The 'Pleasure&Pregnancy' web-based interactive educational programme versus expectant management in the treatment of unexplained subfertility: protocol for a randomised controlled trial.

INTRODUCTION: Many subfertile couples are diagnosed with (relatively) unexplained subfertility and a good prognosis. National professional guidelines (eg, the Netherlands and UK) advise 'expectant management (EM)' for 6-12 months, in which no interaction with healthcare staff is offered. Underpowered studies indicate that face-to-face sex-counselling increases the ongoing pregnancy rates of these couples. In patients with other conditions, web-based interactive educational programmes have the same effect on sexual functioning as face-to-face sex counselling. The 'Pleasure&Pregnancy randomised controlled trial (RCT)' will examine in couples with unexplained subfertility and a good prognosis whether a new web-based interactive educational programme results in a higher chance of naturally conceiving an ongoing pregnancy within 6 months as compared with EM. METHODS AND ANALYSIS: A multicentre RCT with cost-effectiveness analysis will include heterosexual couples diagnosed with (relatively) unexplained subfertility and a good prognosis in Dutch and Belgian secondary or tertiary fertility clinics. Couples will be randomised between 6 months of EM and 6 months of the Pleasure&Pregnancy-programme. This new web-based interactive educational programme includes eight progressive modules of information (on the biology of conception and pleasurable sex) and sensate focus, couple communication and mindfulness exercises. Couples are offered interaction with their coaches via email and can take part in three moderated chat sessions with peers. The primary outcome of this RCT is the probability of naturally conceiving an ongoing pregnancy within 6 months after randomisation. Secondary outcomes include time-to-pregnancy, live birth rate, costs, sexual functioning and personal and relational well-being. Analysis will be according to intention to treat. ETHICS AND DISSEMINATION: This study has been approved by the Medical Ethical Committees of the Academic Medical Centre (the Netherlands) and the Leuven University Hospital (Belgium). The findings of this RCT will be disseminated through presentations at international scientific meetings and peer-reviewed publications. TRAIL REGISTRATION NUMBER: NTR5709; Pre-results.

Ovarian tissue cryopreservation in female-to-male transgender people: insights into ovarian histology and physiology after prolonged androgen treatment.

Female-to-male transgender people (trans men) are faced with the risk of losing their reproductive potential owing to gender-affirming hormone treatment and genital reconstructive surgery. This observational, prospective cohort study investigates the effect of prolonged androgen therapy on their ovarian histology and fertility preservation perspectives. Hormone serum levels, ovarian histology and cumulus-oocyte complexes (COC) of 40 trans men were analysed at the moment of hysterectomy with bilateral oophorectomy in the context of genital reconstructive surgery after testosterone treatment (58.18 ± 26.57 weeks). In the cortex, most follicles were primordial (68.52% total follicle count) compared with 20.26% intermediate and 10.74%primary follicles. Few secondary follicles (0.46%) and a single antral follicle were found in the sections analysed. In total, 1313 COC were retrieved from the medulla of 35 patients (37.51 ± 33.58 COC per patient). Anti-Müllerian hormone serum levels were significantly correlated with number of COC (Rs 0.787, P < 0.001). After 48 h in-vitro maturation, 34.30% metaphase II oocytes were obtained, with 87.10% having a normal spindle structure. In conclusion, the cortical follicle distribution in trans men, after more than a year of testosterone treatment, seems to be surprisingly normal. This work confirms the presence and in-vitro maturation potential of cumulus-oocyte complexes.

The physiology and clinical utility of anti-Mullerian hormone in women.

