Human Small Antral Follicles Research Articles

Expression of genes and enzymes involved in ovarian steroidogenesis in relation to human follicular development.

Granulosa cells (GCs) and theca cells (TCs) play a pivotal role in human ovarian steroidogenesis, facilitating the conversion of cholesterol into sex steroids that regulate normal reproductive function. This study aims to explore the expression patterns of key enzymes that govern human ovarian steroidogenesis throughout follicle development, employing both genomic and immunological methodologies. Follicles and GCs obtained from women undergoing ovarian tissue cryopreservation (OTC) and in vitro fertilisation treatment were utilized. Gene expression data were obtained from a Chinese study using RNA sequencing and from microarray data generated in our laboratory to comprehensively analyse gene expression profiles across distinct stages of follicular development. To corroborate the localisation of key enzymes within GCs and TCs, immunohistochemistry analyses utilizing colourimetric and fluorescent techniques were conducted. Steroidogenesis-related enzymes displayed low gene expression levels during early follicle development. However, a notable upregulation of HSD3B2 was observed in GCs as follicles progressed to the antral/preovulatory stage, confirmed consistently using both microarray and RNA sequencing methodologies. Furthermore, immunohistochemical analyses effectively demonstrated that HSD3B2 were not only expressed in GCs, but co-localised with CYP17A1 within a specific subset of TCs surrounding human small antral follicles. Contributing to an enhanced progesterone production during the second half of the follicular phase was a significant upregulation of CYB5A in both microarray and RNA-seq datasets as follicles transition from the antral stage to the pre-ovulatory stage. Moreover, an augmented expression of DHCR24 and LDLR in both types of data, along with HMGCR expression expression in the microarray data, indicates increased substrate availability for ovarian steroidogenesis. This study confirms and extends that GCs gradually augment expression of HSD3B2 thereby enhancing their capacity for progesterone synthesis as follicles reach the size of selection at around 10 mm in diameter. This is supported by the expression CYB5A and possibly augmented availability of steroid precursors. A subset of TCs exhibit concurrent expression of CYP17A1 and HSD3B2, collectively contributing to the synthesis of 17-hydroxyprogesterone. These data significantly enhance our understanding of the dynamic regulation of progesterone throughout the process of follicular development.

Proteomic Alterations in Follicular Fluid of Human Small Antral Follicles Collected from Polycystic Ovaries-A Pilot Study.

Polycystic ovaries (PCO) contain antral follicles that arrest growing around 3–11 mm in diameter, perturbing the dominant follicle’s selection and the subsequent ovulatory process. Proteomic alterations of PCO follicular fluid (FF) (i.e., microenvironment in which the oocyte develops until ovulation) have been studied from large follicles in connection with oocyte pickup during ovarian stimulation. The present study aimed to detect proteomic alterations in FF from unstimulated human small antral follicles (hSAF) obtained from PCO. After performing deep-sequencing label-free proteomics on 10 PCO and 10 non-PCO FF samples from unstimulated hSAF (4.6–9.8 mm), 1436 proteins were identified, of which 115 were dysregulated in PCO FF samples. Pathways and processes related to the immune system, inflammation, and oxidative stress appeared to be upregulated in PCO, while extracellular matrix receptors interactions, the collagens-containing extracellular matrix, and the regulation of signaling were downregulated. The secreted proteins SFRP1, THBS4, and C1QC significantly decreased their expression in PCO FF, and this downregulation was suggested to affect future oocyte competence. In conclusion, our study revealed, for the first time, evidence of proteomic alterations occurring in the FF of PCO hSAF that may be related to the dysfunction of follicular growth and subsequent oocyte competence.

