Journal of Stem Cells Research Development & Therapy Category: Medical Type: Research Article

Clinical Approach of Novel Autologous Stem Cell Therapy for Primary Ovarian Insufficiency and Endometrial Regeneration

Manoj Bansode1, Shital A Kore2, Younghyun Lee3,4,  Vaman Ghodake2* and Rajneesh Verma3

1 CliniAid Clinical Research Organisation (C.R.O.) Pvt Ltd, Satara, Maharshtra-415002, India
2 IVF Lab, Dr. Ghodake Advanced Laparoscopy Surgery And Test Tube Baby Centre, Sangli, Maharshtra-416416, India
3 VG Biomed Clinic, Polaris Building, Level 6, Sukhumvit 20, Bangkok, Thailand
4 Laboratory Of Reproductive Biotechnology, Building 454, Rm 343, Gyeongsang National University, 501 Jinjudae-ro, Jinju, 52828, South Korea

*Corresponding Author(s):
Vaman Ghodake
IVF Lab, Dr. Ghodake Advanced Laparoscopy Surgery And Test Tube Baby Centre, Sangli, Maharshtra-416416, India
Email:drvamanghodake@gmail.com

Received Date: Nov 14, 2022
Accepted Date: Nov 24, 2022
Published Date: Nov 30, 2022

Abstract

Bone Marrow-Derived Mesenchymal Stem Cell Fraction (BMSCF) therapy has various applications as potential use in regenerative medicine. Here, we present an original case of Primary ovarian insufficiency associated with poor ovarian reserves and thin endometrium in a woman who achieved a live birth with BMSCF supplemented with PRP (platelet rich plasma). The age between 24 to 42-year-old woman (n=10) came to our centre with primary infertility. Her husband's sperm count and quality was normal. The patient was advised for fertility enhancing surgery and advised stem cell therapy for poor ovarian reserve and thin endometrium. Several attempts of IVF with Hormone Replacement Therapy (HRT) were made, but the desired thickness of the endometrium was not achieved. Intra ovarian and sub endometrial injection of BMSCF was given, followed by HRT for three months, which resulted in improved endometrium and ovary function. The treatment resulted in the conception and delivery of a 2.9 kg baby through lower segment caesarean section. Recently, there has been substantial interest in stem cells as a novel regenerative therapy for the regeneration of human endometrium disorders and poor ovarian reserves.

Introduction

Researchers have characterized stem cells and evaluated their therapeutic effects in various animal models of degenerative diseases [1]. Based on the considerable data on the feasibility of stem cells for various degenerative diseases, clinical trials are ongoing to develop new stem cell-based treatment strategies in regenerative medicine. However, there are few reports on the effects of stem cells in reproductive diseases [2]. In reproductive medicine, ovarian function is important in the health of both young and old women. Stem cell therapy has become a promising, particularly for indications not possible to treat medically or surgically to improve the condition. Stem cells have the ability to self-renew and differentiate into specific lineage [3]. MSCs can be isolated from various sources, including bone marrow, placenta, dental pulp [4-6]. The advantages of autologous MSCs are that they do not raise any ethical concerns; they have no immunogenicity and immunomodulatory function. Premature Ovarian Insufficiency (POI) is a clinical syndrome defined by loss of ovarian activity before the age of 40 years. POI is characterized by menstrual disturbance with raised gonadotropins, low Anti-Mullerian Hormone (AMH), and estradiol level [7]. Autologous BMSCF s was tried in POI to increase the follicular recruitment and avoiding the need for oocyte donation program and regeneration of endometrium. This study analyses the role of autologous BMSCF in POI and thin endometrium. In the absence of stem cell therapy, accepting oocyte donor program or adoption would have been the only viable options for this patient for which she was not ready. In particular, POI defines a state of female hypogonadism which causes a loss of ovarian function before the age of 40 years. POI can be caused by side effects of anticancer chemotherapy or by surgical means [8]. This condition can be diagnosed based on a decrease in the number of follicles, abnormalities in the menstrual cycle, and infertility. There are several preclinical studies of stem cell therapy to treat ovarian dysfunction in several animal models and endometrium regeneration [9,10].

