Hum. Reprod. Advance Access originally published online on May 23, 2006
Human Reproduction 2006 21(9):2223-2227; doi:10.1093/humrep/del165
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Anti-Müllerian hormone inhibits initiation of growth of human primordial ovarian follicles in vitro
1Division of Obstetrics and Gynaecology, Department of Clinical Science, Intervention and Technology, Karolinska Institutet, Karolinska University Hospital Huddinge, Stockholm, Sweden 2 Department of Internal Medicine, Erasmus MC, Rotterdam, the Netherlands 3 Programme for Developmental and Reproductive Biology, Biomedicum Helsinki and Department of Bacteriology and Immunology, Haartman Institute, University of Helsinki, Helsinki, Finland
4 To whom correspondence should be addressed at: Department of Obstetrics and Gynaecology K57, Karolinska Institutet, Karolinska University Hospital Huddinge, S-141 86 Stockholm, Sweden. E-mail: inger.britt.carlsson{at}ki.se
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Abstract |
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BACKGROUND: Anti-Müllerian hormone (AMH) inhibits the initiation of the development and early growth of mouse ovarian follicles. Furthermore, the ovarian follicle pool diminishes prematurely in AMH-knockout mice. In this study, we examined whether AMH plays a similar role in humans, controlling ovarian follicle growth. METHODS: Human ovarian cortical tissue biopsy specimens were cut into small pieces and cultured for 7 days in medium containing rat recombinant AMH at 0, 10, 30 or 100 ng/ml. The developmental stages and viability of the follicles were evaluated from histological sections. RESULTS: Similar to previous studies, significant initiation of follicle growth was observed in almost all culture media, as demonstrated by a significantly smaller proportion of primordial follicles (14–26%) compared with non-cultured control tissue (56%). The exception was tissue in medium supplemented with AMH at 100 ng/ml. Here, the proportion of primordial follicles was not significantly different from that in non-cultured tissue; furthermore, it was significantly greater than that in vehicle control cultures and cultures containing AMH at 10 ng/ml, indicating the inhibition of growth initiation. Viability was unaffected by the presence of AMH when compared with tissues in control media. CONCLUSIONS: Recombinant AMH at a concentration of 100 ng/ml has an inhibitory effect on early human ovarian follicular development in vitro, suppressing the initiation of primordial follicle growth.
Key words: AMH/in vitro culture/ovarian follicles/ovary
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Introduction |
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The exact mechanisms controlling the initiation of the growth of small ovarian follicles are unknown. In humans and experimental animals, the removal of tissue from the ovary into culture induces the activation and growth of follicles (Wandji et al., 1996
; Hovatta et al., 1997, 1999). This suggests the removal of unknown inhibitory factors from the tissue that were present in situ to maintain the follicles in a dormant state. It is accepted that many local factors produced in the ovary function concurrently to control follicle development. In contrast to the stimulatory actions of most locally produced transforming growth factor-
superfamily members (Rabinovici et al., 1990; Erickson and Shimasaki, 2000; Yan et al., 2001), anti-Müllerian hormone (AMH) is believed to have an inhibitory effect on the initiation of the development and early stages of follicle growth (Durlinger et al., 1999). AMH was originally identified in connection with its role in male fetal sex differentiation during embryonic development (Josso et al., 1993). It induces the regression of the Müllerian ducts; hence, it is also referred to as Müllerian-inhibiting substance (MIS).
AMH is produced by the granulosa cells in human females from the late antenatal stage to menopause (Rajpert-De Meyts et al., 1999). The development of a monoclonal antibody to human AMH has allowed the detection of AMH expression in the human adult ovary (Weenen et al., 2004). AMH was found not to be expressed in primordial follicles, but 74% of primary follicles showed at least weak staining in their granulosa cells. The most intense staining was localized to pre-antral and small antral follicles, with staining virtually absent in follicles larger than 5 mm. This pattern of expression is analogous to that in rodents, with AMH expressed in all follicles up to the stage when they are selected for dominance (Baarends et al., 1995; Durlinger et al., 2002). On the basis of these results, it has been proposed that the pool of growing follicles produces AMH, acting as a negative paracrine feedback signal on neighbouring primordial follicles, to inhibit their recruitment.
