The effect of serum vitamin D levels on ovarian reserve markers: a prospective cross-sectional study.

Is there any association between serum 25-OH vitamin D levels and ovarian reserve markers in infertile women? Vitamin D is not associated with the ovarian reserve markers, anti-mullerian hormone (AMH) and antral follicle count (AFC), in infertile women. The mechanism underlying the relationship between vitamin D deficiency and reproduction is still unclear; however, evidence indicates a potential direct negative impact on ovarian function. This is mainly due to the fact that gonadal function may be altered by vitamin D deficiency, as observed by the expression of vitamin D receptor mRNA in human ovaries, mixed ovarian cell cultures and granulosa cell cultures. On the other hand, results from clinical studies are conflicting, with some suggesting that vitamin D status is associated with ovarian reserve, whereas other cross-sectional studies have not found any significant correlation between vitamin D and AMH levels. This study was a prospective cross-sectional study from the Centre for Reproductive Medicine at the University Hospital of Brussels. The duration of the study was one year. Overall, the study included 283 consecutive infertile women younger than 42 years old and undergoing their first treatment cycle in our institution. All patients were recruited within a time interval of 12 months from the initiation of the study, before undergoing infertility treatment. Women consuming vitamin D supplements or taking medication for systematic disease or women who had undergone ovarian surgery were excluded from the study. All infertile women had serum AMH and vitamin D sampled on the same day. AFC was measured on the second or third day of the first cycle following the blood sampling for the determination of AMH and 25-OH vitamin D levels. Among all patients, 30.7% (n = 87) were vitamin D deficient (<20 ng/mL) whereas 69.3% (n = 196) had normal vitamin D levels (≥20 ng/mL). The mean AMH and AFC levels did not differ significantly between the two groups: AMH 3.9 μ/L (±3.8) versus 4.3 μ/L (±4.8), (P value = 0.5) and AFC 13.9 (±13.3) versus 12.7 (±11.4), (P = 0.7), respectively. No correlation was observed between 25-O H vitamin D and AMH (spearman's r = 0.02, P value = 0.7) or AFC (spearman's r = -0.02, P value = 0.7). In multiple linear regression analysis, after adjusting for potential confounders (age, BMI, smoking status, infertility cause and season of blood sampling), the regression slope in all participants for total 25OH-D predicting log10 AMH was 0.006 [standard error (SE) = 0.07, P value = 0.9]. Similarly, no significant association was observed between AFC and vitamin D levels, even after controlling for relevant co-variants (regression coefficient -0.09. SE 0.08, P value = 0.2). Although this is the first prospective study to evaluate the relationship between vitamin D and the most important ovarian reserve markers (AMH and AFC), we need to acknowledge that the data used to generate the study findings are cross-sectional in nature. In this regard, we cannot generate or exclude any causal effect hypothesis. Nevertheless, our data support that an association between vitamin D and ovarian reserve markers is highly unlikely to exist. Although data from basic research indicate that vitamin D deficiency may have an effect on steroidogenesis and follicular development, our study, by prospectively recruiting a large number of infertile women, clearly demonstrates that vitamin D deficiency is highly unlikely to have a detrimental effect on ovarian reserve. Ongoing prospective and translational research projects are currently being conducted in order to evaluate the potential effect of vitamin D deficiency on reproductive outcome mediated through either an effect on the oocyte quality or on endometrial receptivity and embryo implantation. No external funding was used for this study. No conflicts of interest are declared. N/A.

Drakopoulos P ，van de Vijver A ，Schutyser V ，Milatovic S ，Anckaert E ，Schiettecatte J ，Blockeel C ，Camus M ，Tournaye H ，Polyzos NP ... -  《-》

Infertile women below the age of 40 have similar anti-Müllerian hormone levels and antral follicle count compared with women of the same age with no history of infertility.