BACKGROUND The measurement of circulating anti-Müllerian hormone (AMH) has been applied to a wide array of clinical applications, mainly based on its ability to reflect the number of antral and pre-antral follicles present in the ovaries. AMH has been suggested to predict the ovarian response to hyperstimulation of the ovaries for IVF and the timing of menopause, and to indicate iatrogenic damage to the ovarian follicle reserve. It has also been proposed as a surrogate for antral follicle count (AFC) in the diagnosis of polycystic ovary syndrome (PCOS). METHODS This paper is a summary of presentations at a European Society of Human Reproduction and Embryology campus workshop on AMH, with literature cited until September 2013. Published peer-reviewed medical literature about AMH was searched through MEDLINE and was subjected to systematic review and critical assessment by the panel of authors. RESULTS Physiologically, recent data confirm that AMH is a follicular gatekeeper limiting follicle growth initiation, and subsequently estradiol production from small antral follicles prior to selection. AMH assays continue to evolve and technical issues remain; the absence of an international standard is a key issue. The dynamics of circulating AMH levels throughout life can be split into several distinct phases, with a peak in the early 20s before a decline to the menopause, with a strong and positive correlation with non-growing follicle recruitment. There is a more complex rise during childhood and adolescence, which is likely to be more reflective of different stages of follicle development. AMH shows limited short-term variability, but the influence of states such as prolonged oral contraceptive use need to be considered in clinical assessment. There are only very limited data on relationships between AMH and natural fertility at different stages of reproductive life, and while it has a relationship to age at menopause the marked variability in this needs further exploration. AMH may be useful in assessing the need for fertility preservation strategies and detecting post-chemotherapy or surgical damage to the ovarian reserve. Long-term follow-up of patients to ascertain fully the value of post-cancer serum AMH in predicting long-term ovarian function is required. There is a linear relationship between AMH and oocyte yield after ovarian stimulation, which is of value in predicting ovarian hyperstimulation. AMH can also identify 'poor responders', but it seems inappropriate at present to withhold IVF purely on this basis. Women with PCOS show markedly raised AMH levels, due to both the increased number of small antral follicles and intrinsic characteristics of those granulosa cells, and this may contribute to anovulation. The value of AMH in the diagnosis of PCOS remains controversial, but it may replace AFC in the future. CONCLUSIONS For the first time in female reproductive biology, it is possible to measure the submerged part of the iceberg of follicle growth, i.e. the intrinsic, so-called 'acyclic' ovarian activity. An international standard for AMH and improved assay validity are urgently needed to maximize the clinical utility of this very promising biomarker of ovarian function in a large array of clinical situations, both in childhood and adulthood.

Mutations in eIF4ENIF1 are associated with primary ovarian insufficiency.

CONTEXT: Primary ovarian insufficiency (POI), or premature ovarian failure, results from ovarian follicle depletion with a consequent elevation of FSH levels before age 40 years. We identified a family in which 9 women in 3 consecutive generations developed menopause at approximately age 30 years. We hypothesized a genetic cause with a dominant mode of inheritance. DESIGN: This was a family-based genetic study and a replicate group of women with POI. SETTING: The study was conducted at an academic medical center. PATIENTS: Seven affected women and an obligate carrier and 7 unaffected family members were genotyped. The genes of interest were also sequenced in 38 unrelated women with POI. INTERVENTION: The DNA from 7 family members was subjected to whole-exome sequencing. The genotypes of interest were confirmed and genotypes of additional family members and unrelated women with POI were determined using Sanger sequencing. MAIN OUTCOME MEASURE: A high-impact, deleterious variant that segregated appropriately with POI in the family was required. RESULTS: A heterozygous stop codon (Ser429X) was identified in the eukaryotic translation initiation factor 4E nuclear import factor 1 (eIF4ENIF1) in the proband and all affected women but not in the unaffected family members. The chance that such a high-impact, deleterious variant would segregate appropriately among the affected and unaffected relatives by chance is very low (P < .05). There were no additional mutations identified in eIF4ENIF1 or eIF4E in 38 unrelated women with POI. CONCLUSION: Data demonstrate a new gene associated with dominantly inherited POI. These results highlight the importance of translation initiation factors and their regulators in ovarian function.

Limited contribution of NR5A1 (SF-1) mutations in women with primary ovarian insufficiency (POI).

OBJECTIVE: To evaluate the significance of NR5A1 mutations in a large, well-phenotyped cohort of women with primary ovarian insufficiency (POI). Mutations in the NR5A1 gene (SF-1) were previously described in disorders of sexual development and adrenal insufficiency. Recently, a high frequency of NR5A1 gene mutations was reported in a small group of women with POI. DESIGN: Cross-sectional cohort study. SETTING: University hospital. PATIENT(S): Well-phenotyped women (n = 386) with secondary amenorrhea and diagnosed with POI, including women with familial POI (n = 77). INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): The entire coding region and splice sites of the NR5A1 gene were PCR-amplified and sequenced. The pathogenicity of identified mutations was predicted in silico by assessing Align-GVGD class and Grantham score. RESULT(S): Sequencing was successful in 356 patients with POI. In total, 9 mutations were identified in 10 patients. Five of these mutations concerned novel nonconservative mutations occurring in 5 patients. Prediction of effect on protein function showed low to intermediate pathogenicity for all nonconservative mutations. The overall NR5A1 gene mutation rate was 1.4%. CONCLUSION(S): The current study demonstrates that mutations in the NR5A1 gene are rare in women with POI. Primary ovarian insufficiency remains unexplained in the great majority of patients; therefore, continued efforts are needed to elucidate its underlying genetic factors.

