Comparative analysis of the use of autologous exosomes and platelet-derived growth factors in women with premature ovarian insufficiency and infertility: A prospective, randomized, observational, analytical study

Abstract

Stromal fibrosis and ovarian aging depend not only on the age of the patients but also on environmental factors, lifestyle and physiological events such as ovulation itself, which leaves repeated scarring in the ovarian stroma, limiting normal tissue vascularization and the provision of biological signals necessary to maintain folliculogenesis and hormone synthesis. In a fibrosed ovarian stroma, there is a decrease in hyaluronic acid concentrations, loss of synthesis and migration proteins, a decrease in microRNAs, in addition to enzymatic alterations that affect mitochondrial kinases, increasing oxygen free radicals, which accelerate cell death.

Objectives

This study seeks to analyze the effectiveness of autologous exosomes in reversing aging and bioregenerating the ovary of patients with ovarian failure and infertility by improving interstitial fibrosis and providing the necessary elements to activate neofolliculogenesis.

Methodology

Prospective, randomized, comparative study in 30 women between 38 and 46 years old, with diminished ovarian reserve and who rejected the egg donation procedure. Three study groups composed of 10 patients each: the first with autologous exosomes, the second with PRP and the third with saline solution.

Results

After completing the study, it was found that women in the Autologous Exosome group showed better ovarian reserve parameters such as FSH, LH, Estradiol, Anti-Müllerian Hormone and antral follicle count, edema of more oocytes collected in Metaphase II, higher fertilization rate, frozen embryos and positive pregnancies.

Conclusion

Ovarian Biostimulation with Autologous Exosomes could be considered a safe and promising therapy to improve markers of low ovarian reserve.

Trial registration

Clinical Trials.gov. Registration Number: NCT06773572. URL: Study Details | Use of Autologous Exosomes vs Platelet Growth Factors to Regenerate the Ovary in Women With Infertility (Exosomas2024-1) | ClinicalTrials.gov.

1. Introduction

Achieving pregnancy has become increasingly challenging due to multiple factors, including environmental and hormonal disruptors [1], as well as the growing tendency to delay childbearing [2,3]. Motherhood after the age of 37 is particularly affected by the decline in ovarian hormone production and interstitial fibrosis resulting from repeated ovulations [4].

The ovary begins its hormonal function as early as day 56 of embryonic development, yet it is also the first organ to undergo aging, which significantly impacts its reproductive capacity [5,6].

By the age of 30, ovarian function begins to decline, leading to infertility and an earlier onset of degenerative, cognitive, and cardiovascular diseases [6].

The aging process of the ovary is primarily driven by the accumulation of fibrotic tissue in the interstitial and extracellular spaces of the ovarian cortex. These changes are influenced by environmental factors and are further exacerbated by repeated ovulatory cycles [6]. As a result, by the age of 35, the ovary may develop approximately 450 physiological scars, preventing the activation of follicles from the reserve pool, as described by Tilly et al. (2012), and the oogonial nests identified by Pepling and Telfer [7,8].

A fibrotic ovarian stroma is unable to receive essential nutrients and interferon signals, impairing its ability to reactivate neofolliculogenesis (Fig. 1).

Fig. 1.

a) A young ovary is evident before the age of 35 with excellent vascularization, large number of antral follicles, a fluid stroma with few collagen fibers but rich in hyaluronic acid and growth factors; multiple oogonia nests (non-exhaustible set of follicles discovered by Johnson et al., in 2008). B) after age 37, We can observe a fibrotic stroma, with little hyaluronic acid and few blood vessels. As a result of the more than 400 scars for each ovulation, we observe the nests of Oogonia in the ovarian cortex trapped in a collagen network and unable to activate due to lack of Growth factors to activate FI3K receptors and initiate the entire follicular development cascade [9].

The ovarian microenvironment and recent studies on the ovarian proteome by Eva de Clercq et al. reveal that at birth, a female infant's ovary contains more than 8800 long-lived proteins, making its protein composition the richest in the human body—even surpassing that of the brain. However, from birth to adulthood (around age 25), the number of long-lived proteins declines drastically, leaving only approximately 383, with an almost total loss of 56 key proteins [10].