Methodology

Ten patients, who had a history of cancelled cycles due to inadequate endometrial growth (less than 7 mm) in the past FET cycles despite standard treatments and POI, were recruited into the study performed in the IVF centre, in 2021. After approval from the Institutional Ethics Committee, 10 patients  was selected  after written informed consent after explaining the pros and cons of this procedure were included in the study. This prospective interventional study was designed for intraovarian instillation and endometrium infusion of BMSCFs along with PRP in 10 patients who are poor responders for fertility. 

Inclusion criteria were as follows:

Age group of 20-45 years

  1. Poor responder (the expected poor responder group where AFC
  2. Normal karyotype
  3. Normal semen parameters 

Exclusion criteria were as follows: 

  1. Abnormal karyotyping
  2. Autoimmune diseases
  3. POD due to chemotherapy or radiotherapy
  4. Active viral infections 

The AFC by transvaginal ultrasound and serum AMH levels were considered markers of ovarian response. Both were recorded in all patients under basal conditions before and 5 weeks after the procedure. 

Bone marrow aspiration 

 Age between 24-45-year-old poor ovarian failures, poor ovarian reserve, thin endometrium female, came to our fertility clinic. Their AMH level was low 0.4 ng/mL and AFC ≤ 2. On ultrasonography, ovaries were unremarkable with antral follicle count of one. It was thought to use autologous BMSCF for the rejuvenation of functioning ovarian tissue and optimizing the success rate of achieving pregnancy through assisted reproduction. Patients with POD, undergo diagnosis and screening confirming diagnosis including history and physical exams, labs and diagnostic procedures. Following final approval and under anaesthesia, bone marrow aspiration with separation of the Bone Marrow Derived Stem Cell Fraction (BMSCF) will be performed. BMSCF harvested from the anterior superior iliac crest region. Bone marrow aspiration procedure (BMAP) was carried out by local anaesthesia with light sedation monitored by an anesthesiologist for access to the posterior superior iliac crest. 

Preparation of Autologous Bone marrow-derived mesenchymal stem cell fraction 

Bone marrow aspiration was done from the posterior superior iliac spine under local anaesthesia using the Jamshidi needle (13G) and 20-ml syringe prewashed with heparin maintaining strict asepsis. Prewashed the syringe avoids clot and coagulation, which can diminish the ultimate yield from the aspiration. Approximately 80-100 mL of bone marrow is aspirated. BMSCF were isolated by using automated closed-circuit centrifugation unit .By this method, we got 20 mL of stem cell concentrate. The stem cell mixed with PRP for the entire patient for transfusion. BMSCF were counted using a Neubauer chamber and cell viability was determined by counting the blue (dead) and transparent (alive) cells by trypan blue (Sigma) staining. 

BMSCF infusion

Considering the small size of ovaries, we preferred laparoscopic instillation of BMSCF in ovaries. Ovaries were held at the cranial and caudal position with forceps and intra-ovarian instillation of about 1–2 ml of BMSCF at 3-4 sites performed bilaterally and sub endometrial 1-2 ml stem cells injected for endometrial regeneration (Figures 1&2).

 Figure 1: Ovary before BMSCF injection. Ovary after BMSCF injection.

 Figure 2: A) Thin endometrium, B) Sub-endometrial PRP+BMSCF Injection.

Results And Discussion

The bone marrow is promising sources of MSCs, and although its aspiration is invasive technique for the MSC isolation, it is the most evaluated approach for cell therapy. MSC differentiation potential and the viability of the bone marrow MSCs decrease with age. PRP is defined as a plasma fraction of autologous blood. It has been shown to improve tissue regeneration with the expression of cytokines and growth factors [11]. A few studies have evaluated the role of intrauterine instillation of PRP in endometrium regeneration [12-14]. It has been shown to improve embryo transfer and vascularity through releasing cytokines and growth factors, containing Vascular Endothelial Growth Factor (VEGF), transforming growth factor, platelet-derived growth factor, and epidermal growth factor [15]. These factors regulate cell proliferation and differentiation, and promote extracellular matrix accumulation. 