Assessment of androgen concentration in women: liquid chromatography-tandem mass spectrometry and extraction RIA show comparable results.

OBJECTIVE: The measurement of serum testosterone in women is challenging due to lack of trueness, precision, and sensitivity of various available testosterone assays. Accurate assessment of testosterone in women is crucial especially in conditions associated with alleged over- or under-production of testosterone, such as in polycystic ovary syndrome (PCOS) or primary ovarian insufficiency (POI). The aim of this study was to measure and compare androgen concentrations in women with PCOS, POI, and female controls and to evaluate the performance of extraction RIA and liquid chromatography-tandem mass spectrometry (LC-MS/MS) in these women. DESIGN: Cross-sectional study. METHODS: Carefully phenotyped women with POI (n=208) or PCOS (n=200) and 45 healthy, regularly cyclic female controls were included. Method comparison analyses were performed for total testosterone, androstenedione (AD), and DHEA, as measured by LC-MS/MS and extraction RIA. RESULTS: All androgen levels were significantly elevated in women with PCOS compared with POI patients (P<0.05) and controls (P<0.05). Women with POI presented with similar androgen concentrations as controls, except for AD. Compared with measurements by extraction RIA, testosterone, DHEA, and AD concentrations measured by LC-MS/MS were systematically lower. However, using extraction RIA and LC-MS/MS, testosterone, DHEA, and AD measurements were shown to have good agreement as assessed by Bland-Altman analysis and intraclass correlation coefficient: 0.95 (95% confidence interval 0.94-0.91), 0.83 (0.79-0.86), and 0.96 (0.95-0.97) respectively. CONCLUSIONS: LC-MS/MS, compared with a labor-intensive extraction RIA, shows good precision, sensitivity, and high accuracy for measuring female testosterone, DHEA, and AD concentrations under various clinical conditions. LC-MS/MS, therefore, represents a convenient and reliable assay for both clinical and research purposes, where androgen measurement in women is required.

Similar phenotype characteristics comparing familial and sporadic premature ovarian failure.

OBJECTIVE: Premature ovarian failure (POF) is characterized by secondary amenorrhea before the age of 40 years, along with repeated increased follicle-stimulating hormone and low estrogen concentrations. POF is considered a complex genetic disease with a familial presentation in 12% to 50% of cases. POF may originate from different genes and various gene-environment interactions. The aim of this study was to identify possible differences in phenotype comparing women with familial and women with sporadic POF. METHODS: A multicenter study was initiated in the Netherlands using standardized phenotyping. For each woman, medical history, menstrual cycle, and fertility and smoking status were assessed and a standardized examination was performed. Based on a detailed three-generation family history, women were identified as having either familial (defined as having at least one relative with POF) or sporadic POF. RESULTS: A total of 58 familial cases and 142 sporadic cases of POF were identified. Maternal age at menopause was significantly lower in the women with familial compared with the women with sporadic POF (41.0 +/- 7.5 and 49.7 +/- 2.6 y, respectively; P < 0.001). Sex hormone-binding globulin concentration was significantly higher in the women with familial than in the women with sporadic POF (73.6 +/- 37.1 and 55.2 +/- 26.9 nmol/L, respectively; P = 0.002). All other characteristics, such as parity, bone mineral density, and serum follicle-stimulating hormone and lipid levels were similar, as was the incidence of autoimmunity and cytogenetic abnormalities. CONCLUSIONS: Familial and sporadic POF do not differ in phenotype except for maternal menopause age and sex hormone-binding globulin concentration. Future studies are needed to unravel the genotype-phenotype interactions in POF.

Noninvasive metabolomic profiling as an adjunct to morphology for noninvasive embryo assessment in women undergoing single embryo transfer.