O-081 High variability of molecular isoforms of AMH in follicular fluid and granulosa cells from human small antral follicles

Abstract Study question Is the composition of AMH isoforms different in follicular fluids (FF) and granulosa cells (GCs) from human small antral follicles? Summary answer There is a high viability of AMH isoforms in FFs and GCs. Even between same size follicles from the same women, the isoform composition differs. What is known already Anti Müllerian Hormone (AMH) is a member of the TGF-β superfamily produced by follicular granulosa cells (GCs) in women from late gestation to the end of reproductive live. AMH is suggested to inhibit aromatase (i.e. CYP19) expression and thereby decreasing the conversion of androgens to oestrogens in humans, especially in small antral follicles before dominance is achieved and thereby act as a gatekeeper of ovarian steroidogenesis. However, the exact function and processing of AMH in human follicles is still not clarified. Study design, size, duration This retrospective study measured AMH isoforms in human FF and GCs from small antral follicles using ELISA, Western blot, and immunofluorescence analysis. A total of 41 female adolescents and women aged 15 to 38 years (mean age: 29.7 years), who underwent ovarian tissue cryopreservation (OTC) at the University Hospital of Copenhagen, Rigshospitalet were included between year 2006 and 2020 included. Participants/materials, setting, methods Donated human ovarian medulla tissue were FFs and GCs were obtained in connection with OTC. The following isoforms were evaluated in FFs using ELISA analysis: full-length AMH precursor (proAMH), cleaved associated AMH (AMHN,C), N-terminal pro-region (AMHN), and active C-terminal (AMHC) AMH. Antibodies specific for the N-terminal and the C-terminal AMH were used in both Western blot and immunofluorescence analysis of FFs and GCs. Main results and the role of chance A negative correlation between follicle diameter and the mentioned AMH forms were detected. Moreover, Western blot analysis detected various AMH forms in both FFs and GCs, which did not match the above-mentioned consensus forms suggesting an unknown proteolytic processing of AMH. The presence of these new molecular weight isoforms of AMH differs between individual follicles of identical size from the same woman. Limitations, reasons for caution The study group is limited and the significance of the variable AMH isoforms compositions between follicles cannot not be clarified from this data. Wider implications of the findings Collectively, these data suggest that intrafollicular processing of AMH is complex and variable, and thus, it may be difficult to develop an antibody based AMH assay that detect all AMH isoforms. Furthermore, the variability between follicles suggests that designing a proper standard will be difficult. Trial registration number not applicable

High Variability of Molecular Isoforms of AMH in Follicular Fluid and Granulosa Cells From Human Small Antral Follicles.

Anti-Müllerian hormone (AMH) is a member of the TGF-β superfamily produced by follicular granulosa cells (GCs) in women from late gestation to the end of reproductive life. AMH is thought to inhibit aromatase (i.e., CYP19) expression and decrease the conversion of androgens to oestrogens, especially in small antral follicles before dominance is achieved. Thus, AMH acts as a gatekeeper of ovarian steroidogenesis. However, the exact function and processing of AMH has not been fully elucidated. The present study measured and determined AMH isoforms in human follicular fluid (FF) from small antral follicles and in human GCs using four ELISAs, western blot, and immunofluorescence analysis. We evaluated the presence of the following isoforms: full-length AMH precursor (proAMH), cleaved associated AMH (AMHN,C), N-terminal pro-region (AMHN), and active C-terminal (AMHC) AMH. A negative correlation between follicle diameter and the AMH forms was detected. Moreover, western blot analysis detected various AMH forms in both FFs and GCs, which did not match our consensus forms, suggesting an unknown proteolytic processing of AMH. The presence of these new molecular weight isoforms of AMH differs between individual follicles of identical size in the same woman. This study detected several AMH forms in FF and GCs obtained from human small antral follicles, which suggests that intrafollicular processing of AMH is complex and variable. Thus, it may be difficult to develop an antibody-based AMH assay that detects all AMH isoforms. Furthermore, the variability between follicles suggests that designing a recombinant AMH standard will be difficult.

Proteome of fluid from human ovarian small antral follicles reveals insights in folliculogenesis and oocyte maturation.

