi.org/10.1093/humrep/dex271

Abstract:
How common were children among infertile couples? A total of 61.7% of infertile couples presenting for care subsequently had live born children 13.1 years after first being clinically assessed, with a mean of 1.7 children among those who had at least one. While the prognoses for infertile couples undertaking specific treatments have been well described, less is known about those not undergoing these treatments or the total number of children. This information is necessary for decision-making in many individual cases; not knowing this has been cited by patients and clinicians as impeding implementation of care. The sole provider of specialist fertility care for the two southern-most regions in New Zealand enroled 1386 infertile couples from 1998 to 2005 in a longitudinal study with follow-up on all births until the end of 2014. Couples were followed in care for a median of 1.1 years and median follow-up for births was 13.1 years. Clinic-collected data were linked to national maternity data to extend follow-up past the end of clinical contact. The primary outcome was the total number of live born children. Hurdle regression was used to investigate factors associated with resolving infertility and the total number of children. Infertility was resolved with a live birth by 61.7% (95% CI 59.1–64.2%) of couples; just over half of all first births were treatment-dependent. Among couples who resolved their infertility, 55.6% (52.2–58.9%) had at least one additional child and the mean number of children was 1.7. While female age strongly influenced outcomes, one-third of women aged 40–41 years had a child, not significantly less than those in their late 30s. The lowest levels of resolution occurred in women aged ≥42 years, couples who were infertile for >4 years and women with a BMI ≥ 35 kg/m2. Moderate obesity did not affect outcomes. The main limitation of this study was insufficient data to investigate male factor infertility outcomes. It is also possible that treatment-dependent resolution could be higher in more recent cohorts with the increased use of ART. Outcomes in these couples are comparable to those seen in other studies in high-income countries despite the relatively low contribution of ART. The prognosis for most infertile couples is positive and suggests many will not require treatment. Further research is needed to inform best practice for women in their early forties or with moderate obesity, and to develop prediction models that are more relevant for the initial management of infertility. This study was co-funded by a University of Otago PhD Scholarship and the Department of Women's and Children's Health, University of Otago. There were no competing interests to declare.
L. Boucret, C. Bris, V. Seegers, D. Goudenège, , M. Domin-Bernhard, V. Ferré-L'hotellier, P.E. Bouet, P. Descamps, , et al.
Human Reproduction, Volume 32, pp 2101-2109; https://doi.org/10.1093/humrep/dex268

Abstract:
Does ovarian ageing increase the number of heteroplasmic mitochondrial DNA (mtDNA) point mutations in oocytes? Our results suggest that oocytes are not subject to the accumulation of mtDNA point mutations during ovarian ageing. Ageing is associated with the alteration of mtDNA integrity in various tissues. Primary oocytes, present in the ovary since embryonic life, may accumulate mtDNA mutations during the process of ovarian ageing. This was an observational study of 53 immature oocyte–cumulus complexes retrieved from 35 women undergoing IVF at the University Hospital of Angers, France, from March 2013 to March 2014. The women were classified in two groups, one including 19 women showing signs of ovarian ageing objectified by a diminished ovarian reserve (DOR), and the other, including 16 women with a normal ovarian reserve (NOR), which served as a control group. mtDNA was extracted from isolated oocytes, and from their corresponding cumulus cells (CCs) considered as a somatic cell compartment. The average mtDNA content of each sample was assessed by using a quantitative real-time PCR technique. Deep sequencing was performed using the Ion Torrent Proton for Next-Generation Sequencing. Signal processing and base calling were done by the embedded pre-processing pipeline and the variants were analyzed using an in-house workflow. The distribution of the different variants between DOR and NOR patients, on one hand, and oocyte and CCs, on the other, was analyzed with the generalized mixed linear model to take into account the cluster of cells belonging to a given mother. There were no significant differences between the numbers of mtDNA variants between the DOR and the NOR patients, either in the oocytes (P = 0.867) or in the surrounding CCs (P = 0.154). There were also no differences in terms of variants with potential functional consequences. De-novo mtDNA variants were found in 28% of the oocytes and in 66% of the CCs with the mean number of variants being significantly different (respectively 0.321, SD = 0.547 and 1.075, SD = 1.158) (P< 0.0001). Variants with a potential functional consequence were also overrepresented in CCs compared with oocytes (P = 0.0019). N/A. Limitations may be due to the use of immature oocytes discarded during the assisted reproductive technology procedure, the small size of the sample, and the high-throughput sequencing technology that might not have detected heteroplasmy levels lower than 2%. The alteration of mtDNA integrity in oocytes during ovarian ageing is a recurring question to which our pilot study suggests a reassuring answer. This work was supported by the University Hospital of Angers, the University of Angers, France, and the French national research centers, INSERM and the CNRS. There are nocompeting interests.
, F. Lolicato, , , H. Van Ranst, , E. Anckaert,
Human Reproduction, Volume 32, pp 2056-2068; https://doi.org/10.1093/humrep/dex262