After BMSCs transplantation in 45-year-old female to gives successful birth to a healthy baby. There are reports of pregnancy after intraovarian stem cell instillation, the mean duration for which their effect remains is still not fully understood. In our cases, the patient underwent cycle of IVF after stem cell therapy. The sonographic findings were suggestive of better ovarian volume, blood flow and antral follicle count. The patients were counselled to keep on trying naturally with the hope that the effect of stem cell therapy will remain in the subsequent cycles. The patients were followed up for 6-8 months with ultrasonography and conceived spontaneously in the subsequent years. But this therapy comes with some limitations such as genetic and karyotype abnormalities. The present report described 10 IVF cycles in patients who underwent BMSCF therapy with PRP. For successful implantation, the endometrium needs to regenerate and proliferate properly. In this complex process, different type of cell such as endothelial, epithelial, and stem cells and fibroblasts are involved. Growth factors, pro-inflammatory cytokines, and local chemokines also have important role in tissue regeneration. The bone marrow-derived mesenchymal stem cells (BMSCs) could restore the structure and function of injured tissues [16]. Recent studies suggest that stem cell therapy holds promise in treatment of variety of diseases including reproductive dysfunction. In a recent study, BMSC treated animals resumed ovarian function. In another investigation, granulosa cell apoptosis induced by cisplatin was reduced when BMSCs were migrated to granulosa cells in vitro [9]. In this study, chemotherapy-induced POI rats were injected with BMSCs. The BMSCs treatment group's antral follicle count and estradiol levels increased after 30 days, compared with untreated POI group. In a recent clinical trial evaluated the therapeutic potential of autologous mesenchymal bone marrow stem cells transplantation in women with POI [17]. Patients with POI were selected and their ovaries were injected with autologous BMSC at time of laparoscopy. The results shown continuation of menstruation in after 3 months; showed focal secretory changes after having atrophic endometrium. According to these results, BMSC seem to have the ability to resuscitate prematurely failed ovaries both in their hormonal and follicular development abilities. 

Researchers recently reported that stem cell transplantation improves ovarian function which suggests a therapeutic potential [18,19]. The therapeutic effects of stem cells can be measured by several factors, such as folliculogenesis, the Granulosa Cells (GC) apoptosis rate, vascular formation, the pregnancy rate and the regulation of hormone levels. Folliculogenesis can be endorsed by the stem cells transplantation, which can trigger oogenesis. ADSCs are known to augment the severity of theca cells and GCs, resulting in an ideal environment for folliculogenesis [20,21]. PBMC contain various multipotent progenitor cells and have the ability to restore organ function and regenerate tissues. The combination of Platelet-Rich Plasma (PRP) and PBMCs has a synergistic effect on restoring folliculogenesis when transplanted into the induced Premature Ovarian Insufficiency (POI) model [21]. Our study shown that synergetic effect of autologous BMSCF and PRP therapy in a 24-year-old blighted ovum poor ovarian reserve helped in procuring a pregnancy and delivery of a healthy 2.9 kg baby through assisted reproduction. It also help to improve endometrium thicken for embryo implantation for IVF treatment (Table 1). 

No

Pt / age / married

Diagnosis

USG

USG RT OVARY

USG LT OVARY

LAPAROSCOPY

HYSTEROSCOPY

Clinical

Stem cell

result

1

KMC/27 /4

Twice aborted – blighted ovum

Poor ovarian reserve

Uterus normal

 

19x13x16 mm= VOLUME  2.3 ml

AFC = 0-2

13x19x20 mm= VOLUME 2.9 ml

AFC 0-2

uterus normal

ovaries smaller atrophic 

Endometrial cavity normal

Non responding well ovulation induction  drugs

Poor follicular response

Endometrium thin not growing more than 6 mm

Intra ovarian

Subendometrial

 

opu - -4 eoocystes

 

3 embryos

IVF 2950 gms

Baby

 