STUDY QUESTIONIs it possible to identify by mass spectrometry a wider range of proteins and key proteins involved in folliculogenesis and oocyte growth and development by studying follicular fluid (FF) from human small antral follicles (hSAF)?SUMMARY ANSWERThe largest number of proteins currently reported in human FF was identified in this study analysing hSAF where several proteins showed a strong relationship with follicular developmental processes.WHAT IS KNOWN ALREADYProtein composition of human ovarian FF constitutes the microenvironment for oocyte development. Previous proteomics studies have analysed fluids from pre-ovulatory follicles, where large numbers of plasma constituents are transferred through the follicular basal membrane. This attenuates the detection of low abundant proteins, however, the basal membrane of small antral follicles is less permeable, making it possible to detect a large number of proteins, and thereby offering further insights in folliculogenesis.STUDY DESIGN, SIZE, DURATIONProteins in FF from unstimulated hSAF (size 6.1 ± 0.4 mm) were characterised by mass spectrometry, supported by high-throughput and targeted proteomics and bioinformatics. The FF protein profiles from hSAF containing oocytes, capable or not of maturing to metaphase II of the second meiotic division during an IVM (n = 13, from 6 women), were also analysed.PARTICIPANTS/MATERIALS, SETTING, METHODSWe collected FF from hSAF of ovaries that had been surgically removed from 31 women (∼28.5 years old) undergoing unilateral ovariectomy for fertility preservation.MAIN RESULTS AND THE ROLE OF CHANCEIn total, 2461 proteins were identified, of which 1108 identified for the first time in FF. Of the identified proteins, 24 were related to follicular regulatory processes. A total of 35 and 65 proteins were down- and up-regulated, respectively, in fluid from hSAF surrounding oocytes capable of maturing (to MII). We found that changes at the protein level occur already in FF from small antral follicles related to subsequent oocyte maturation.LIMITATIONS, REASONS FOR CAUTIONA possible limitation of our study is the uncertainty of the proportion of the sampled follicles that are undergoing atresia. Although the FF samples were carefully aspirated and processed to remove possible contaminants, we cannot ensure the absence of some proteins derived from cellular lysis provoked by technical reasons.WIDER IMPLICATIONS OF THE FINDINGSThis study is, to our knowledge, the first proteomics characterisation of FF from hSAF obtained from women in their natural menstrual cycle. We demonstrated that the analysis by mass spectrometry of FF from hSAF allows the identification of a greater number of proteins compared to the results obtained from previous analyses of larger follicles. Significant differences found at the protein level in hSAF fluid could predict the ability of the enclosed oocyte to sustain meiotic resumption. If this can be confirmed in further studies, it demonstrates that the viability of the oocyte is determined early on in follicular development and this may open up new pathways for augmenting or attenuating subsequent oocyte viability in the pre-ovulatory follicle ready to undergo ovulation.STUDY FUNDING/COMPETING INTEREST(S)The authors thank the financial support from ReproUnion, which is funded by the Interreg V EU programme. No conflict of interest was reported by the authors.TRIAL REGISTRATION NUMBERN/A

Transcriptome Analysis of Long Non-coding RNAs and Genes Encoding Paraspeckle Proteins During Human Ovarian Follicle Development.

Emerging evidence indicated that many long non-coding (lnc)RNAs function in multiple biological processes and dysregulation of their expression can cause diseases. Most regulatory lncRNAs interact with biological macromolecules such as DNA, RNA, and protein. LncRNAs regulate gene expression through epigenetic modification, transcription, and posttranscription, through DNA methylation, histone modification, and chromatin remodeling. Interestingly, differential lncRNA expression profiles in human oocytes and cumulus cells was recently assessed, however, lncRNAs in human follicle development has not previously been described. In this study, transcriptome dynamics in human primordial, primary and small antral follicles were interrogated and revealed information of lncRNA genes. It is known that some lncRNAs form a complex with paraspeckle proteins and therefore, we extended our transcriptional analysis to include genes encoding paraspeckle proteins. Primordial, primary follicles and small antral follicles was isolated using laser capture micro-dissection from ovarian tissue donated by three women having ovarian tissue cryopreserved before chemotherapy. After RN sequencing, a bioinformatic class comparison was performed and primordial, primary and small antral follicles were found to express several lncRNA and genes encoding paraspeckle proteins. Of particular interest, we detected the lncRNAs XIST, NEAT1, NEAT2 (MALAT1), and GAS5. Moreover, we noted a high expression of FUS, TAF15, and EWS components of the paraspeckles, proteins that belong to the FET (previously TET) family of RNA-binding proteins and are implicated in central cellular processes such as regulation of gene expression, maintenance of genomic integrity, and mRNA/microRNA processing. We also interrogated the intra-ovarian localization of the FUS, TAF15, and EWS proteins using immunofluorescence. The presence and the dynamics of genes that encode lncRNA and paraspeckle proteins may suggest that these may mediate functions in the cyclic recruitment and differentiation of human follicles and could participate in biological processes known to be associated with lncRNAs and paraspeckle proteins, such as gene expression control, scaffold formation and epigenetic control through human follicle development. This comprehensive transcriptome analysis of lncRNAs and genes encoding paraspeckle proteins expressed in human follicles could potentially provide biomarkers of oocyte quality for the development of non-invasive tests to identify embryos with high developmental potential.