Abstract:
Are meiotic and developmental competence of human oocytes from small (2–8 mm) antral follicles improved by applying an optimized IVM method involving a prematuration step in presence of C-Type Natriuretic Peptide (CNP) followed by a maturation step in presence of FSH and Amphiregulin (AREG)? A strategy involving prematuration culture (PMC) in the presence of CNP followed by IVM using FSH + AREG increases oocyte maturation potential leading to a higher availability of Day 3 embryos and good-quality blastocysts for single embryo transfer. IVM is a minimal-stimulation ART with reduced hormone-related side effects and risks for the patients, but the approach is not widely used because of an efficiency gap compared to conventional ART. In vitro systems that enhance synchronization of nuclear and cytoplasmic maturation before the meiotic trigger are crucial to optimize human IVM systems. However, previous PMC attempts have failed in sustaining cumulus-oocyte connections throughout the culture period, which prohibited a normal cumulus-oocyte communication and precluded an adequate response by the cumulus-oocyte complex (COC) to the meiotic trigger. A first prospective study involved sibling oocytes from a group of 15 patients with polycystic ovary syndrome (PCOS) to evaluate effects of a new IVM culture method on oocyte nuclear maturation and their downstream developmental competence. A second prospective study in an additional series of 15 women with polycystic ovaries characterized and fine-tuned the culture conditions. Fifteen women with PCOS (according to Rotterdam criteria) underwent IVM treatment after 3–5 days of highly purified human menopausal gonadotropin (HP-hMG) stimulation and no human chorionic gonadotropin (hCG) trigger before oocyte retrieval. A first study was designed with sibling oocytes to prospectively evaluate the impact of an IVM culture method: 24 h PMC with CNP + 30 h IVM with FSH and AREG, on embryo yield, in comparison to the standard (30 h) IVM clinical protocol (Group I, n = 15). A second prospective study was performed in 15 women with polycystic ovaries, to characterize and optimize the PMC conditions (Group II, n = 15). The latter study involved the evaluation of oocyte meiotic arrest, the preservation of cumulus-oocyte transzonal projections (TZPs), the patterns of oocyte chromatin configuration and cumulus cells apoptosis following the 24 and 46 h PMC. Furthermore, oocyte developmental potential following PMC (24 and 46 h) + IVM was also evaluated. The first 20 good-quality blastocysts from PMC followed by IVM were analysed by next generation sequencing to evaluate their aneuploidy rate. PMC in presence of CNP followed by IVM using FSH and AREG increased the meiotic maturation rate per COC to 70%, which is significantly higher than routine standard IVM (49%; P ≤ 0.001). Hence, with the new system the proportion of COCs yielding transferable Day 3 embryos and good-quality blastocysts increased compared to routine standard IVM (from 23 to 43%; P ≤ 0.001 and from 8 to 18%; P ≤ 0.01, respectively). CNP was able to prevent meiosis resumption for up to 46 h. After PMC, COCs had preserved cumulus-oocyte TZPs. The blastocysts obtained after PMC + IVM did not show increased aneuploidy rates as compared to blastocysts from conventional ART. The novel IVM approach in PCOS patients was tested in oocytes derived from small antral follicles which have an intrinsically low developmental potential. Validation of the system would be required for COCs from different (larger) follicular sizes, which may involve further adjustment of PMC conditions. Furthermore, considering that this is a novel strategy in human IVM treatment, its global efficiency needs to be confirmed in large prospective randomized controlled trials. The further application in infertile patients without PCOS, e.g. cancer patients, remains to be evaluated. The findings of this pilot study suggest that the efficiency gap between IVM and conventional IVF can be reduced by fine-tuning of the culture methods. This novel strategy opens new perspectives for safe and patient-friendly ART in patients with PCOS. IVM research at the Vrije Universiteit Brussel has been supported by grants from: the Institute for the Promotion of Innovation by Science and Technology in Flanders (Agentschap voor Innovatie door Wetenschap en Technologie—IWT, project 110680); the Fund for Research Flanders (Fonds Wetenschappelijk Onderzoek–Vlaanderen—FWO, project G.0343.13), the Belgian Foundation Against Cancer (HOPE project, Dossier C69). The authors have no conflicts of interest.
, D. Tsolakidis, V. Theodoulidis, A. Makedos, T. Zaramboukas, B. Tarlatzis
Human Reproduction, Volume 32, pp 2036-2041; https://doi.org/10.1093/humrep/dex258