2

VRM

24/ 2

Premature ovarian failure

AMH = below 0.1

Fsh =70

Lh 24.56

Secondary amenorrhoea

Responding well to medications

Atrophic  smaller 35x25x17 mm

Thin central endometrial echo

Smaller

Could not be accessed properly

Smaller

Could not be accessed properly

streak like smaller atrophic ovaries

cavity smaller

twice metroplasty done

Ovarian failure

 Secondary amenorrhea

 Hormones correlating with post-menopausal age group

Endometrium thin 2-3 mm

Only spotting after withdrawal bleeding after medication

Sub-endometrial stem cells

 

Oocyte donation

Husband sperms

Ivf done

 1stivf aborted at 6 wks

2ndivf –

Maximum endometrial thickmess – 6.5 mm

Presently  14 wks viable pregnancy

3

STJ

32 /

17

Poor responder

Uterus normal

20x25x29 mm

Vol = 5.8 ml

25x17x21 mm

Vol = 5.2 ml

Uterus normal

Ovaries appears smaller

Endometrial cavity normal

Poor endometrial quality

Endometrium is poor and took almost 2 yrs for endometrial preparation

Finally endometrium was 7.4 mm thick with zone 5 vascularity

IVF – FET done

Sub endometrial stem cells

Successful IVF  conception

4

Spp

33 /

8

Poor responder

 Very poor response to the oral and injectable ovulation agents

Uterus

Normal

16x17x16 mm

Vol 2.4 ml

AFC= 1-2

17x20x21 mm

Vol 4.11 ml

AFC= 0- 2

Uterus

Normal

 Both ovaries appears smaller atrophic

Endometrial cavity normal

Poor endometrial quality

Endometrium is poor and took almost 6 months  for endometrial preparation

Finally endometrium was 7.3 mm thick with zone 5 vascularity

IVF – FET done

Intra ovarian stem cells

 

One blast formed

IVF

FET

DONE

FAILED

5

MPG

35/13

Very poor response to the endometrial preparation

 Embryo are ready but endometrium is not well responding

Uterus normal

Thin endometrium

Smaller in size atrophic

Smaller in size atrophic

Uterus

Normal

 Both ovaries appears smaller atrophic 

Endometrial cavity normal

Poor endometrial quality

Embryos are ready but endometrium is not responding well

Suggestive of poor endometrial response

Intraovarian and sub endometrial stem cells

Planning for endometrial preparation

6

SPM

24/4

Poor responder

Uterus rt unicornuate

Poor responder

AMH-0.8

FSH -8.5

UTERUS 31X23 MM UNICORNUATE

ovary small atrophic

ovary small atrophic

ENDOMETRIUM IS GOOD

Non responding well ovulation induction  drugs

Poor follicular respoce

 

Intraovarian

Stem cells

 

Opu  done

6 oocytes

3- M2

And

3 - abnormal oocytes

 

3 day 3 embryos

FET done

Positive

7

SCG

29/7

Poor responder

Uterus normal

Smaller 26x17x20 mm

Smaller 23x18x21 mm

Uterus normal

Both ovaries appears smaller atrophic

Endometrial cavity good

Non responding well ovulation induction  drugs

Poor follicular respoce

 

Intraovarian

Stem cells

 

Opu  done

3 oocytes

2- M2

And

1 - abnormal oocytes

 

1 day 3 embryos

FET DONE

8

Vsp

41/12

Poor responder

Three abortions

Two blighted ovum and one missed abortion

Uterus bulky adenomyotic

One anterior wall fibroid

 One posterior wall fibroid

Fibroids non indentiong the endometrial cavity

Smaller 18x16x20 mm

Smaller

15x18x19 mm

Uterus bulky adenomyotic

Smaller atrophic ovaries

Endometrial cavity appears normal

Intraovarian stem cells planned

3 oocytes

2 abnormal empty ooplasm

One good m2

Failed fertilization

 