Hallmarks of Human Small Antral Follicle Development: Implications for Regulation of Ovarian Steroidogenesis and Selection of the Dominant Follicle.

Regulation of human ovarian steroidogenesis differs from other species and precise knowledge on how human small antral follicles (hSAF) develop and acquire competence for continued growth and steroid output is still incomplete. The present study has characterized almost 1,000 normal hSAF collected in connection with cryopreservation of ovarian tissue for fertility preservation. The antral follicles (ranging from 3 to 13 mm) were generally aspirated from one ovary surgically removed during the natural cycle, and the follicular fluid (FF) and the granulosa cells (GC) were isolated and snap-frozen. In FF, the following hormones were measured: inhibin-B, inhibin-A, AMH, follistatin, PAPP-A, estradiol, progesterone, testosterone, and androstenedione. In GC, mRNA gene expressions using q-PCR were measured for the following genes: FSHR, AMH, CYP19, and AR. All samples in which one of the abovementioned parameters was measured were included, but typically multiple parameters were measured. Highly significant differences in concentration and follicular content in relation to follicular diameter were found for all measured hormones despite massive variability in-between follicles for any given diameter. The results demonstrate that profound changes take place in the hormonal microenvironment around follicular diameters of 8–11 mm corresponding to when follicular selection occurs. At this point, inhibin-B and inhibin-A showed distinct peaks concomitant with a significant reduction in both AMH protein and mRNA expression. Concentrations of inhibins, androgens, FSHR, and AR were intimately associated, and it is suggested that inhibin-B in combination with PAPP-A and thereby IGF2 activity exerts important paracrine signaling at follicular selection. At the same time upregulation of estradiol synthesis and CYP19 mRNA expression increased steroid output profoundly. Furthermore, the highly significant association between FSHR and AR mRNA gene expression enforces important functions of androgens in follicular development. Collectively, these data reintroduce the understanding of the follicular phase as two parted in which regulation of steroidogenesis differs. The profound changes taking place around follicular selection highlight important paracrine actions of TGF-β family members and IGFs for securing dominance of the selected follicle.

In Vitro Maturation and Culture of Human Oocytes.

We describe the collection, culture, and ex vivo, in vitro maturation of germinal vesicle (GV) oocytes obtained from human small antral follicles (hSAFs). hSAFs contain fully grown GV oocytes and have the advantages that they are more numerous than large or mature follicles, which are used in IVF treatment. hSAFs can be obtained directly from human ovarian tissue without exogenous gonadotrophin stimulation and therefore allows studies of oocytes even from young women and girls. The method described here was developed to study human female meiosis but could in theory also be used for fertility treatment.

The polymorphic insertion of the luteinizing hormone receptor “insLQ” show a negative association to LHR gene expression and to the follicular fluid hormonal profile in human small antral follicles

The luteinizing hormone receptor (LHCGR) has a little studied polymorphic 6 bp insertion (rs4539842/insLQ). This study has evaluated the insLQ polymorphism in relation to potential associations with hormonal characteristics of human small antral follicles (hSAFs).In total, 310 hSAFs were collected from 86 women undergoing fertility preservation. Analysis included hormonal profile of 297 follicular fluid (FF) samples and 148 corresponding granulosa cells samples were evaluated by qPCR for selected genes.Significantly reduced and non-detectable mRNA levels of anti-Müllerian hormone receptor II (AMHR2) and LHCGR, respectively, were observed for insLQ/insLQ compared to –/insLQ and the −/− genotypes. Moreover, LHCGR and CYP19a1 together with oestradiol and inhibin-B were significantly increased in –/insLQ compared to the −/− genotype. The homozygous insLQ genotype showed strong significant associations to GC specific genes LHCGR and CYP19a1, which may translate into significant changes in FF hormone profiles and an altered LH signaling.

Effect of pregnancy-associated plasma protein-A (PAPP-A) single-nucleotide polymorphisms on the level and activity of PAPP-A and the hormone profile in fluid from normal human small antral follicles.

