P-439 Human Platelet Lysate improve the growth and survival of isolated human pre-antral follicles in vitro

Abstract

Study question Can human Platelet Lysate (hPL) and umbilical cord plasma (UCP) be used as alternative serum sources in the culture of isolated human pre-antral follicles? Summary answer hPL significantly increased the growth and survival of human follicles cultured for 8 days compared to fetal bovine serum (FBS) and human serum albumin (HSA). What is known already Culture of human ovarian follicles is a potential new source of mature oocytes for fertility preservation and an important model system to study basic biology. However, only a few studies have produced mature human oocytes after culturing pre-antral stage follicles. Moreover, optimising culture systems is challenging due to the scarcity of human material. Platelet-rich plasma solutions have been used extensively in regenerative medicine due to their high platelet content, which upon activation, release a multitude of growth factors, hormones, and cytokines. Clinically cell-based therapies have used platelet-rich solutions, such as hPL or UCP, successfully as an animal-free serum. Study design, size, duration Human pre-antral follicles (n = 378; mean diameter: 78 µm; range: 46-237 µm) were isolated from ovarian medulla tissue. The follicles were encapsulated in 0.5% alginate and cultured for 8 days in media supplemented with one of four sources of serum; 5% FBS (n = 74), 2.5% HSA (n = 74), 5% hPL (n = 140), and 5% UCP (n = 90). The primary endpoints were follicular growth and survival. Secondary endpoints included follicular gene expression analysis and media hormone concentrations. Participants/materials, setting, methods Ovarian surplus tissue was donated by 7 women (aged 19-32 years) undergoing unilateral oophorectomy and ovarian tissue cryopreservation for fertility preservation. Pre-antral follicles were isolated enzymatically and growth and survival were assessed every second day during culture by microscopy. AMH and Estradiol concentrations were measured by ELISA in media collected at day 4 and 8. At day 8, surviving follicles were snap-frozen and the expression of AMH, FSHR, GDF9, and BMP15 was analysed by qPCR. Main results and the role of chance After 8 days in culture, the follicle survival rate in the hPL group (86%; n = 120/140) was significantly higher compared to the FBS group (60%; n = 45/74; p < 0.0003), but comparable to the HSA group (76%; n = 56/74; p = 0.292). On the contrary, the follicle survival rate in the UCP group (33%; n = 30/90) was significantly lower compared to any of the other groups (p < 0.0001 for all three groups). The average diameter of surviving follicles was statistically significantly larger in the hPL and UCP group compared to the FBS and HSA groups (hPL: 149 ±4.6 µm; UCP: 138 ±7.6 μm; FBS: 110 ±3.8 μm; HSA: 115 ±4.2 μm; p < 0.001 for hPL compared to FBS and HSA; p = 0.0129 for UCP compared to HSA). The relative growth of the follicles compared to their initial diameter was 45% and 33% higher in the hPL group compared to the FBS and HSA groups, respectively, which was statically significant in both groups (p < 0.001). In the UCP group, follicle growth was also significantly higher by 25% compared to the FBS group (p < 0.001) and by 14% compared to the HSA group (p = 0.0376). Hormone measurements from the culture media and follicle gene expression analysis are awaited. Limitations, reasons for caution One limitation is the variability in the initial follicle sizes. Extrapolating results according to primordial, primary, and secondary follicles would require more follicles but could provide valuable insight. Moreover, long term culture studies are needed to evaluate the effects of hPL and UCP on antral follicle development and oocyte maturation. Wider implications of the findings Our findings show that hPL can be used as an alternative serum source in the culture of human pre-antral follicles as it increased follicle growth and survival compared to FBS and HSA. UCP also increased follicle growth but had detrimental effects on follicle survival which needs further studies. Trial registration number Not applicable

Reducing 3D Hydrogel Stiffness, Addition of Oestradiol in a Physiological Concentration and Increasing FSH Concentration Improve In Vitro Growth of Murine Preantral Follicles

“…This difference is most likely explained by differences in media composition: Xu and co-workers used 3 mg/mL bovine serum albumin (BSA) and 1 mg/mL fetuin during the culture, while we employed 5% foetal bovine serum (FBS). Evidence in mice and humans has demonstrated that various serum-derived products impact preantral follicle growth differently in vitro [32][33][34]. In particular, FBS contains more than a thousand components, including proteins, hormones, growth factors, enzymes, and other undefined constituents, which generate potential batchto-batch variations, making comparing results from different studies difficult.…”

The Clinical Application of Platelet-Rich Plasma in the Female Reproductive System: A Narrative Review

“…In vitro, culture media supplemented with PRP was shown to support the growth and survival of early preantral follicles significantly better than media without PRP [61]. In comparison to other growth supplements routinely used in culture media, such as human serum albumin and fetal bovine serum, human platelet lysate (hPL), a derivative of PRP, has demonstrated improvement in follicular growth and survival of isolated primary and secondary human ovarian follicles [76].…”

Cultivo del tejido ovárico para el desarrollo folicular in vitro: futura estrategia de restauración de la fertilidad

“…Los métodos de aislamiento folicular se dividen en tres categorías: enzimáticos [36][37][38] , mecánicos 18,39,40 , o una combinación de ambas [41][42][43][44][45] .…”

“…Aunque requiere más tiempo, este enfoque da como resultado una mejor conservación de la integridad y morfología folicular. Por otro lado, varios estudios [41][42][43][44][45] informaron resultados efectivos utilizando una combinación de ambos protocolos que incluían una breve digestión enzimática, seguida de un aislamiento por microdisección individual.…”

“…En este estadio, los folículos se vuelven dependientes de las gonadotropinas 63 , ya que las CG comienzan a expresar receptores de FSH. Otros aditivos de cultivo han demostrado mejorar la viabilidad y desarrollo folicular, como el factor de crecimiento de fibroblastos básico (bFGF) 42 , y plasma rico en plaquetas, que contiene altas concentraciones de factores de crecimiento 38,45 .…”