Do infertile patients below the age of 40 years have a lower ovarian reserve, estimated by anti-Müllerian hormone (AMH) and total antral follicle count (AFC), than women of the same age with no history of infertility? Serum AMH and AFC were not lower in infertile patients aged 20-39 years compared with a control group of the same age with no history of infertility. WHAT IS KNOWN ALREADY?: The management of patients with a low ovarian reserve and a poor response to controlled ovarian stimulation (COS) remains a challenge in assisted reproductive technologies (ART). Both AMH levels and AFC reflect the ovarian reserve and are valuable predictors of the ovarian response to exogenous gonadotrophins. However, there is a large inter-individual variation in the age-related depletion of the ovarian reserve and a broad variability in the levels of AMH and AFC compatible with conception. Women with an early depletion of the ovarian reserve may experience infertility as a consequence of postponement of childbearing. Thus, low ovarian reserve is considered to be overrepresented among infertile patients. A prospective cohort study including 382 women with a male partner referred to fertility treatment at Rigshospitalet, Copenhagen, Denmark during 2011-2013 compared with a control group of 350 non-users of hormonal contraception with no history of infertility recruited during 2008-2010. Included patients and controls were aged 20-39 years. Women with polycystic ovary syndrome were excluded. On Cycle Days 2-5, AFC and ovarian volume were measured by transvaginal sonography, and serum levels of AMH, FSH and LH were assessed. Infertile patients had similar AMH levels (11%, 95% confidence interval (CI): -1;24%) and AFC (1%, 95% CI: -7;8%) compared with controls with no history of infertility in an age-adjusted linear regression analysis. The prevalence of very low AMH levels (<5 pmol/l) was similar in the two cohorts (age-adjusted odds ratio: 0.9, 95% CI: 0.5;1.7). The findings persisted after adjustment for smoking status, body mass index, gestational age at birth, previous conception and chronic disease in addition to age. The comparison of ovarian reserve parameters in women recruited at different time intervals could be a reason for caution. However, all women were examined at the same centre using the same sonographic algorithm and AMH immunoassay. This study indicates that the frequent observation of patients with a poor response to COS in ART may not be due to an overrepresentation of women with an early depletion of the ovarian reserve but rather a result of the expected age-related decline in fertility. The study received funding from MSD and the Interregional European Union (EU) projects 'ReproSund' and 'ReproHigh'. The authors have no conflict of interest. Not applicable.

Hvidman HW ，Bentzen JG ，Thuesen LL ，Lauritsen MP ，Forman JL ，Loft A ，Pinborg A ，Nyboe Andersen A ... -  《-》

Serum anti-Mullerian hormone production is not correlated with seasonal fluctuations of vitamin D status in ovulatory or PCOS women.

Is there a relationship between serum anti-Mullerian hormone (AMH) levels and seasonal variations in serum vitamin D in ovulatory and polycystic ovary syndrome (PCOS) women? Serum AMH levels were not associated with serum vitamin D status even after controlling for relevant co-variants, with this finding being consistent for all causes of infertility. As expected, seasonal variations in serum vitamin D were observed between summer and winter. AMH plays an important role in maintaining ovarian reserve and modifying follicle sensitivity to FSH stimulation. Studies suggest that vitamin D has the ability to modify AMH production in vitro, yet only one clinical study reports the influence of vitamin D on AMH levels. This was a retrospective cohort study analyzing the potential interaction of AMH and vitamin D for 340 women (58 PCOS and 282 ovulatory women) aged less than 40 years collected as part of their routine fertility assessment between January and December 2013 at a private fertility clinic in Adelaide, South Australia. Patient data including age, BMI, cause of infertility, antral follicle counts (AFC), serum AMH and vitamin D levels, smoking status, and menstrual cycle length for women aged less than 40 years of age, with serum AMH and vitamin D sampled within the same 4-week period were retrieved from a database. The hours of sunlight per day and daily UV index were extracted from a database at the South Australian Bureau of Meteorology, South Australia. Serum vitamin D (25-hydroxyvitamin D) levels were analyzed against seasonal variation in sunlight and UV exposure and serum AMH levels, while controlling for relevant co-variants. Seasonal variations in serum vitamin D were observed between summer and winter (30% variance; P < 0.001), while serum AMH levels (mean ± SEM) remained unaffected by season status (36.9 ± 3.3 versus 38.5 ± 2.7 pmol/l; P > 0.05), even after controlling for relevant co-variants. Overall, no correlation was observed between serum AMH and vitamin D levels, in either the PCOS or ovulatory cohort. Serum vitamin D levels were not significantly related to the underlying cause of infertility (PCOS, diminished ovarian reserve, 'fertile' ovulatory controls). The data used to generate the study findings are cross sectional in nature. While we acknowledge that a longitudinal study monitoring the relationship between serum AMH and vitamin D in individuals over the four seasons would have been ideal, we believe the current findings are robust as our four seasonal groups did not differ for any significant co-variant for serum AMH or vitamin D (age, BMI, PCOS status or AFC) and that there is no significant association between serum vitamin D concentration and AMH production. At present, while in vitro studies suggest vitamin D has the potential to modify AMH production, clinical study findings are conflicting. If vitamin D does influence AMH production, this could have important therapeutic implications. K.G. was supported through a University of South Australia summer scholarship. The authors have no competing interests.