To reveal a possible relationship between two single nucleotide polymorphisms (SNPs) in PAPP-A-1224 (rs7020782) and 327 (rs12375498)-and the level and activity of PAPP-A in follicular fluid (FF) of human small antral follicles, and to analyze the intrafollicular hormone levels. Laboratory investigation. University hospital. Fifty volunteer women who contributed a total of 210 samples of FF from normal small antral follicles. Genotyping and measurement of antigen levels of steroids, PAPP-A, stanniocalcin-2 (STC2), and antimüllerian hormone (AMH) plus activity of PAPP-A toward insulin-like growth factor binding protein 4 (IGFBP-4). Measurement of PAPP-A levels and hormones with enzyme-linked immunosorbent assay (ELISA) and PAPP-A activity toward radiolabeled IGFBP-4. Women homozygous for the minor C allele of the 1224 SNP showed a statistically significantly lower level of PAPP-A protein and activity in FF compared with women carrying the major A allele. These women also displayed nonsignificant reduced levels of estradiol and increased levels of AMH and androgen. A statistically significant correlation between FF levels of PAPP-A activity and the molar ratio of PAPP-A/STC2 was obtained. The 327 SNP did not show statistically significant associations. This study presents a statistically significant effect of the 1224 SNP on the level and activity of PAPP-A in human follicles, suggesting that the FF level of bioactive insulin-like growth factor depends on the genotype. We observed STC2 to be an important regulator of PAPP-A in human FF. The 1224 SNP has previously been associated with recurrent pregnancy loss, so further evaluation of an underlying mechanism including aberrant control of insulin-like growth factor activity is warranted.

Size matters: Associations between the androgen receptor CAG repeat length and the intrafollicular hormone milieu

Granulosa cell (GC) expressed androgen receptors (AR) and intrafollicular androgens are central to fertility. The transactivating domain of the AR contains a polymorphic CAG repeat sequence, which is linked to the transcriptional activity of AR and may influence the GC function. This study aims to evaluate the effects of the AR CAG repeat length on the intrafollicular hormone profiles, and the gene expression profiles of GC from human small antral follicles.In total, 190 small antral follicles (3–11 mm in diameter) were collected from 58 women undergoing ovarian cryopreservation for fertility preservation. The biallelic mean of the CAG repeat lengths were calculated for each woman, and grouped in three groups: Long CAG repeats (23–26 mean CAG); medium CAG repeats (20.5–22.5 mean CAG) and short CAG repeats (17.5–20.0 mean CAG). The following parameters were measured: follicle diameter, intrafollicular levels of Anti-Müllerian Hormone (AMH), progesterone, oestradiol, testosterone and androstenedione, and GC gene expression levels of FSHR, LHR, AR, CYP19A1, and AMH.The long CAG repeat lengths were associated with significantly decreased testosterone levels, as compared to medium CAG repeats (P = 0.05) and short CAG repeats (P = 0.003). Furthermore, in follicles 3–6 mm in diameter, the long CAG repeats were associated with significantly increased LHR and CYP19A1 gene expression levels compared to short CAG repeat lengths (P = 0.004 and P = 0.04 respectively), and significantly increased LHR expression compared to medium CAG repeat lengths (P = 0.03).In conclusion, long CAG repeat lengths in the AR were associated to significant attenuated levels of androgens and an increased conversion of testosterone into oestradiol, in human small antral follicles.

Effect of the FSH receptor single nucleotide polymorphisms (FSHR 307/680) on the follicular fluid hormone profile and the granulosa cell gene expression in human small antral follicles.

