Stine Gry Kristensen

Which follicles make the most anti-Mullerian hormone in humans? Evidence for an abrupt decline in AMH production at the time of follicle selection.

Anti-Müllerian hormone (AMH) is exclusively produced by granulosa cells (GC) of the developing pre-antral and antral follicles, and AMH is increasingly used to assess ovarian function. It is unclear which size follicles make the most AMH (total content) and are the main contributors to circulating AMH concentrations. To determine AMH gene expression in GC (q-RT-PCR) and follicular AMH production (Elisa and RIA) in relation to follicular development, 87 follicles (3-13 mm diameter) including both GC and the corresponding follicular fluid (FF) were collected in connection with fertility preservation of human ovaries. Further, follicle number and diameter, graded in 1 mm increments, were determined by 3D ultrasound in 113 women in their natural menstrual cycle to determine follicle number and diameter in relation to circulating AMH levels. This study demonstrates for the first time a positive association between AMH gene expression in human and both total follicular fluid AMH (P < 0.02) and follicular fluid AMH concentration (P < 0.01). AMH gene expression and total AMH protein increased until a follicular diameter of 8 mm, after which a sharp decline occurred. In vivo modelling confirmed that 5-8 mm follicles make the greatest contribution to serum AMH, estimated for the first time in human to be 60% of the circulating concentration. Significant positive associations between gene expression of AMH and FSHR, AR and AMHR2 expression (P < 0.00001 for all three) and significant negative association between follicular fluid AMH concentration and CYP19a1 expression were found (P < 0.0001). Both AMH gene expression (P < 0.02) and follicular fluid concentration of AMH (P < 0.00001) correlated negatively with estradiol concentration.

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.

Vitrification of in vitro matured oocytes collected from surplus ovarian medulla tissue resulting from fertility preservation of ovarian cortex tissue

PurposeThe aim of the study was to investigate the maturation rate of immature oocytes collected from ovarian medulla tissue normally discarded during preparation of ovarian cortical tissue for fertility preservation. Further we evaluated survival of derived MII oocytes following vitrification and warming.Methods36 patients aged from 8 to 41xa0years who had one ovary excised for fertility preservation were included. Oocytes were collected from the medulla tissue and matured in vitro 44–48xa0h followed by vitrification. Number of oocytes collected, the rates of maturation and post-warming survival were assessed.ResultsOn average, 11 immature oocytes were collected per patient. The overall maturation rate was 29xa0% irrespective of whether the ovary was transported 4–5xa0h on ice or obtained immediately after oophorectomy. The maturation rate in patients below 20xa0years of age (55xa0%) was significantly higher than that of patients aged 20–30 years (29xa0%) and above 30xa0years (26xa0%). The post-warming survival rate was 64xa0%. No significant relationship was observed between the number of collected oocytes and the age of patients.ConclusionsApproximately three MII oocytes were obtained per patient following in vitro maturation (IVM) of immature oocytes collected from medulla tissue, of which two survived vitrification and warming. This approach represents an add-on method to potentially augment the fertility opportunity for cancer patients, especially in young women with cancer where transplantation of cortical tissue may pose a risk of relapse, but the IVM approach is currently too inefficient to be the only method used for fertility preservation.

Optimizing outcomes from ovarian tissue cryopreservation and transplantation; activation versus preservation

Ovarian tissue cryopreservation and transplantation (OTCP) is gaining increasing traction in the field of fertility preservation as a result of accumulated successes. We now have a decade of experience with the technique, with tens of live births and greater than 90% return of ovarian function in graft recipients. Recently, a novel method of OTCP has been described, termed in vitro activated OTCP which proposes significant changes to the standard protocol. This method aims to stimulate activation of dormant follicles within the grafts prior to transplantation and ensure that mature oocytes can be generated in the immediate short term after transplantation. By contrast, conventional OTCP seeks to maintain dormancy and thus preserve the follicle reserve in the graft with the aim of maximizing graft lifespan. This opinion paper will compare the two methods of OTCP, highlighting their respective advantages and disadvantages, and provide suggestions as to when to apply either one of these methods in a clinical setting.

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.

Transplantation of frozen-thawed ovarian tissue: an update on worldwide activity published in peer-reviewed papers and on the Danish cohort

PurposeThe purpose of the study is to review all peer-reviewed published reports of women receiving ovarian tissue transplantation (OTT) with frozen/thawed tissue (OTC) with respect to age, diagnosis, transplantation site, fertility outcome, and potential side effects, including data from all women in the Danish program.MethodsA systematic review of the literature was performed in PubMed combined with results from all patients who had received OTT in Denmark up to December 2017.ResultsOTT has been reported from 21 different countries comprising a total of 360 OTT procedures in 318 women. In nine women, malignancy was diagnosed after OTT; none were considered to be directly caused by the OTT. Despite a potential under reporting of cancer recurrence, there is currently no evidence to suggest that OTT causes reseeding of the original cancer. Renewed ovarian endocrine function was reported in 95% of the women. Half of all children born following OTT resulted from natural conception, and newborns were reported to be healthy except for one neonate with a chromosome anomaly with a family disposition. Women who conceived after OTT were significantly younger than those who failed.ConclusionThis study found no indications of sufficient numbers of malignant cells present in the ovarian tissue to cause recurrence of cancer after OTT. Further, it is unlikely that OTC affects the well-being of children born. OTC is now an established method of fertility preservation in Denmark with public reimbursement. The current data encourage that women who require gonadotoxic treatment should be offered an individual evaluation considering fertility preservation.

Defining quality assurance and quality control measures in connection with ovarian tissue cryopreservation and transplantation: a call to action

Freezing of ovarian tissue for fertility preservation has been gaining ground as a valid method in recent years. More than 100 children have been born from this procedure worldwide. As a result, many fertility clinics are now implementing this method. However, the practical procedures that need to be mastered to successfully implement the freezing of ovarian tissue are different in many aspects from those normally used in fertility clinics and are not well defined. Furthermore, success is difficult to measure since patients usually do not return for transplantation until several years after freezing, which puts extra emphasis of good quality control and quality assurance measures to secure a transplantation of tissue with surviving follicles that can sustain fertility. The present paper describes the procedures and a checklist implemented in Denmark in order to secure a successful clinical service. To standardize and implement uniform measures for this new method, we suggest a consensus conference to collectively agree on the best technical and clinical practice.

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 13u2009mm) 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 8–11u2009mm 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.

Temporal expression pattern of genes during the period of sex differentiation in human embryonic gonads

The precise timing and sequence of changes in expression of key genes and proteins during human sex-differentiation and onset of steroidogenesis was evaluated by whole-genome expression in 67 first trimester human embryonic and fetal ovaries and testis and confirmed by qPCR and immunohistochemistry (IHC). SRY/SOX9 expression initiated in testis around day 40u2009pc, followed by initiation of AMH and steroidogenic genes required for androgen production at day 53u2009pc. In ovaries, gene expression of RSPO1, LIN28, FOXL2, WNT2B, and ETV5, were significantly higher than in testis, whereas GLI1 was significantly higher in testis than ovaries. Gene expression was confirmed by IHC for GAGE, SOX9, AMH, CYP17A1, LIN28, WNT2B, ETV5 and GLI1. Gene expression was not associated with the maternal smoking habits. Collectively, a precise temporal determination of changes in expression of key genes involved in human sex-differentiation is defined, with identification of new genes of potential importance.

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.