Among the lost proteins are those dependent on kinase activity, including PI3K/AKT, mTOR, AMPK, and sirtuins (SIRT), as well as 15 Bone Morphogenetic Factors, all of which play vital roles in cellular energy regulation, metabolic signaling pathways, and aging. Additionally, there is a reported decrease in CD63 and CD81 proteins, along with heat shock proteins Hsp8 and Hsp90, which are essential for meiosis activation, cell cycle maintenance, and the prevention of accelerated apoptosis [11].

One of the most crucial biochemical aspects of aging is undoubtedly the expression of mitochondrial sirtuins, particularly SIRT3, SIRT4, and SIRT5 (Fig. 2). These proteins belong to a family with versatile functions, including metabolic control, epigenetic modifications, and lifespan extension. They are essential for ATP-to-ADP conversion, mitochondrial activation, cell division, and hyaluronate synthesis, all of which are vital for folliculogenesis [11].

Fig. 2.

Mitochondrial regulation in young and aged ovaries: Requires coordination of signaling networks, including the PI3K/AKT pathway, mTOR, AMPK, and sirtuins, in response to environmental or intracellular fluctuations. In the young ovary there are sufficient nutrients and energy resources that, under hormonal effects and adequate vascularization conditions, activate anabolic regulation pathways; releasing growth factors with high affinity for the Protein Kinase receptors (RTK) of the follicular cells, which allows the phosphorylation of PI3K, with the activation of the mTORC1 and mTORC2 factors that stimulate the intracellular synthesis of lipids and proteins. Furthermore, Sirtuins (SIRT3 and SIRT4-SIRT5) are activated within the mitochondria, which regulate energy production by activating the Krebs Cycle and the metabolism of Pyruvate from glucose [12].

One of the most critical biochemical aspects of aging is the expression of mitochondrial sirtuins, particularly SIRT3, SIRT4, and SIRT5 (Fig. 2). These proteins belong to a versatile family with key functions, including metabolic regulation, epigenetic modification, and lifespan extension. They play a crucial role in ATP-to-ADP conversion, mitochondrial activation, cell division, and hyaluronate acid synthesis, all of which are essential for folliculogenesis [11].

The normal proteostasis of oocytes and ovaries requires extreme protein longevity and the harmonious function of all cellular systems. The ovary is the most important endocrine organ in women, as more than 95 % of female sex hormones are produced in its cortex [13]. It serves as a key determinant of female health and longevity, protecting against disease [7].

Moreover, the ovary is responsible for generating over 95 % of the female sex hormones, which regulate numerous endocrine functions. Its dysfunction has been linked to conditions such as osteopenia, sarcopenia, metabolic disorders, cardiovascular diseases, as well as degenerative and cognitive impairments [14].

Physiological conditions of the ovary can be improved through biorregenerative techniques that help preserve reproductive and hormonal functions, which are otherwise lost due to aging and the physiological fibrosis that accompanies each ovulation [15].

In the follicular fluid of young women, approximately 37 miRNAs have been identified that are not present in peripheral blood but are exclusive to follicular fluid. Among these, 32 are encoded in CD63 and CD81 proteins, which are involved in protein migration and synthesis, membrane integrity, and signal transduction. In contrast, in the follicular fluid of older women, only about nine miRNAs have been detected, with significantly fewer GP1-anchored proteins and an almost complete loss of CD63 and CD81 [16].

These proteins and miRNAs could potentially be restored through molecular mechanisms aimed at increasing the longevity of long-lived proteins and enhancing proteostasis. This could be achieved through the use of molecular chaperones and cellular antioxidants, ultimately supporting the maintenance of the female germline over extended periods [17].

It is now known that a pool of dormant, non-depletable follicles exists within the ovarian cortex, capable of reactivating oogenesis even after birth and throughout female aging [18]. However, their activation is hindered by ovarian fibrosis, which accumulates with each ovulation, as well as by chronological and environmental aging. These factors prevent the ovarian stroma from receiving the essential substances and growth factors required for their biological activation [19,20].

It is now known that platelet-derived growth factors provide approximately 10,000 essential factors for the proper functioning of biological systems [21,22]. However, platelet-derived exosomes contain around 40,000 factors, including various types of microRNAs, structural and migratory proteins such as CD63 and CD81, chaperones, chaperonins, lysines, and enzymatic stimulators of kinases and mitochondrial sirtuins. These exosomes act on target organs as multipotent stem cell-like entities due to their origin from megakaryocytes. They play a crucial role in tissue repair and functional regeneration by either stimulating or supplying the necessary factors, depending on the specific biological context [23].