The most pronounced effects of FSH signalling are potentially displayed in the follicle fluid, which acts as a reservoir for FSH-induced granulosa cell (GC) secreted hormones. This study investigates the effects of two common polymorphisms of FSHR, FSHR 307 (rs6165) and FSHR 680 (rs6166), by evaluating the hormone and gene expression profiles of human small antral follicles collected under physiological conditions in connection with fertility preservation. In total 69 women at various time during the menstrual cycle were included in this study. The intrafollicular hormone content of 179 follicular fluid samples and the gene expression levels of 85 GC samples were correlated to the genotype of both FSHR polymorphisms. The following parameters were evaluated: follicle diameter, levels of Anti-Müllerian hormone (AMH), progesterone, estradiol, testosterone and androstenedione and gene expression levels of FSHR, luteinizing hormone receptor (LHR), androgen receptor, aromatase cytochrome p450 (CYP19A1), AMH and AMH receptor II (AMHR2). There was 100% concordance between the FSHR 307 and the FSHR 680 genotypes: A/A (p.307Thr/Thr and p.680Asn/Asn), A/G (p.307Thr/Ala and p.680Asn/Ser) and G/G (p.307Ala/Ala and p.680Ser/Ser). Considering all follicles, compared with the other genotypes the G/G genotype was associated with significantly elevated gene expression levels for LHR, while AMHR2 gene expression levels were significantly reduced. In follicles 3-6 mm in diameter LHR gene expression was significantly increased, whereas AMH gene expression was significantly reduced for the G/G genotype. In follicles >6 mm, estradiol and CYP19A1 gene expression levels were significantly higher for the G/G genotype. In conclusion, significant changes were observed between the FSHR 307/680 polymorphisms in human small antral follicles collected under physiological FSH conditions.

Granulosa cells from human small antral follicles changes gene expression during culture

Granulosa cells from human small antral follicles changes gene expression during culture

Effects of common FSH receptor single-nucleotide polymorphisms on the follicular fluid hormone profile and the granulosa cell gene expression in human small antral follicles

Effects of common FSH receptor single-nucleotide polymorphisms on the follicular fluid hormone profile and the granulosa cell gene expression in human small antral follicles

Developmental competence of oocytes isolated from surplus medulla tissue in connection with cryopreservation of ovarian tissue for fertility preservation

Evaluating the developmental competence of immature oocytes collected from surplus medulla tissue in connection with ovarian tissue cryopreservation for fertility preservation. Cohort comparative study. University laboratory in Denmark from 2011-2012. 69 girls and women (0-38 years of age) who each had one ovary cryopreserved for fertility preservation. Ovaries were obtained directly from the local hospital or from collaborating hospitals (two to five hours' transport on ice). Immature oocytes were aspirated from large antral follicles visible on the ovaries, and collected from the saline solution, containing surplus medulla tissue, following dissection of the ovarian cortical tissue for cryopreservation. The immature oocytes were cultured for 48 h in an Embryoscope™ Time-lapse System or in culture dishes overlaid with liquid paraffin using commercial and in-house supplemented culture media. Maturation rate for immature oocytes reaching metaphase II. With a maturation rate of 3.1%, only 21 of 682 immature oocytes reached metaphase II. Immature oocytes from ovaries that had been transported on ice for two to five hours performed significantly poorer than those recovered immediately after surgery. Addition of epidermal growth factor and follicle fluid from human small antral follicles to the culture medium did not augment the maturation rate. Immature oocytes cultured in the Embryoscope performed significantly better than those in conventional culture dishes. In vitro maturation of immature oocytes should only be attempted clinically from visible antral follicles and where the ovary is not subjected to a cooling period prior to recovery of immature oocytes.

Concentration of activin A and follistatin in follicular fluid from human small antral follicles associated to gene expression of the corresponding granulosa cells

The present study correlated concentrations of activin A and follistatin in follicular fluid (FF) from human small antral follicles to FF concentrations of AMH, inhibin B, progesterone, and oestradiol and to the mRNA expression of FSH-receptor (FSHR), LH-receptor (LHR), AMH-receptor2 (AMHR2), CYP19a, and androgen-receptor (AR) in the corresponding granulosa cells (GC). FF from 144 follicles (3–12mm in diameter) was included whereas mRNA expression profiles were established in GC from 66 of the 144 follicles.Levels of follistatin remained constant in relation to follicular diameter, whereas activin A levels increased in follicles exceeding 10mm in diameter. Levels of activin A and inhibin B showed a highly significant inverse association. Follistatin showed highly significant positive associations with AMH and inhibin B levels and with FSHR and AR gene expression in GC.This study revealed unexpected associations that probably reflect the complicated regulatory mechanisms governing human folliculogenesis.