Pearce K ，Gleeson K ，Tremellen K 《-》

Ovarian reserve assessment in users of oral contraception seeking fertility advice on their reproductive lifespan.

To what extent does oral contraception (OC) impair ovarian reserve parameters in women who seek fertility assessment and counselling to get advice on whether their remaining reproductive lifespan is reduced? Ovarian reserve parameters defined by anti-Müllerian hormone (AMH), antral follicle count (AFC) and ovarian volume were found to be significantly decreased by 19% (95% CI 9.1-29.3%), 18% (95% CI 11.2-24.8%) and 50% (95% CI 45.1-53.7%) among OC users compared with non-users. AMH and AFC have proved to be reliable predictors of ovarian ageing. In women, AMH declines with age and data suggest a relationship with remaining reproductive lifespan and age at menopause. OC may alter parameters related to ovarian reserve assessment but the extent of the reduction is uncertain. A cross-sectional study of 887 women aged 19-46 attending the Fertility Assessment and Counselling Clinic (FACC) from 2011 to 2014 comparing ovarian reserve parameters in OC users with non-OC users. The FAC Clinic was initiated to provide individual fertility assessment and counselling. All women were examined on a random cycle day by a fertility specialist. Consultation included; transvaginal ultrasound (AFC, ovarian volume, pathology), a full reproductive history and AMH measurement. Women were grouped into non-users and users of OC (all combinations of estrogen-progestin products and the contraceptive vaginal ring). Non-users included women with an intrauterine device (IUD) or no hormonal contraception. Of the 887 women, 244 (27.5%) used OC. In a linear regression analyses adjusted for age, ovarian volume was 50% lower (95% CI 45.1-53.7%), AMH was 19% lower (95% CI 9.1-29.3%), and AFC was 18% lower (95% CI 11.2-24.8%) in OC users compared with non-users. Comparison of AMH at values of <10 pmol/l OC was found to have a significant negative influence on AMH (OR 1.6, 95% CI 1.1; 2.4, P = 0.03). Furthermore, we found a significant decrease in antral follicles sized 5-7 mm (P < 0.001) and antral follicles sized 8-10 mm (P < 0.001) but an increase in antral follicles sized 2-4 mm (P = 0.008) among OC users. The two groups (OC users versus non-users) were comparable regarding age, BMI, smoking and maternal age at menopause. The study population comprised women attending the FAC Clinic. Recruitment was based on self-referral, which could imply a potential selection bias. Ovarian reserve was examined at a random cycle day. However, both AMH and AFC can be assessed independently of the menstrual cycle. The accuracy in predicting residual reproductive lifespan is still needed in both users and non-users of OC. OC has a major impact on the ovarian volume, and a moderate impact on AFC and AMH with a shift towards the smaller sized antral follicle subclasses. The most evident reduction occurs in the antral follicles of 5-7 and 8-10 mm with the highest number of AMH secreting granulosa cells. It is essential to be aware of the impact of OC use on ovarian reserve parameters when guiding OC users on their fertility status and reproductive lifespan. The FAC Clinic was established in 2011 as part of the ReproHigh collaboration. This study received funding through the Capital Region Research Fund and by EU-regional funding. There are no competing interests. The biobank connected to FAC Clinic is approved by the Scientific Ethical Committee (H-1-2011-081).