Finally, mechanical signals play a crucial role in regulating cell proliferation and apoptosis to maintain optimal organ size. It is increasingly evident that most ovarian follicles are physiologically restricted in their growth due to local Hippo signaling [24].

Therefore, we know that by performing transvaginal ovarian cortex puncture and applying 0.9 % saline solution as another alternative for ovarian biostimulation, it is possible to interrupt Hippo signaling and enhance AKT signaling, which could somehow reactivate ovarian function secondary to the stimulation of a local inflammatory process that attracts blood vessels and nutrients necessary for the stimulation of follicleogenesis. This, in turn, leads to an increase in growth factors, a reduction in apoptosis, and greater stimulation of the growth of dormant primordial follicles [25,26].

Based on all the previously analyzed studies, we ask the following questions: Can autologous exosomes reactivate oogenesis in patients with low ovarian reserve or premature ovarian aging? Is it possible that exosomes provide the biochemical mediators necessary to improve the physiological and mechanical conditions of the ovary and reactivate ovulation?

This study seeks to evaluate the ability of autologous exosomes obtained from patients' own platelets to biostimulate the ovary by providing proteins, microRNAs, enzymes, and growth factors necessary to restore ovarian function lost over time. This study seeks to reinforce the theory that ovarian aging is not due to a decrease in oocytes, but rather to damage to the ovarian stroma, as described by Tilly and Johnson in 2006 [27].

2. Methodology and development of the study

An observational, prospective, randomized, comparative, and analytical study was conducted at a fertility clinic in Caracas, Venezuela, between January 2024 and September 2024. The study consisted of an ovarian biostimulation or bioregeneration procedure performed on a total of 30 patients between 38 and 46 years of age with low ovarian reserve who wished to conceive with their own eggs and declined egg donation.

The study was reviewed and approved by the Scientific and Ethics Review Committee of the Center for Women and Fertility Care, and submitted to and approved by the General Directorate of Research and Teaching of the Ministry of People's Power for Health and Research in Caracas, Venezuela. The study followed all ethical principles of research and adhered to the Declaration of Helsinki for research involving human subjects.

The procedure consisted of assessing the parameters of diminished ovarian reserve prior to the start of the study, as determined by: hormone levels on day three of the cycle: FSH (follicle-stimulating hormone) > 12 mIU/ml, estradiol <35 pg/ml, and anti-Müllerian hormone <0.7 ng/ml, in addition to a total antral follicle count <5 in both ovaries, confirming low ovarian reserve or resistance, and rejection of egg donation.

A total of 52 patients between the ages of 38 and 46 were enrolled in the study, 9 of whom expressed their desire not to undergo Biostimulation, 3 had thrombophilia or some alteration in the platelet count, 6 were obese with a BMI> 35 % and 4 had only one avarice, so only 30 patients met the inclusion criteria to belong to the study (Fig. 3). The 30 patients were distributed into three groups of 10 patients each, the first group for Ovarian Biostimulation with autologous Exosomes, the second for Ovarian Biostimulation with PRP and the third for Biostimulation with 0.9 % saline solution. All participants had similar ethnic, biological and geographical characteristics. The patients were assigned to each group (1, 2 or 3) using an Excel-type random group generator and the base group was generated by their personal identification number (Table 1).

Fig. 3.

Distribution of the groups.

Table 1.

Patient randomization table.

Patients	Patient coding in the study based on the Identification Number	Distribution of patients according to simple randomization by identity document generated by Excel.	Distribution of patients by group
1	EX001	EX019	GRUPO 3
2	EX002	EX007	GRUPO 2
3	EX003	EX029	GRUPO 2
4	EX004	EX022	GRUPO 1
5	EX005	EX018	GRUPO 1
6	EX006	EX001	GRUPO 2
7	EX007	EX016	GRUPO 3
8	EX008	EX004	GRUPO 2
9	EX009	EX012	GRUPO 2
10	EX010	EX023	GRUPO 1
11	EX011	EX014	GRUPO 3
12	EX012	EX026	GRUPO 1
13	EX013	EX003	GRUPO 3
14	EX014	EX008	GRUPO 2
15	EX015	EX005	GRUPO 3
16	EX016	EX006	GRUPO 1
17	EX017	EX010	GRUPO 1
18	EX018	EX009	GRUPO 2
19	EX019	EX011	GRUPO 3
20	EX020	EX021	GRUPO 3
21	EX021	EX025	GRUPO 3
22	EX022	EX028	GRUPO 1
23	EX023	EX015	GRUPO 2
24	EX024	EX020	GRUPO 3
25	EX025	EX002	GRUPO 1
26	EX026	EX027	GRUPO 3
27	EX027	EX024	GRUPO 2
28	EX028	EX017	GRUPO 1
29	EX029	EX030	GRUPO 2
30	EX030	EX013	GRUPO 1