Abstract:
What is the prevalence of leiomyosarcomas and atypical leiomyomas after laparoscopic morcellation of fibroids in reproductive age women? No case of leiomyosarcomas but seven atypical leiomyomas were found in 1216 subjects. Although uterine sarcoma is a rare entity affecting usually older peri- or post-menopausal women, the Food and Drug Administration discourages use of laparoscopic power morcellation of uterine fibroids. Retrospective review of data extracted from a single center database of 1216 consecutive women who underwent laparoscopic morcellation of 2582 unsuspicious leiomyomas between June 2003 and December 2015 and were followed-up until December 2016. A total of 1216 women, aged 18–45 years, underwent laparoscopic morcellation of 2582 apparently benign leiomyomas by the same surgeon and all specimen slides were examined by the same experienced pathologist. The prevalence of leiomyosarcomas and atypical leiomyomas was 0% (95% CI: 0–0.3%) and 0.6% (95% CI: 0.23–1.18%) (six atypical-bizarre and one mitotically active leiomyoma) respectively. In addition, there were identified 34 cases of adenomyomas, 45 leiomyomas with infarcts, 81 cellular leiomyomas and 133 degenerated leiomyomas. No morcellator-associated complication was recorded and none of the patients included in this study required conversion to laparotomy. Retrospective and single referral center study design. Laparoscopic morcellation of unsuspicious leiomyomas after careful preoperative work up seems to be safe in women of reproductive age. None.
, Marian G. Showell, Emily A.E. Showell, Penny Beetham, Nora Baak, Selma Mourad, Vanessa M.B. Jordan
Human Reproduction, Volume 32, pp 1827-1834; https://doi.org/10.1093/humrep/dex251

Abstract:
What is the prevalence and source of prospectively and retrospectively registered and unregistered trials in fertility treatments? Trial registration is low and does not appear to be changing over the 5 years studied. Trial registration is associated with lower risk of bias than in unregistered trials. The Cochrane Gynaecology and Fertility Group's specialised register was searched on 5 November 2015 for randomised controlled trials (RCTs) published from January 2010 to December 2014. Eligible trials included randomised women or men for fertility treatments, were published in full text, and written in English. Two reviewers independently assessed trial registration status for each trial, by searching the publication, trial registries, and by contacting the original authors. Of 693 eligible RCTS, only 44% were registered trials. Of 309 registered trials, 21.7% were prospectively registered, 15.8% were registered within 6 months of first patient enrolment and 62.5% were retrospectively registered trials. Prospective trial registration by country varied from 0% to 100%. The highest frequency of prospective trial registration amongst the top 10 publishing countries was 31% in the Netherlands. Only English language trials were included in this review. Prospective trial registration is still low. Journals, funders and ethics committees could have a greater role to increase trial registration. University of Auckland. No competing interests.
, Martin Gosnell, Ayad G. Anwer, Melissa White, Malcolm Purdey, Andrew D. Abell, Ewa M. Goldys,
Human Reproduction, Volume 32, pp 2016-2025; https://doi.org/10.1093/humrep/dex261