Female AMH-knockout mice were originally believed to be reproductively normal when first generated (Behringer et al., 1994). However, further investigation revealed that AMH is involved in inhibiting follicular development (Durlinger et al., 1999). At 4 months of age, AMH-deficient mice had a reduced number of primordial follicles and an elevated number of growing follicles, resulting in an increase in ovarian weight. At 13 months of age, the primordial follicle pool was almost completely depleted. Cultures of neonatal mouse ovaries with AMH present in the medium showed 40% fewer growing follicles than control cultured ovaries (Durlinger et al., 2002), with no effect on the concentrations of growth differentiation factor-9 or AMH receptor type II mRNA. In later stage follicles, AMH attenuated the FSH-stimulated growth of pre-antral follicles (Durlinger et al., 2001). Therefore, it was suggested that AMH might play a role in determining FSH sensitivity and hence in cyclic recruitment and selection of the dominant follicle for ovulation. Species variability exists, with AMH acting differently in rats, enhancing rather than attenuating FSH-stimulated growth of pre-antral follicles (McGee et al., 2001). In human tissues, Schmidt et al. (2004) cultured ovarian slices for 4 weeks in the presence of recombinant human AMH (rhAMH) at 300 ng/ml, with and without testosterone, using tissues from six women. The results showed that AMH, alone and with testosterone, advanced the presence of primary and secondary follicles, although the diameter of the oocytes did not increase.
We have established a human ovarian cortical tissue-culture system that offers the possibility to study the regulation of early ovarian follicular growth (Hovatta et al., 1997, 1999; Hreinsson et al., 2002; Scott et al., 2004a). The culture system was developed with the goal of obtaining oocytes from frozen-thawed ovarian cortical tissue for fertility preservation. Women benefiting from this procedure include those receiving treatments that could potentially induce sterility and those with haematological or ovarian malignancies for whom the re-transplantation of ovarian tissue presents a high risk. Once this system is established, all women requiring cryopreservation of ovarian cortical tissue, such as those with Turner’s syndrome, may benefit. At the same time, this method offers an excellent model to study the development of early human ovarian follicles. Using this culture system, we examined the effect of AMH on the initiation and regulation of the growth of human ovarian primordial follicles.
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Human ovarian tissue
Fifteen women aged 26–41 years (mean ± SD 33.7 ± 3.6 years) donated small ovarian cortical biopsy specimens, when undergoing routine Caesarean sections (n = 14) or gynaecological laparoscopies (n = 1) at Karolinska University Hospital Huddinge, Sweden. Informed consent was obtained before surgery, and the study was approved by the Ethics Committee of the Karolinska Institutet, Sweden.
Tissue culture
The biopsy specimens (n = 15) were placed in pre-equilibrated HEPES-buffered oocyte collection medium (Gamete-100; Vitrolife, Kungsbacka, Sweden) and transferred immediately to the culture laboratory. Ovarian tissue samples were cut into small pieces, approximately 1–2 mm3, using a needle and scalpel. The tissue pieces were then either directly fixed for histological analyses (non-cultured control at day 0) or placed inside well inserts and cultured for 7 days, as parallel cultures from the same biopsy specimen. Two to five (mean 3.3) pieces of tissue from each specimen were added to each well.