Source: data provided by researchers.

The groups were as follows.

• Group I: Patients who would receive transvaginal ovarian biostimulation with autologous exosomes obtained from the patients' platelets.

• Group II: Patients who would receive transvaginal ovarian biostimulation with autologous activated platelet growth factors.

• Group III: Patients who would receive ovarian biostimulation with 0.9 % saline solution (Table 2)

Table 2.

Population studied (30 patients) distribution by groups and age.

GROUP I AUTOLOGOUS EXOSOMES
PATIENTS	AGES	Fertilized embryos	Positive pregnancy test	Abortions	Frozen embryos on day III	Frozen Embrio on day V	Pregnancy > be 12 weeks
EX022	38	4	1			2	1
EX010	38	3
EX018	39	3			1
EX028	39	3	1				1
EX026	40	3
EX006	40	5	1	1	2
EX023	41	5	1		1		1
EX002	45	4	1				1
EX017	46	2	1	1
EX013	46	1
Total		33	6	2	4	2	4
Average age group 1	41,89	Stándar deviation G1	3,22
GROUP II PRP
PATIENTS	AGES	Fertilized embryo	Positive pregnancy test	Abortions	Frozen embryo day III	Frozen Embrio day V	Pregnancy > be 12 weeks
EX001	38	3	1	1
EX009	38	2
EX008	39	4	1		1		1
EX015	39	2
EX030	39	1
EX024	40	3	1	1	1
EX012	41	1
EX004	42	2
EX029	43	3	1				1
EX007	46	1
Total		22	4	2	2	0	2
Average age group 2	42,35	Stándar deviation G2	2,54
GROUP III PHYSIOLOGICAL SOLUTION
PATIENS	AGES	Fertilized embryo	Positive pregnancy test	Abortions	Frozen embryo day III	Frozen Embrio day V	Pregnancy > be 12 Weeks
EX005	38	4	1	1
EX003	39	1
EX021	39	2
EX025	40	0
EX016	42	0
EX014	42	0
EX020	43	1
EX019	44	0
EX011	46	0
EX027	46	0
Total		8	1	1	0	0	0
Average age group 3	44.98	Stándar deviation G3	2,84
Average age population n = 30	42	Sample Stándar D n = 30	2857	Population Standard D n = 30	2809

Source: data provided by researchers.

All patients underwent a complete clinical evaluation, and admission to the study was defined according to the following inclusion and exclusion criteria:

Inclusion criteria.

• −Female patients between 38 and 46 years of age.
• −Patients with a desire to become a mother
• −Patients with Body Mass Index (BMI) between 23 and 30.
• −Patients with proven ovarian insufficiency, according to the parameters mentioned above.
• −Patients who refuse egg donation.
• −Patients with full willingness to participate in the study and who have understood and signed the informed consent.
• −Nulliparous patients without a live baby at home
• −Patients with both ovaries without oncological pathologies, active endometriomas or suspicious ovarian or other tumors.
• −Patients with platelet count over 250 thousand.
• −Patients with no history of hematological diseases.
• −Patients without treatment with anticoagulants.
• −Patients with no active infection at the time.

Exclusion criteria.

• −Female patients outside the age range of 38–46 years old.
• −Patients who are not willing to become mothers.
• −Patients with a BMI outside the range of 23–30
• −Patients without ovarian insufficiency
• −Patients who do not wish to participate in the procedure and have not signed the informed consent form.
• −Primiparous or multiparous patients
• −Patients with suspected or diagnosed oncological diseases.
• −Patients with platelet count of less than 250 thousand
• −Patients with a history of hematological diseases.
• −Patients with anticoagulant treatment.
• −Patients with any type of active infection.

