Human primordial follicles are not regenerative resources, age, genetic mutations and environmental factors all impact on the size and quality of follicle pool [4]. During women’s reproductive lifespan, it’s simultaneity for ageing of hypothalamus-pituitary-gonad (HPG) axis and exhaust of follicle pool [26]. Reductions in oocyte quantity and quality with advanced age (typically in the mid-40s) are a normal physiologic occurrence termed DOR. Some women experiencing DOR much earlier and becoming prematurely infertile classify the pathologic DOR [27]. In clinic, young women with DOR still have an boosted fecundity over elderly women that indicate it’s not synchronous recession between quantity and quality of oocytes. To investigate this query, the current study was a prospective, cohort study. Study group were recruited from patients with number of retrieved oocytes within 5, and divided into young group and ageing group. Those patients also met the standard of DOR [20]. Because DOR is a normal physiologic process when it occurs in the mid − 40s and is pathologic at younger patients [27], so young women with biologically older ovarian, and we call this group young DOR; ageing group represented the situation of elderly women with chronologucal ovarian age. control group were young women with the normal functional ovarian reserve [28]. In current study, the ovarian reserve were not significant different between young DOR and aging group, hence according to assessment of biological ovarian age [29, 30], young DOR and aging group have same biological ovarian age. However, significantly higher MII oocyte rates and implantation rates were observed in young DOR than ageing group. Next, we carried out further study to explore the factors in follicular microenvironment presumably cause to different IVF outcomes between young DOR and ageing group, since two groups have the same ovarian reserve and ovarian age.
Recent researches have shown that exosomes secreted by cells through unconventional exocytosis have been found in follicular fluid. Follicular fluid is the microenvironment to support oocytes survive, so we speculate that except cytokines, exosomes might play an important role in communication between germ cells and somatic cells in follicular microenvironment. In current study, exosomes were isolated from human FF by Exo-quick precipitation [31] from all three groups, then isolated exosomes were observed by scanning electron microscope (SEM), and a few deferent sized membranous vesicles were observed in every sample. To identity what difference in expression of exosomes in FF were among three groups, we adopted transmission electron microscopy (TEM) to measure the concentration and size of exosomes. The results revealed that the concentration of exosomes in FF was highest in control group, and was exceedingly lowest in ageing group. Young DOR has medium level of concentration of exosomes, but significantly higher than ageing group. In line with a decline trend in concentration of exosome among three groups, the uniform of Exosomes sizes also reduced in sequence among control group, young DOR, and ageing group. The distribution of different size of exosomes were completely different among three groups. Exosomes with a diameter of 30 to 100 nm were in the majority in control group and young DOR. In ageing group, a population of extracellular vesicle (EVs) with a diameter higher than 100 nm could be observed, and the proportion of exosomes with a diameter of 30 to 100 nm was fewer than other groups. Pioneering research reported that exosomes and micro-vesicles are membrane bound vesicles that differ based on their process of biogenesis and biophysical properties, including size and surface protein markers. Exosomes are homogenous small particles ranging from 40 to100nm in size and have a variety of common exosomes surface markers such as CD9, CD63, and CD81 [32]. To further identify the isolated vesicles in FF, we detected the CD63 and CD81 by western blot analysis. Our results showed isolated vesicles from three groups all expressed those exosomes mark. The changes of expression of above exosomes mark were consist with the concentration of exosomes among three groups. For the first time, we confirmed there exist different sized vesicles in FF, of which exosomes with diameter of 30 to 100 nm have positively correlation with age and oocytes’ quality, and the concentration of exosomes can predict the situation of follicular microenvironment ageing. The large variation in oocyte’s quality or IVF outcomes among three groups is evident from the variability of distribution and concentration of exosomes among them. In addition, we found that there were completely deferent in the size and concentration of exosomes between the larger follicles (diameter > 14mm) and smaller follicles(diameter < 14mm) from an individual (data not shown). Furthermore, the recent studies have shown that the exosomes of boar seminal plasma could preserve sperm function by combining to the sperm membrane and transferring proteins to sperm [33]. Combined with those results, we conclude that exosomes in follicle microenvironment may play different role in the different stage of gametes’ growth and development.