In human granulosa cells from small antral follicles, androgen receptor mRNA and androgen levels in follicular fluid correlate with FSH receptor mRNA

Human small antral follicles (diameter 3-9 mm) were obtained from ovaries surgically removed for fertility preservation. From the individual aspirated follicles, granulosa cells and the corresponding follicular fluid were isolated in 64 follicles, of which 55 were available for mRNA analysis (24 women). Expressions of androgen receptor (AR) mRNA levels in granulosa cells, and of androstenedione and testosterone in follicular fluid, were correlated to the expression of the FSH receptor (FSHR), LH receptor (LHR), CYP19 and anti-Müllerian Hormone-receptor II (AMHRII) mRNA in the granulosa cells and to the follicular fluid concentrations of AMH, inhibin-B, progesterone and estradiol. AR mRNA expression in granulosa cells and the follicular fluid content of androgens both showed a highly significant positive association with the expression of FSHR mRNA in granulosa cells. AR mRNA expression also correlated significantly with the expression of AMHRII, but did not correlate with any of the hormones in the follicular fluid. These data demonstrate an intimate association between AR expression in immature granulosa cells, and the expression of FSHR in normal small human antral follicles and between the follicular fluid levels of androgen and FSHR expression. This suggests that follicular sensitivity towards FSH stimulation may be augmented by stimulation of androgens via the AR.

Concentrations of AMH and inhibin-B in relation to follicular diameter in normal human small antral follicles

The aim of the present study was to determine the intrafollicular concentrations of anti-Müllerian hormone (AMH), inhibin-B and steroids in normal human small antral follicles and to relate them to follicular size. A group of 103 women having one ovary removed for fertility preservation by cryopreservation prior to gonadotoxic treatment served as a source of a total of 272 human small antral follicles. Prior to cryopreservation of the ovarian cortex, fluid from small antral follicles were collected. On the basis of the follicular volume, the diameter was calculated and follicles with diameters from 3 to 12 mm were included. Concentrations of AMH decreased significantly (P < 0.0005) from 1124 +/- 158 ng/ml (mean +/- SEM) in follicles with a diameter of 3 mm to a concentration of 392 +/- 98 ng/ml in 9 mm follicles, followed by a reduction to below 100 ng/ml in 12 mm follicles. The concentrations of inhibin-B rose from 57 +/- 10 ng/ml (mean +/- SEM) in 3 mm follicles to 142 +/- 10 ng/ml in 12 mm follicles (P < 0.0005) with a peak concentration of almost 200 ng/ml in 9-10 mm follicles. Relating hormone concentrations with age showed that even follicles from girls younger than 10 years showed the same range of AMH concentrations as those from older girls or women. The intrafollicular concentrations of AMH become progressively lower with increasing follicle diameters. In contrast, concentrations of inhibin-B increased with increasing follicle diameter with peak values at around 9 mm in diameter. This suggests that AMH and inhibin-B undertake important intrafollicular functions around the time of normal follicular selection in the mid-follicular phase of the menstrual cycle.

Concentrations of anti-Mullerian hormone in fluid from small human antral follicles show a negative correlation with CYP19 mRNA expression in the corresponding granulosa cells

Ovaries surgically removed for fertility preservation from a total of 24 women served as a source of human small antral follicles, including the follicular fluid (FF) and the corresponding granulosa cells (GC). The FF was used to evaluate the intrafollicular concentrations of anti-Müllerian hormone (AMH), inhibin-B, estradiol, progesterone, androstenedione and testosterone. In GC mRNA expression of the AMH type II receptor (AMH-r2) was determined and correlated to the mRNA expression of CYP19 (aromatase), FSH-receptor (FSH-r) and LH-receptor (LH-r) and to the hormonal profiles of the corresponding FF. GC and FF from a total of 64 follicles (diameter of 3-9 mm) were evaluated. Concentrations of AMH in FF showed a highly significant inverse correlation with CYP19 mRNA expression in the corresponding GC and with concentrations of estradiol, progesterone and inhibin-B in the FF. However, a small subgroup of follicles exhibited high levels of AMH simultaneously with relative high levels of CYP19 mRNA. In contrast to AMH, mRNA expression of AMH-r2 was significantly positively correlated to the mRNA expression of FSH-r and CYP 19, but failed to correlate to any other measured parameters. These data confirms an intimate correlation between follicular AMH levels, AMH-r2, FSH-r expression and estradiol secretion in the developing human follicle.