After the initial evaluation, informed consent was obtained, the relevant paraclinical studies were performed, and the patients were scheduled for the procedure according to randomization.

The protocol consisted of transvaginal intraovarian administration of 2 cc of autologous exosomes, autologous platelet growth factors, or saline solution into each ovary, depending on the study group to which the patient was previously assigned. Administration was performed in all patients in the early follicular phase on day 7 or 8 of the cycle, once a month for four consecutive months, and the assisted fertility procedure was performed in the fifth month.

Before starting the first biostimulation, peripheral blood samples were taken from each patient on the third day of the cycle to determine: FSH, LH, estradiol, anti-Müllerian hormone, and antral follicle count for each ovary. These same tests were performed after the fourth biostimulation and before starting the fertility procedure. All blood samples taken before and after the procedure were frozen and sent simultaneously to a clinical laboratory in Caracas for analysis to avoid bias in the results.

All patients were instructed 5 days before and 5 days after each procedure to follow a diet free of dairy products and processed carbohydrates and were recommended to include unflavored gelatin (2 times a day), in order to obtain better quality platelets[[28], [29], [30]].

The procedure was performed in the operating room of our fertility clinic with the patient under controlled sedation. Peripheral blood was drawn from all patients in the three study groups using a PRP kit (4 tubes with separator gel per patient) for each biostimulation session. A total of 120 kits were used for 30 patients, who underwent biostimulation for four consecutive cycles.

Once the blood was drawn from the internal cubital vein, applying gentle compression with elastic tape (never a tourniquet) and using the Vacutainer system, it was centrifuged at 270 g for 10 min.

All procedures were performed under controlled sedation.

In the PRP group (Group 1), the injection was administered transvaginally using a follicular aspiration needle. All biostimulation procedures were performed during the early follicular phase (days 7 or 8) depending on the technique assigned to each group.

For the Autologous Exosome group (Group 2), the same blood collection procedure was performed using the selected PRP kit. In this case, the entire PRP sample was transferred into two 20 cc syringes and filtered using the EXOSMAT kit for platelet-derived autologous exosomes. This process yielded a total of 5–6 cc of platelet-derived exosome-rich fluid, with a concentration of 5–6 trillion exosomes per milliliter. A volume of 2 cc was then injected into each ovary via the transvaginal route [31].

Finally, the control group (Group 3) received 2 cc of 0.9 % BEHRENS physiological saline solution in each ovarian cortex, following the same four-cycle protocol as the other two groups.

All procedures were performed under controlled surgical sedation using a transvaginal follicular aspiration needle, guided by transvaginal ultrasound imaging.

The results of the procedures and all the variables generated after the paraclinical tests were processed for analysis using descriptive statistical techniques, through the use of the IBM - SPSS Computerized Statistical Program, last version, to describe the variables of this research. Inferential statistical techniques were also used: Kolmogorov - Smirnov normality test, Student T in related samples, Analysis of Variance of a Factor (ANOVA) and Tukey's Post Hoc Follow-up Test with a value of p < 0.05 considered as statistically significant.

3. Results

This study was conducted in a total of 30 patients, with an average age of 41.20 ± 2.86 years, for a minimum age of 38 and a maximum age of 46 years (53.3 % ≤ 40 years and 46.7 % > 40 years), willing to become mothers, with previous diagnosis of ovarian resistance or insufficiency and who also refuse egg donation.

These patients with the characteristics described above, were divided, under statistical and methodological criteria, into three research groups, to receive autologous therapy with PRP, another with Autologous Exosomes and another with physiological solution, in order to separately evaluate the efficacy of these treatments in the regeneration of ovarian folliculogenesis.

In order to establish initial group equivalence, an intergroup analysis was performed before ovarian biostimulation, by means of ANOVA (one-factor analysis of variance) (Table 3).

Table 3.

Comparison of FSH, Estradiol, AMH and Follicular Count in each of the groups before ovarian biostimulation.