OBJECTIVE: To determine whether metabolomic profiling of spent embryo culture media correlates with reproductive potential of human embryos. DESIGN: Retrospective study. SETTING: Academic and a private assisted reproductive technology (ART) programs. PATIENT(S): Women undergoing single embryo transfer after IVF. INTERVENTION(S): Spent embryo culture media were collected after single embryo transfer on day 3 (n = 304) or day 2 (n = 181) and analyzed by near infrared spectroscopy. Near infrared spectral regions were correlated to reproductive potential using a genetic algorithm optimization. Models of these spectral regions were used to calculate viability indices, and were validated by blinded analysis of a subset (n = 60) of samples. Implantation rates were also compared between embryos of higher (>or=0.3) and lower (<0.3) viability indices, and within each morphology grade. MAIN OUTCOME MEASURE(S): Viability index and embryo viability. RESULT(S): Mean viability indices of embryos that resulted in positive fetal cardiac activity were significantly higher compared with embryos that did not for both day 2 and day 3 embryos. Blinded validation of the day 2 model proved to be significant. Increasing viability index values correlated with an increase in pregnancy. Viability indices were found to be independent of morphology for both day 2 and day 3 embryos. Implantation rates were significantly higher among embryos with viability indices >or=0.3. CONCLUSION(S): Metabolomic profiling of human embryo culture media using near infrared spectroscopy is independent of morphology and correlates with reproductive potential of embryos.

Body composition, smoking, and spontaneous dizygotic twinning.

OBJECTIVE: To examine differences in body composition and smoking between mothers of spontaneous monozygotic and dizygotic twins, while taking into account maternal age, gravidity, and educational attainment. DESIGN: Retrospective cohort study. SETTING: The Netherlands Twin Register. PATIENT(S): Mothers of twins (n = 19,357) registered with the Netherlands Twin Register. Data were selected from mothers of spontaneous monozygotic twins (MZ; n = 5663) and mothers of spontaneous dizygotic twins (DZ; n = 8515). INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): The odds of having spontaneous DZ twins versus spontaneous MZ twins as a function of height, body mass index (BMI), and smoking before pregnancy, after accounting for age, gravidity, and educational attainment. RESULT(S): Compared with spontaneous MZ twinning, spontaneous DZ twinning is significantly associated with increasing height (odds ratio, 1.6; 95% confidence interval [CI], 1.5-1.8 for the tallest versus the shortest height quartile), an increased BMI (odds ratio, 1.3; 95% CI, 1.1-1.4 for overweight vs. normal weight), and smoking before the twin pregnancy (odds ratio, 1.4; 95% CI, 1.3-1.5 for smoker vs. nonsmoker). Maternal age and gravidity, but not educational attainment, had to be included in the model. CONCLUSION(S): Spontaneous dizygotic twinning is associated with body composition and smoking.

Genome-wide association study in premature ovarian failure patients suggests ADAMTS19 as a possible candidate gene.

BACKGROUND: Spontaneous premature ovarian failure (POF) occurs in 1% of women and has major implications for their fertility and health. Besides X chromosomal aberrations and fragile X premutations, no common genetic risk factor has so far been discovered in POF. Using high-density single nucleotide polymorphism (SNP) arrays, we set out to identify new genetic variants involved in this condition. METHODS: A genome-wide association study involving 309 158 SNPs was performed in 99 unrelated idiopathic Caucasian POF patients and 235 unrelated Caucasian female controls. A replication study on the most significant finding was performed. We specifically focused on chromosomal areas and candidate genes previously implicated in POF. RESULTS: Suggestive genome-wide significant association was observed for rs246246 (allele frequency P = 6.0 x 10(-7)) which mapped to an intron of ADAMTS19, a gene known to be up-regulated in the female mouse gonads during sexual differentiation. However, replication in an independent Dutch cohort (60 POF patients and 90 controls) could not confirm a clear association (P = 4.1 x 10(-5) in a joint analysis). We did not observe strong evidence for any of 74 selected POF candidate genes or linkage regions being associated with idiopathic POF in Caucasian females, although suggestive association (P < 0.005) was observed for SNPs that mapped in BDNF, CXCL12, LHR, USP9X and TAF4B. CONCLUSION: We observed a possible association between POF and a SNP in a biologically plausible candidate gene. Although limited by sample size, this proof-of-principle study's findings reveal ADAMTS19 as a possible candidate gene for POF and thus a larger follow-up study is warranted.

Anti-Mullerian hormone, inhibin B, and antral follicle count in young women with ovarian failure.