Abstract:
Can we separate embryos cultured under either 7% or 20% oxygen atmospheres by measuring their metabolic heterogeneity? Metabolic heterogeneity and changes in metabolic profiles in morula exposed to two different oxygen concentrations were not detectable using traditional fluorophore and two-channel autofluorescence but were detectable using hyperspectral microscopy. Increased genetic and morphological blastomere heterogeneity is associated with compromised developmental competence of embryos and currently forms the basis for embryo scoring within the clinic. However, there remains uncertainty over the accuracy of current techniques, such as PGS and time-lapse microscopy, to predict subsequent pregnancy establishment. The impact of two oxygen concentrations (7% = optimal and 20% = stressed) during post-fertilisation embryo culture was assessed. Cattle embryos were exposed to the different oxygen concentrations for 8 days (D8; embryo developmental competence) or 5 days (D5; metabolism measurements). Between 3 and 4 experimental replicates were performed, with 40–50 embryos per replicate used for the developmental competency experiment, 10–20 embryos per replicate for the fluorophore and two-channel autofluorescence experiments and a total of 21–22 embryos used for the hyperspectral microscopy study. In-vitro produced (IVP) cattle embryos were utilised for this study. Post-fertilisation, embryos were exposed to 7% or 20% oxygen. To determine impact of oxygen concentrations on embryo viability, blastocyst development was assessed on D8. On D5, metabolic heterogeneity was assessed in morula (on-time) embryos using fluorophores probes (active mitochondria, hydrogen peroxide and reduced glutathione), two-channel autofluorescence (FAD and NAD(P)H) and 18-channel hyperspectral microscopy. Exposure to 20% oxygen following fertilisation significantly reduced total blastocyst, expanded and hatched blastocyst rates by 1.4-, 1.9- and 2.8-fold, respectively, compared to 7% oxygen (P< 0.05), demonstrating that atmospheric oxygen was a viable model for studying mild metabolic stress. The metabolic profiles of D5 embryos was determined and although metabolic heterogeneity was evident within the cleavage stage (i.e. arrested) embryos exposed to fluorophores, there were no detectable difference in fluorescence intensity and pattern localisation in morula exposed to the two different oxygen concentrations (P > 0.05). While there were no significant differences in two-channel autofluorescent profiles of morula exposed to 7% and 20% oxygen (main effect, P > 0.05), morula that subsequently progressed to the blastocyst stage had significantly higher levels of FAD and NAD(P)H fluorescence compared to arrested morula (P< 0.05), with no change in the redox ratio. Hyperspectral autofluorescence imaging (in 18-spectral channels) of the D5 morula revealed highly significant differences in four features of the metabolic profiles of morula exposed to the two different oxygen concentrations (P< 0.001). These four features were weighted and their linear combination revealed clear discrimination between the two treatment groups. Metabolic profiles were assessed at a single time point (morula), and as such further investigation is required to determine if differences in hyperspectral signatures can be detected in pre-compaction embryos and oocytes, using both cattle and subsequently human models. Furthermore, embryo transfers should be performed to determine the relationship between metabolic profiles and pregnancy success. Advanced autofluorescence imaging techniques, such as hyperspectral microscopy, may provide clinics with additional tools to improve the assessment of embryos prior to transfer. This study was funded by the Australian Research Council Centre of Excellence for Nanoscale BioPhotonics (CE140100003). The Fluoview FV10i confocal microscope was purchased as part of the Sensing Technologies for Advanced Reproductive Research (STARR) facility, funded by the South Australian Premier's Science and Research Fund. The authors declare there are no conflict of interest.
S. Hendriks, K. Peeraer, H. Bos, ,
Human Reproduction, Volume 32, pp 2076-2087; https://doi.org/10.1093/humrep/dex256

Abstract:
Do men and women beginning to attend a fertility clinic prefer genetic over non-genetic parenthood? Nearly, all infertile men and women prefer genetic parenthood. Clinicians assume that all infertile couples prefer genetic parenthood over non-genetic parenthood and, therefore, consider treatments with donor gametes an option of last resort. Previous studies of the desire for parenthood identified 30 motivations for genetic parenthood, and 51 motivations for which having a genetically related child is not strictly necessary but might be deemed required. The exact strength of the preference of infertile men and women for genetic parenthood remains unclear, as does the importance of the various motivations. A questionnaire was developed based on a literature review. It was assessed by professionals and pilot tested among patients. The coded paper–pencil questionnaire was disseminated among both partners of 201 heterosexual infertile couples after their first consultation at one of two Belgian fertility clinics between October 2015 and May 2016. The survey addressed: (i) the preference for genetic parenthood for themselves and for their partner, (ii) the importance of 30 motivations for genetic parenthood and (iii) the importance of 51 other motivations for parenthood and whether these motivations require being the genetic parent of their child to be fulfilled. To simplify presentation of the results, all 81 motivations were grouped into reliable categories of motivations using psychometric analyses. The survey was completed by 104 women and 91 men (response rate: 49%). Almost all respondents (98%) favored genetic over non-genetic parenthood for both their partner and themselves. One-third of the respondents stated they only wanted to parent their own genetically related child. Achieving genetic parenthood for their partner was considered significantly more important than achieving genetic parenthood for themselves. Within couples, men had a stronger preference for genetic parenthood (P = 0.004), but this was not significant after correction for educational level, which was significantly associated with the preference of both men and women. The 30 motivations for becoming a genetic parent clustered into 11 categories of which ‘to experience a natural process’ was deemed most important. The 51 motivations for becoming a parent for which having a genetically related child is not strictly necessary clustered into 14 categories of which ‘to contribute to a child's well-being’ and ‘to experience the love of a child’ were most important. Respondents deemed they would need to be the genetic parent of their child to fulfill nearly all their motivations for parenthood. We included couples that visited the fertility clinic for the first time, and the preference for genetic parenthood might change throughout a fertility treatment trajectory. Moreover, what prospective parents expect to be important for their future well-being might not really define parents’ well-being. The presumed preference of couples for genetic parenthood was confirmed. Resistance against using donor gametes is more likely among lower educated individuals. Researching whether non-genetic parents actually feel they cannot fulfill the 51 motivations for parenthood, could be a basis for developing patient information. Funded by the Parkes Foundation, the University of Amsterdam and the Leuven University Hospital. No conflict of interest.
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