VARIABLES	OVARIAN BIOSTIMULATION
	COMPLETE SAMPLE
SAMPLE	30
AVERAGE AGE	41.20 ± 2.86
	AUTOLOGOUS EXOSOMES	PRP	PHYSIOLOGICAL SOLUTION	F Value	P Value	Statistical significance
FSH	22.80 ± 9.82	25.40 ± 13.48	23.80 ± 12.13	0.121	0.886	P > 0.05
ESTRADIOL	38.10 ± 12.48	42.10 ± 13.30	48.10 ± 15.41	1.332	0.281	P > 0.05
AMH	0.38 ± 0.21	0.56 ± 0.25	0.50 ± 0.38	0.950	0.399	P > 0.05
FOLLICULAR COUNT	2.90 ± 0.74	4.40 ± 1.50	3.50 ± 1.27	2.867	0.063	P > 0.05

Source: data provided by researchers

At the end of the study a total of 120 biostimulations were conducted, 4 to each patient, the average age of the patients participating in the study was 41.20 years and all had in common the previous diagnosis of resistance or ovarian insufficiency, none wanted ovodonation so they agreed to join the ovarian biostimulation program to try to rescue the ovaries from aging and reactivate their biological functions, including folliculogenesis.

When evaluating the first parameters of the study referring to fertility hormones and antral follicle count before and after the intervention, significant statistical improvements were observed in the Exosomes and PRP groups, but not in the physiological solution group, as shown in Table 4, Graph 1, Graph 2, Graph 3.

Table 4.

FSH, Estradiol, AMH and Follicular Count results in each of the groups, before and after ovarian biostimulation.

GROUPS	OVARIAN BIOSTIMULATION
	FSH		ESTRADIOL		AMH		FOLLICULAR COUNT
	BEFORE	AFTER	BEFORE	AFTER	BEFORE	AFTER	BEFORE	AFTER
AUTOLOGOUS EXOSOMES	22.80 ± 9.82	7.05 ± 1.70	38.10 ± 12.48	76.20 ± 11.78	0.38 ± 0.21	1.50 ± 0.35	2.90 ± 0.74	6.10 ± 1.52
Student t	5.123		7.272		5.494		7.686
P value	0.001		0.000		0.000		0.000
Statistical significance	P < 0.05		P < 0.05		P < 0.05		P < 0.05
PRP	25.40 ± 13.48	9.38 ± 1.84	42.10 ± 13.30	76.00 ± 10.22	0.56 ± 0.25	1.13 ± 0.22	4.40 ± 1.50	7.70 ± 2.11
Student t	4.154		5.618		10.421		9.000
P value	0.001		0.000		0.000		0.000
Statistical significance	P < 0.05		P < 0.05		P < 0.05		P < 0.05
PHYSIOLOGICAL SOLUTION	23.80 ± 2.13	21.60 ± 11.25	48.10 ± 15.41	49.20 ± 18.09	0.50 ± 0.38	0.55 ± 0.40	3.50 ± 1.27	4.00 ± 1.70
Student t	1.377		0.646		2.186		2.236
P value	0.202		0.634		0.057		0.052
Statistical significance	P > 0.05		P > 0.05		P > 0.05		P > 0.05

Source: data provided by researchers

Graph 1.

Average comparison for FSH, Estradiol, AMH and antral follicle count before and after biostimulation in the Exosomes group.

Graph 2.

Average Comparison for FSH, Estradiol, AMH and antral follicle count, before and after Biostimulation, in the PRP group.

Graph 3.

Average comparison for FSH, Estradiol, AMH and antral follicle count before and after biostimulation in the physiological solution group.

The evaluation of the parameters of the second control was conducted, including: antral follicle count, collected oocytes, immature oocytes or in Metaphase I and mature oocytes or Metaphase II, fertilization rate, haploid and triploid embryos, and those with fragmentation greater than 35 %, number of clinical pregnancies, miscarriages and frozen embryos (Table 5).

Table 5.

Oocytes collected, metaphase I and II, III and V embryos, transferred and discarded embryos, positive pregnancy test, miscarriage, pregnancies ≥12 weeks, and frozen embryos (day III and V), after ovarian biostimulation in the different research groups.