About the exosomes within the ovarian follicle, little is known about the source of exosomes in FF. Human ovarian follicle is a unit comprised of different cell types such as oocyte, theca, granulosa and cumulus cells. We collected oocytes of fertilization failure in IVF, rinsing thoroughly with PBS, then fixed and observed. We first demonstrated exosomes are binding on oocyte’s surface by SEM. To verify the source of exosomes in FF, we analyzed the exosome-derived amplified cDNA by PCR. The results showed that Growth differentiation factor 9 (GDF9) and bone morphogenetic protein 15(BMP15) mRNA band could be detected in all three groups. And the expression of above ovular marks in exosomes were vary significantly among three groups. GDF9 and BMP15 are two secreted proteins selectively expressed in the oocyte which are necessary for the normal fertility in mammals [34, 35]. Throughout follicular development, GDF9 and BMP15 are co-expressed in oocytes and keep exceptionally high levels in FF [36]. The latest study demonstrated BMP15 up regulate AMHR2 and AMH expression by effecting on AMH promoter activity in human GCs, moreover GDF9 enhanced above procession [37]. Together these provide solid support for the concept that Exosomes containing GDF9 and BMP15 are the principal mediating the paracrine effects of oocytes on granulosa/cumulus cells. In present study, we only collected and isolated exosomes from mature follicles (diameter > 14mm), during the stage of follicle’s development, we do not know when exosomes start to appear, where come form and to go in FF. Anyway, based on these findings, the ovular origin of exosomes has been confirmed by the presence of GDF9 and BMP15 gene transcripts. Exosome exchange may be suggested as an emerging way of communication between oocyte and granular cells in the mature follicles.
Increasing evidence suggests that aging is associated with dynamic changes in the hypothalamic components of the reproductive axis that are independent of changes in gonadal hormone secretion. Hall JE [38] reported that GnRH pulse frequency and interval in older postmenopausal women (PMW) decreased and got longer than young women. Hence, the changes in level of pituitary components such as FSH, FSH/LH do not really represent the situation of reproductive axis ageing. Therefore, in current research, although the young DOR have biologically older ovarian age as same as the ageing group’s, they still have healthy reproductive axis and follicle environment to supply excellent oocytes. That’s also can help to explain a clinical study by Seckin B [39] which illustrated elevated day 3 FSH/LH ratio is useful in predicting IVF outcome in older women but does not seem to be an accurate predictor in younger women. Combining previous researches and our present study, we fist come up with a concept that the changes related-age of reproductive axis simultaneously accelerate the declining in follicle pool and degenerating in follicle microenvironment, there are interactive regulations among reproductive axis, follicle pool and follicle microenvironment, two later respectively charge in the reproductive lifespan and reproductive quality, and then both of them crucially determine female fecundity. However, any aggregate estimates of ovarian reserve may be misleading as they represent a mixture of different trends seen in individuals’ fecundity. As previously described, those ovarian reserve marks, the levels of FSH, AMH and AFC, also be used to asses biological ovarian age [40–42], so “biological ovarian age” just represent reproductive lifespan rather than female fecundity, because those marks do not independently reflect oocyte’s quality charging in reproductive quality. Follicle microenvironment is formed starting at preantral follicles, and gradually change ingredient of cytokines and exosomes along with the oocyte maturation of folliculogenesis under the regulation of gonadal hormone [14]. In current research, exosome is found to be the only factor which is different between young DOR and ageing group, and exosome positively correlate with age and oocyte’s quality. The results demonstrated that exosomes may relatively reflect the situation of follicular microenvironment aging and also expose the real reason why young DOR still have better IVF outcomes than elderly group although they had same ovarian age. As for whether dose the decreasing in exosomes’ concentration in ageing group attribute to the age-related changes of hypothalamus? and whether is there any correlation between exosomes and age-associated oocyte aneuploidy? All those are needed to further study. Next, -we will detect other markers to identify cells secreting exosomes and further explore the specific mechanism of exosomes by means of microarray and qRT-PCR to assess LncRNA, circRNA and miRNA expression in exosomes. This study improved knowledge of the exosome function in follicular microenvironment and may be a prediction of clinic outcomes in IVF and manipulation of the early steps of folliculogenesis to extend fertility lifespan.