We used an established tissue-culture method as previously described (Hovatta et al., 1997, 1999; Hreinsson et al., 2002; Scott et al., 2004a,b). Cultures were performed in 24-well low evaporative lid plates (Falcon, Becton Dickinson, Bedford, MA, USA) fitted with Millicell CM inserts (12 mm diameter, 0.4 µm pore size, Millipore, Sundbyberg, Sweden). The inserts were coated with 100 µl of growth factor-reduced extracellular matrix (GFR MatrigelTM, Becton Dickinson), pre-diluted 1:3 with alpha minimal essential medium with Glutamax (
MEM, Gibco Invitrogen, Lindingö, Sweden). The base culture medium was MEM supplemented (10%) with human serum albumin (HSA, Pharmacia Upjohn, Stockholm, Sweden). Further supplements were recombinant human FSH (0.5 IU/ml, Gonal-F, Serono, Solna, Sweden) (Wright et al., 1999), 8-bromoguanosine 3',5'-cyclic monophosphate (8-br-c-GMP, 1.1 mg/ml, Sigma-Aldrich, Stockholm, Sweden) (Scott et al., 2004a), 1% ITS-G (Gibco Invitrogen) (with a final concentration of 10 µg insulin/ml, 5.5 µg transferrin/ml and 6.7 ng/ml of sodium selenite in the media) and 0.5% antibiotic/antimycotic solution (50 IU/ml penicillin, 50 µg/ml of streptomycin sulphate, 0.125 µg/ml of amphotericin B; Gibco Invitrogen). To test the dose–response to AMH, we added recombinant rat AMH (rrAMH) to the culture medium at four different concentrations: 10, 30, 100 and 300 ng/ml (n = 15). Only five biopsies were cultured at the highest concentration of AMH (300 ng/ml) owing to the scarcity of purified hormone, thus making this part of the work a pilot study only. rrAMH was produced and purified as described previously and prepared in stock solution [Hanks’ balanced salt solution (HBSS)/0.1% bovine serum albumin] (Weenen et al., 2004). Two control media containing no AMH were included in the cultures, one serving as a vehicle control containing 102.5 µl of stock solution used to dilute the AMH. Culture medium (500 µl) was added to each well; 100 µl was pipetted into the insert with the remaining 400 µl in the well outside the insert. Every second day, 110 µl of culture medium was removed and replaced with fresh medium (in the inserts). Before use, medium was pre-equilibrated for 45 min at 37°C in a 95% air, 5% CO2 humidified environment.
Histology and follicle counts
Fresh non-cultured ovarian biopsy material (day 0) and cultured specimens were fixed in Bouin’s solution (Sigma-Aldrich) for 4–5 h at room temperature, dehydrated in 70% ethanol, embedded in paraffin and serially sectioned at 4 µm thickness. To prevent double counting of follicles, we omitted eight sections between those sections mounted on slides. Following staining with haematoxylin and eosin, cells were analysed for viability using the method previously described by our group (Hovatta et al., 1997, 1999; Hreinsson et al., 2002; Scott et al., 2004a,b). Briefly, the presence of pyknotic granulosa cells, eosinophilia of the ooplasm and clumping of the chromatin material identified atretic follicles. Follicles were counted and their developmental stages recorded according to the classification described by Gougeon (1986). Those follicles containing a single layer of flattened granulosa cells were regarded as primordial, those having cuboidal granulosa cells in a single layer identified as primary and those having two or more layers of cuboidal granulosa cells identified as secondary. A digital imaging analysis system (Easy Image Mätning; Tekno Optik, Huddinge, Sweden) was used to measure the areas of the tissue pieces, from which the volumes were calculated by multiplying the area by the known section thickness of 4 µm. The density of the follicles was then determined as the total number of follicles at all stages of development per cubic millimetre of ovarian tissue.
Statistical analyses
The proportions of follicles at different developmental stages, density of follicles and proportions of viable follicles within each patient sample were analysed. Statistical comparisons between groups for all data were carried out using one-way analysis of variance (ANOVA) followed by Fisher’s calculation of least significant differences. Before analysis, the data were tested for normality and homogeneity of variance. Significance is reported at the 0.05 level. Cultures containing 300 ng/ml of AMH were excluded from statistical analyses owing to the small number of samples (n = 5). Data are presented as mean ± SEM.
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Developmental stages of the follicles
The mean proportions of the developmental stages of the follicles changed significantly during the 7-day culture period in most groups when compared with fresh non-cultured control tissue (Figure 1). In non-cultured fresh tissue, the proportion of primordial follicles was 56%, whereas after 7 days, it had significantly decreased to levels between 14 and 26% in all cultures except those containing AMH at 100 ng/ml. In these cultures, the proportion of primordial follicles (40%) did not differ statistically from that in the non-cultured control tissue, and it was also significantly greater than the proportions observed in vehicle control cultures and cultures containing AMH at 10 ng/ml. The proportions of primary and secondary follicles increased similarly in the majority of cultures, with significantly more primary follicles observed in control, vehicle control and 10 ng/ml of cultures when compared with the non-cultured tissue. Figure 2 illustrates a typical histological view of the follicle-containing tissue cultured in control medium (A) and medium containing AMH at 100 ng/ml (B). More primordial follicles were seen in the cultures containing AMH at 100 ng/ml.
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Viability of the follicles
The proportion of viable follicles was significantly reduced in all cultured tissues (40–52%) when compared with that in non-cultured tissue (83%, Figure 3). No differences were observed between the culture groups.
