In vitro activation and maturation of primordial follicles of trans men is currently limited by inadequate support of the preantral follicle growth

Ovarian tissue cryopreservation is an interesting fertility preservation option for trans men as it does not require controlled ovarian stimulation, involving female hormone rises and vaginal ultrasound monitoring. Future use... [ view full abstract ]

Ovarian tissue cryopreservation is an interesting fertility preservation option for trans men as it does not require controlled ovarian stimulation, involving female hormone rises and vaginal ultrasound monitoring. Future use by transplanting thawed tissue includes unwanted side effects by restoring female hormone activity. In theory, an alternative to transplantation could be in vitro maturing the residing immature follicle pool. However, attempts of in vitro activation and maturation of primordial follicles are not yet satisfactory.

In order to follow the primordial follicle activation and maturation during in vitro culture of frozen thawed ovarian tissue of trans men, detailed histological follicle observations were performed during the in vitro culture. [ view full abstract ]

In order to follow the primordial follicle activation and maturation during in vitro culture of frozen thawed ovarian tissue of trans men, detailed histological follicle observations were performed during the in vitro culture.

Cryopreserved ovarian tissue, donated for research, of 3 trans men (23.3 +/- 1.53 years old) was thawed and prepared for in vitro culture. All of them underwent a hysterectomy with bilateral oophorectomy after a period of... [ view full abstract ]

Cryopreserved ovarian tissue, donated for research, of 3 trans men (23.3 +/- 1.53 years old) was thawed and prepared for in vitro culture. All of them underwent a hysterectomy with bilateral oophorectomy after a period of intramuscular testosterone undecanoate (Nebido®) (55.3 +/- 1.15 weeks). After thawing, the ovarian cortex (2.5*5*1mm) was pulled mechanically using the blunt end of a scalpel to flatten out the tissue and to minimize the underlying stroma. The tissue was then cut into small interconnecting strips. Two pieces were taken and fixed in 10% paraformaldehyde for histological evaluation. The cortical strips were transferred individually in 24-well cell culture plates (Corning B.V. Life Sciences Europe, Amsterdam, The Netherlands) containing 300 µl of McCoy's 5a medium with bicarbonate supplemented with HEPES (20 mM), HSA (0.1%) (Red Cross, Belgium), glutamine (3 mM) (Thermo Scientific, Erembodegem, Belgium), penicillin G (0.1 mg/ml)-streptomycin (0.1 mg/ml) (Thermo Scientific), transferrin (2.5 µg/ml), selenium (4 ng/ml), insulin (10 ng/ml) and ascorbic acid (50 µg/ml), all obtained from Sigma-Aldrich (Bornem, Belgium), unless stated otherwise. As described by E. Telfer (2008), they were cultured for 6 days at 37°C in humidified air with 5% CO2 with medium change and collection of 2 pieces every second day.

The paraffin embedded ovarian cortical tissue piece was serially sectioned at 5µm, resulting in 10 slices, and stained with (Mayer) hematoxylin (Merck, Overijse, Belgium) and eosin (Thermo Scientific, Erembodegem, Belgium).... [ view full abstract ]

The paraffin embedded ovarian cortical tissue piece was serially sectioned at 5µm, resulting in 10 slices, and stained with (Mayer) hematoxylin (Merck, Overijse, Belgium) and eosin (Thermo Scientific, Erembodegem, Belgium). Follicles were analyzed using an inverted microscope with a 40x magnification. Follicles were classified according to the Gougeon (1986) classification for human follicles: primordial follicle (an oocyte surrounded by a single layer of flattened granulosa cells (GC)); intermediate follicle (a single layer of flattened and cubical GCs surrounds the oocyte); primary follicle (a single layer of exclusively cubical GCs surrounds the oocyte); secondary follicle (an intact second layer of cubical GCs surrounds the oocyte); antral follicle (more than 2 layers of cubical GCs surrounds the oocyte in presence of an antrum) (Gougeon, 1986). Follicles were classified on the section containing the nucleus to avoid double counting. Two independent observers analyzed the follicles and the mean of the 2 observations was used for further analysis. The follicle number and classification on culture day 0, 2, 4 and 6 were compared. Statistical analysis was performed with IBM SPSS Statistics 23 (New York, USA). Values of P <0.05 were considered to be statistically significant.

The mean count of the 2 observers showed a total of 2099.00 follicles, with a mean of 699.67 + 242.06 follicles per patient (N=3) and a mean of 524.75 +/- 166.04 follicles per culture day (N=4). The total number of follicles... [ view full abstract ]

The mean count of the 2 observers showed a total of 2099.00 follicles, with a mean of 699.67 + 242.06 follicles per patient (N=3) and a mean of 524.75 +/- 166.04 follicles per culture day (N=4). The total number of follicles did not differ significantly over the different culture days (P=0.230). A progressive decrease in number of primordial follicles was seen with a significant difference between culture day 0 and 6 (P=0.011). For the other follicle classification, a shift towards the more mature stages was visible, however no significant change per follicle class was seen. Unfortunately, limited secondary follicle and no antral follicle growth was observed

The current culture model, as described by E. Telfer (2008), nicely supports the primordial follicle activation in in vitro culture of frozen thawed ovarian tissue of trans men. However, a clinically useful culture system... [ view full abstract ]

The current culture model, as described by E. Telfer (2008), nicely supports the primordial follicle activation in in vitro culture of frozen thawed ovarian tissue of trans men. However, a clinically useful culture system should not only allow in vitro activation of primordial follicles but should also support the subsequent preantral follicle growth. The current findings revealed the limited preantral follicle development as the most important restriction and identified the focus for further culture optimalisation.