VARIABLES	OVARIAN BIOSTIMULATION			REMARKS
	AUTOLOGOUS EXOSOMES	PLATELET-DERIVED GROWTH FACTOR	PHYSIOLOGICAL SOLUTION
COLLECTED OOCYTES	56	43	22	> No. WITH AUTOLOGOUS EXOSOMES
METAPHASE I	12	18	12	> No. WITH PRP
METAPHASE II	44	25	10	> No. WITH AUTOLOGOUS EXOSOMES
FERTILIZED EMBRYOS	33	22	8	> No. WITH AUTOLOGOUS EXOSOMES
DISCARDED (HAPLOID)	2	1	1	> No. WITH AUTOLOGOUS EXOSOMES
DISCARDED (TRIPLOIDS)	1	2	2	SAME No. WITH PRP and PHYSIOLOGICAL SOLUTION
DISCARDED (FRAGMENTATION >35 %)	2	3	2	> No. WITH PRP
DAY III EMBRYOS	17	13	3	> No. WITH AUTOLOGOUS EXOSOMES
DAY V EMBRYOS	5	1	0	> No. WITH AUTOLOGOUS EXOSOMES
TRANSFERRED EMBRYOS	22	18	3	> No. WITH AUTOLOGOUS EXOSOMES
PREGNANCY TEST (+)	6	4	1	> No. WITH AUTOLOGOUS EXOSOMES
MISCARRIAGES	2	2	1	SAME No. WITH PRP and AUTOLOGOUS EXOSOMES
PREGNANCY ≥12 WEEKS	4	2	0	> No. WITH AUTOLOGOUS EXOSOMES
FROZEN EMBRYOS	6	2	0	> No. WITH AUTOLOGOUS EXOSOMES
DAY III FROZEN	4	2	0	> No. WITH AUTOLOGOUS EXOSOMES
DAY V FROZEN	2	0	0	> No. WITH AUTOLOGOUS EXOSOMES

Source: data provided by researchers

According to the results obtained, the autologous exosomes group had the highest number of oocytes collected after ovarian stimulation (56), followed by the PRP group with 43 and the physiological solution group with the lowest number with only 22 oocytes collected (p < 0.05). Regarding the oocytes collected in metaphase I, the PRP group exhibited the highest number (18), followed by autologous exosomes (12) and the same number with physiological solution (p > 0.05). In metaphase II, the autologous exosomes group performed better (44); the PRP group had 25 and the physiological solution group 10 (p < 0.05).

It must be noted that a total of 63 embryos were fertilized, distributed as follows: 33 from the autologous exosomes group; 22 from the PRP group and 8 from the physiological solution group. It should be noted that in the autologous exosomes group 5 embryos were discarded (2 haploid; 1 triploid and 2 with fragmentation >35 %). In the PRP group, 6 embryos were discarded (1 haploid; 2 triploid and 3 with fragmentation >35 %) and in the physiological solution group 5 embryos were discarded (1 haploid; 2 triploid and 2 with fragmentation >35 %), Graph 4.

Graph 4.

Oocytes collected, metaphase I and II, fertilized and discarded embryos after ovarian biostimulation in the different research groups.

Regarding day III embryos, 17 were counted with autologous exosomes; 13 with PRP and 3 with physiological solution. Regarding embryos on day V, the results were as follows: 5 (autologous exosomes); 1 (PRP) and 0 with physiological solution (p > 0.05). In relation to the embryos transferred, it was observed that 22 embryos were transferred in the autologous exosomes group; 22 embryos were transferred in the PRP group, Graph 5.

Graph 5.

Day III and V embryos and embryos transferred after ovarian biostimulation in the different research groups.

Of this number of transferred embryos, the following resulted in positive pregnancy tests: 6 with autologous exosomes; 4 with PRP and 1 with physiological solution. It is worth mentioning that in the autologous exosomes group, 2 miscarriages occurred; also 2 with PRP and 1 with physiological solution. On ultrasound follow-up, 4 pregnancies ≥12 weeks were recorded in the autologous exosomes group; 2 with PRP and none with physiological solution. n 18 and in the physiological solution group 3 embryos were transferred, Graph 6.

Graph 6.

Positive pregnancy test, abortions and pregnancies ≥12 weeks after ovarian biostimulation in the different research groups.

Regarding the number of frozen embryos, the results were as follows: 6 embryos were frozen in the autologous exosomes group, 4 of which were from day III and 2 were from day V; 2 embryos were frozen in the PRP group (both day III) and none in the physiological solution group, Graph 7.

Graph 7.

Frozen embryos (day III and V) after ovarian biostimulation in the different research groups.

Regarding the fertility rate and embryos not suitable for transfer, the following information was recorded (Table 6).

Table 6.

Rates of fertilization and rate of embryos not suitable for transfer after ovarian biostimulation in the different research groups.