Birch Petersen K ，Hvidman HW ，Forman JL ，Pinborg A ，Larsen EC ，Macklon KT ，Sylvest R ，Andersen AN ... -  《-》

Diminished ovarian reserve is not observed in infertility patients with high normal CGG repeats on the fragile X mental retardation 1 (FMR1) gene.

Does an association exist between high normal numbers of CGG trinucleotide repeats on the fragile X mental retardation 1 (FMR1) gene and diminished ovarian reserve (DOR)? This large data set demonstrated that a high normal number of CGG repeats (35-54 repeats) on the FMR1 gene was not significantly correlated with DOR. The FMR1 premutation (55-200 repeats) is a known cause of primary ovarian insufficiency. However, the relationship between high normal CGG repeat numbers (35-54 repeats) and ovarian reserve has yet to be conclusively demonstrated. This is a retrospective data analysis conducted between January 2012 and February 2014 that included 1287 women. Over 1140 women had complete data. All women, excluding oocyte donors, who presented to a large private practice specializing in reproductive endocrinology and infertility for treatment and who underwent both fragile X and ovarian reserve testing were included. All fragile X testing was performed using triplet repeat PCR, with confirmation of positives by Southern blot. CGG repeat numbers from both alleles were recorded, and the allele with the higher number of repeats was used for statistical calculations. We did not differentiate between patients with one or two high normal alleles. Women with >54 CGG repeats were excluded from the analysis. For our analysis, we considered both a 'high normal' number of CGG repeats (35-44) and an intermediate number of GCC repeats (45-54) as 'high normal'. Ovarian reserve testing was carried out on Cycle Day 2 or 3 and included measurements of FSH, anti-Müllerian hormone (AMH) and antral follicle count (AFC). A generalized linear regression model assuming gamma distribution and log link function that controlled for age was used to assess correlation between CGG repeat number and FSH, AMH and AFC. As expected, there was a significant correlation between increasing age and increasing FSH and decreasing AFC and AMH for the patients in this study. For every 1-year increase in age, FSH increased by a factor of 1.04, AFC decreased by a factor of 0.93 and AMH decreased by a factor of 0.89. After controlling for age, there was no significant correlation between FMR1 CGG trinucleotide repeat number and FSH (P = 0.23), AFC (P = 0.14) or AMH (P = 0.53). Three subgroup analyses were also performed. We found a significant relationship between increasing CGG repeat number and decreasing AMH levels (P = 0.01) in women >44 years old. The second subgroup analysis included only Caucasian patients and found no significant correlation between CGG repeat number and DOR. In a subgroup analysis comparing women with at least one allele <26 repeats, at least one allele >35 and women with both alleles between 29 and 32, there were no significant associations regarding ovarian reserve in any of these groups. One limitation of this study is that it involved a heterogeneous population of infertile women with mixed diagnoses. Factors that could affect ovarian reserve, such as medical comorbidities, prior surgeries, family history and endometriosis, were not accounted for. Finally, there was a lack of racial diversity, with Caucasians representing 67.8% of the total population. The findings of this study are generalizable to an infertility population and are in line with several previously published studies. Women who are found to have high normal CGG repeat numbers can be counseled that this is not causative for DOR. Further studies are needed to investigate whether increasing CGG repeat numbers are associated with ovarian responsiveness to gonadotrophin stimulation or IVF outcome.

Schufreider A ，McQueen DB ，Lee SM ，Allon R ，Uhler ML ，Davie J ，Feinberg EC ... -  《-》