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Density of follicles
The density of follicles did not significantly differ between the non-cultured control tissues and the cultured tissues, or between the culture groups, as shown in Figure 4. Great variation in follicle densities within each biopsy specimen accounts for the wide error margins observed.
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Discussion |
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In this study, we demonstrated that rrAMH exerts an effect on the early stages of human ovarian follicular development. At a concentration of 100 ng/ml, AMH suppressed the initiation of the growth of primordial follicles, although having no detrimental effect on viability or follicle density in the tissue. In the pilot study in which 300 ng/ml of AMH was used (n = 5), the proportions of viable follicles were as follows: primordial (26.5 ± 18.7%), primary (57.4 ± 16.5%) and secondary (16.1% ± 9.3%). Follicle viability was 47% (±16.6) and follicle density 157.9 (±55.9) follicles per cubic millimetre. No conclusions could be drawn regarding the 300 ng/ml concentration owing to the small sample number, but we cannot exclude the possibility that a higher concentration of AMH may have a more pronounced or different effect. Hence, further dose–response studies are required to elucidate its exact role in controlling follicle growth.
The proportions of the various developmental stages of the follicles changed in a manner similar to that in our earlier studies (Hovatta et al., 1997, 1999; Hreinsson et al., 2002; Scott et al., 2004a,b) in all cultures except those supplemented with rrAMH at 100 ng/ml. In those particular cultures, the proportion of primordial follicles was not significantly different from that in the non-cultured tissues; furthermore, it was significantly greater than that in vehicle control and 10 ng/ml of cultures, indicating the inhibition of growth initiation. The lack of significant primordial follicle activation is in accordance with results obtained in mouse follicle cultures, where treatment with AMH appeared to inhibit early follicular development (Durlinger et al., 2002). These results further support the theories first presented in connection with AMH-knockout mice, i.e. AMH is necessary to slow down or halt the initiation and growth of primordial follicles (Durlinger et al., 1999). Hence, the proposal that AMH is produced by the pool of growing follicles to act as a negative paracrine feedback signal on neighbouring primordial follicle initiation is strengthened (Visser and Themmen, 2005).
On the basis of the present results, we can state that AMH may play a role in inhibiting and controlling the recruitment of primordial follicles and their early growth in humans, as has been shown in mice (Durlinger et al., 2002). Studying and identifying the roles of AMH, in addition to those of other factors, will lead to greater understanding of follicle recruitment and growth. Although obvious species differences exist, even between rats and mice (Durlinger et al., 2001, 2002; McGee et al., 2001), our results on human ovarian tissue are in accordance with those observed in mice.
In a study by Schmidt et al. (2004), long-term cultures of up to 4 weeks were carried out for a small number of samples. They observed that 300 ng/ml of AMH increased the proportion of primary and secondary follicles during 4-week culture, but not the diameter of the oocytes. In the present study, ovarian tissue was cultured for only 7 days to pinpoint and observe the recruitment and initial growth of the primordial follicles. Our interest was centred on the role of AMH in inhibiting primordial follicle recruitment. However, longer-term cultures would be interesting to observe any differences in the growth rate of human oocytes and follicles. It is possible that prolonged exposure to high levels of AMH could down-regulate AMH receptors and over time render AMH treatment ineffective. Schmidt et al. (2004) used rhAMH, whereas in the present study, we used rrAMH. Both hormones were similarly active, and it is difficult to say whether a greater dose or longer treatment would change the initiation of follicle growth.
The initial recruitment and growth of primordial follicles is a complex process, involving a multitude of factors with both positive and negative effects. AMH is one of the hormones involved in the inhibition of primordial follicle recruitment, as demonstrated in this study. There remain several unknown or unidentified factors implicated in the control of early follicle growth, highlighting the importance of continuing studies to further our understanding.
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We are grateful to the surgical staff at the Department of Obstetrics and Gynaecology, Karolinska University Hospital Huddinge, for their enthusiastic co-operation in collecting ovarian biopsy specimens. The Swedish Research Council and The Swedish Society for Medical Research supported this work. JV, AT and OR were funded by the European Commission (grant QLK6-CT-2000-00338). Finally, we thank Nicholas Bolton for revising the language.
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Submitted on June 27, 2005; resubmitted on September 28, 2005; resubmitted on December 16, 2005; resubmitted on March 13, 2006; accepted on April 20, 2006.
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