RATES	OVARIAN BIOSTIMULATION
	AUTOLOGOUS EXOSOMES	PLATELET-DERIVED GROWTH FACTOR	PHYSIOLOGICAL SOLUTION
RATE OF FERTILIZATION	58.93 %	51.16 %	36.36 %
RATE OF EMBRYOS NOT SUITABLE FOR TRANSFER	15.15 %	27.27 %	62.50 %

Source: data provided by researchers

The fertilization rate in the autologous exosomes group was 58.93 %; in the RPR group it was slightly lower with 51.16 % and in the physiological solution group it was only 36.36 % (Graph 8).

Graph 8.

Rates of fertilization, following ovarian biostimulation in the different research groups.

Regarding the rate of embryos not suitable for transfer, the physiological solution group exhibited the highest rate (62.50 %), followed by the PRP group (27.27 %) and in a lower percentage the autologous exosomes group (15.15 %), Graph 9.

Graph 9.

Rates of embryos not suitable for transfer, following ovarian biostimulation in the different research groups.

4. Discussion

It is possible to rescue the ovarian microenvironment from aging if we provide the necessary elements from the molecular and biochemical point of view to regenerate the stromal matrix, providing nutrients, proteins and reactivating cellular communication so that the cells activate their defense mechanisms against oxidation and the biological processes are resumed to give rise to the development of follicles present in the ovarian cortex, which is still aged but requires perfect ovarian microenvironment conditions.

In short, the data processed in this research allow us to point out that in the autologous exosomes group there were better results: greater number of oocytes collected in metaphase II, greater number of embryos fertilized and transferred, greater number of positive pregnancy tests and greater number of frozen embryos.

These results allow us to state with a broad statistical criterion that ovarian biostimulation with autologous exosomes, considered as multipotent autologous stem cell therapy, are effective and safe to improve the markers of low ovarian reserve. This is because they improve ovarian hormone production, improve intra-ovarian fibrosis because the nutrients and biochemical signals present in the lipid vesicles (Exosomes) have the desired effect allowing to reactivate the follicular function and stimulate the follicles of the oogonium nests (pool of dormant follicles) so that they wake up and reactivate their development to follicles capable of giving quality oocytes in women with diminished ovarian reserve. This technique represents a safe and very promising alternative to rescue the ovary from aging and reactivate fertility.

However, it should be considered that this experience is only a pilot study, so other case control studies, with a larger number of patients, different age groups and communities are required. Ovarian biostimulation with autologous exosomes can be considered as a safe therapy to improve the markers of low ovarian reserve by providing miRNA, improve interstitial fibrosis, stimulate protein and hyaluronic acid synthesis that reactivates follicular function and stimulate the oogonium nests (pool of dormant follicles) to reactivate fertility. This will help protect the ovary from aging and allow many women the opportunity to become mothers with their own eggs.

5. Conclusion

Ovarian biostimulation with autologous exosomes could be considered a safe therapy to improve markers of low ovarian reserve; however, many more studies must be conducted with larger groups of patients to obtain a more accurate conclusion. We know that exosomes provide a number of growth factors, proteins, and microRNAs that could contribute to improving the ovarian stromal microenvironment to reactivate biological functions lost over time, due to environmental factors, and lifestyle. However, it would be very interesting to better understand the immunohistochemical behavior of the ovaries before and after these therapies, so we remain open to further studies.

Disclosure statement

The authors declare that there are no financial, personal, academic, or professional conflicts of interest that could have influenced the preparation of this manuscript.

Furthermore, we confirm that no financial support has been received from any institution, organization, or individual that could be perceived as influencing the results or interpretations presented in this article.

Attestation statement

It is attached.

Data Sharing Statement

It is attached (For reports of clinical trials, authors are required to provide a Data Sharing Statement to indicate if data will be shared or not with other researchers for further analysis. This information will be published in a Data Sharing Statement. Please see Instructions for Authors for data sharing details).

Capsule

Ovarian biostimulation with autologous exosomes improves ovarian reserve by reactivating follicular function and stimulating fertility in women with diminished reserve, safely and promisingly.

Funding statement

This study is funded with our own resources.

Declaration of competing interest

The authors declare no financial, personal, academic, or professional conflicts of interest that could have influenced the preparation of this manuscript.

Furthermore, we confirm that no financial support has been received from any institution, organization, or individual that could be perceived as influencing the results or interpretations presented in this article.