CONTEXT: Ovarian dysfunction is classically categorized on the basis of cycle history, FSH, and estradiol levels. Novel ovarian markers may provide a more direct insight into follicular quantity in hypergonadotropic women. OBJECTIVE: The objective of the study was to investigate the distribution of novel ovarian markers in young hypergonadotropic women as compared with normogonadotropic regularly menstruating women. DESIGN: This was a nationwide prospective cohort study. SETTING: The study was conducted at 10 hospitals in The Netherlands. PATIENTS: Women below age 40 yr with regular menses and normal FSH (controls; n = 83), regular menstrual cycles and elevated FSH [incipient ovarian failure (IOF); n = 68]; oligomenorrhea and elevated FSH [referred to as transitional ovarian failure (TOF); n = 79]; or at least 4 months amenorrhea together with FSH levels exceeding 40 IU/liter [premature ovarian failure (POF); n = 112]. MAIN OUTCOME MEASURES: Serum levels of anti-Müllerian hormone (AMH), inhibin B, and antral follicle count (AFC) was measured. RESULTS: All POF patients showed AMH levels below the fifth percentile (p(5)) of normoovulatory women. Normal AMH levels (>p(5)) could be identified in 75% of IOF, 33% of TOF patients, and 98% of controls. AFC and AMH levels changed with increasing age (P < 0.0001), whereas inhibin B did not (P = 0.26). AMH levels were significantly different between TOF and IOF over the entire age range, whereas AFC became similar for TOF and IOF at higher ages. CONCLUSIONS: Compared with inhibin B and AFC, AMH was more consistently correlated with the clinical degree of follicle pool depletion in young women presenting with elevated FSH levels. AMH may provide a more accurate assessment of the follicle pool in young hypergonadotropic patients, especially in the clinically challenging subgroups of patients with elevated FSH and regular menses (i.e. IOF) and in hypergonadotropic women with cycle disturbances not fulfilling the POF diagnostic criteria (i.e. TOF).

Dizygotic twinning.

The tendency to conceive spontaneous dizygotic (DZ) twins is a complex trait with important contributions from both environmental factors and genetic disposition. Twins are relatively common and occur on average 13 times per 1000 maternities, though the twinning frequency varies over time and geographic location. This variation is mostly attributed to the differences in DZ twinning rate, since the monozygotic twinning rate is relatively constant. DZ twinning is in part under genetic control, with mothers of DZ twins reporting significantly more female family members with DZ twins than mothers of monozygotic twins. Maternal factors such as genetic history, advanced age and increased parity are known to increase the risk of DZ twins. Recent research confirmed that taller mothers and mothers with a high body mass index (30>) are at greater risk of DZ twinning. Seasonality, smoking, oral contraceptive use and folic acid show less convincing associations with twinning. Genetic analysis is beginning to identify genes contributing to the variation in twinning. Mutations in one of these genes (growth differentiation factor 9) are significantly more frequent in mothers of DZ twins. However, the mutations are rare and only account for a small part of the genetic contribution for twinning.

Endometrial biopsy during induction of ovulation with clomiphene citrate in polycystic ovary syndrome.

BACKGROUND: The dichotomy between ovulation rates and pregnancy rates for women with polycystic ovary syndrome (PCOS) treated with clomiphene citrate (CC) prompted the present study to determine the effect of CC on endometrial maturity. METHODS: Retrospective case-control study of anovulatory women with PCOS (n = 119) on their third ovulatory cycle of CC and controls, 238 healthy regularly ovulating women whose partners had abnormal sperm, all of whom had an endometrial biopsy in the late luteal phase. RESULTS: Endometrial histology classified according to the classical Noyes criteria revealed out-of-phase endometrium in 19/119 (16%) of the CC group compared with 7/238 (3%) in controls (p < 0.0001). Duration of the luteal phase was not influenced by histological age of the endometrium. Endometrial biopsy performed during 138 conception cycles extracted from the database did not increase the miscarriage rate significantly (23.9%). CONCLUSIONS: CC treatment significantly increases the prevalence of out-of-phase endometrium and this could explain, in part, the large difference between ovulation and pregnancy rates. There was no correlation between the results of the endometrial biopsy and the duration of the luteal phase. Performing an endometrial biopsy during a conception cycle does not seem to have a significant negative effect on the outcome of pregnancy.

Polycystic ovary syndrome in adolescence--a therapeutic conundrum.

The polycystic ovary syndrome (PCOS) often presents in adolescence with menstrual disorders, acne and hirsutism. The early diagnostic signs are sometimes dismissed as 'normal' changes of adolescence, and the opportunity to save the teenager from the stigmata of the syndrome is missed. The finding that the metabolic syndrome is a possible long-term sequela of PCOS now presents a challenge to make an early diagnosis, educate patients regarding the importance of weight control and exercise, and treat accordingly both symptomatically and prophylactically. The use of long-term insulin sensitizers, particularly metformin, for these purposes in adolescents is now the subject of an inter-disciplinary debate. Good, hard supportive data are not yet forthcoming but, as in the adult, the establishment of metformin treatment for the hyperinsulinaemic adolescent with PCOS may precede the evidence.