9

MSS

25/5

Poor responder

Premature ovarian failure

AMH – 0.6

FSH – 9.7

UTERUS NORMAL

Smaller

AFC = 1-2

SMALLER

AFC = 0-1

uterus slightly bulky

ovaries smaller atropic

endometrial cavity appears normal

Non responding well ovulation induction  drugs

Poor follicular respoce

 

intraovarin stem cell

opu done

3 m 2 oocytes

One ady 3 good embryo

FET

 Failed

10

Ssp

37/17

Poor responder

Severe adenomyotic uterus

Kinked endometrial cavity

Non responding endometrium

Massive adenomyosis with kinked cavity poor endomyometrial differentiation

Thin hazy non triple line  endometrium with poor vascularity

Smaller atrophic

Smaller atrophic

Severe adenomyoic bulky uterus with smaller atrophic ovaries

Normal cavity capacity but poor endometrial quality

Nonresponding , thin hazy , nontriple line , typical adenomyotic endometrial response

Subendometrial stem cells

FET DONE

TWIN PREGNANCY

DELIVERED

BABY -1 – 2.5KG

AND

BABY -2 – 2.3KG

Table 1: Table showing details of the synergetic effects of stem cells and PRP enhance and restoring various aspects of ovarian function, such as folliculogenesis, vascular formation, genetic stability and its clinical outcomes in various study subjects.

**Note: Atrophic is a term given to signify a structure that is shrunken or diminished in size and function. In the case of the ovary this implies a decreased in the ovarian tissue volume and may be indicative of ovarian failure or interrupted ovarian blood supply.

Conclusion

It is evident that combine effect of BMSCF and PRP therapies have potential in treating POD. The synergetic effects of stem cells and PRP enhance and restoring various aspects of ovarian function, such as folliculogenesis, vascular formation, and genetic stability. Since stem cells are a proven new therapeutic strategy for the future cure of ovarian dysfunction.

Acknowledgment

The author thanks to Dr. Sandeep Nemani, Haematologist for his support in getting patient’s bone marrow during this various studies of patients. Also we thank to all IVF staff of Dr Ghodake IVF and fertility centre, Sangli for their support.

References

  1. Aly RM (2020) Current state of stem cell-based therapies : an overview. Stem Cell Investig 7: 8.
  2. Mousaei Ghasroldasht M, Seok J, Park HS, Liakath Ali FB, Al-Hendy A (2022) Stem Cell Therapy : From Idea to Clinical Practice. Int J Mol Sci 23: 2850.
  3. Kharat A, Padmanabhan U, Gadre SV (2017) Effect of growth hormone–releasing hormone and IGF-1 on human placenta-derived mesenchymal stem cells’ proliferation and differentiation. J Stem Cells 12: 7-15.
  4. Polymeri A, Giannobile WV, Kaigler D (2016) Bone Marrow Stromal Stem Cells in Tissue Engineering and Regenerative Medicine. Horm Metab Res 48: 700-713.
  5. Kharat A, Chandravanshi B, Gadre S, Patil V, Bhonde R, et al. (2019) IGF-1 and somatocrinin trigger islet differentiation in human amniotic membrane derived mesenchymal stem cells. Life Sci 216: 287-294.
  6. Patil VR, Kharat AH, Kulkarni DG, Kheur SM, Bhonde RR (2018) Long term explant culture for harvesting homogeneous population of human dental pulp stem cells. Cell Biol Int 42: 1-20.
  7. Torrealday S, Kodaman P, Pal L (2017) Premature Ovarian Insufficiency - an update on recent advances in understanding and management. F1000Res 6: 2069.
  8. Chon SJ, Umair Z, Yoon MS (2021) Premature Ovarian Insufficiency : Past , Present , and Future. Front Cell Dev Biol 9: 672890.
  9. Yang Z, Du X, Wang C, Zhang J, Liu C, et al. (2019) Therapeutic effects of human umbilical cord mesenchymal stem cell-derived microvesicles on premature ovarian insufficiency in mice. Stem Cell Res Ther 10: 250.
  10. de Miguel-Gómez L, López-Martínez S, Francés-Herrero E, Rodríguez-Eguren A, Pellicer A, et al. (2021) Stem Cells and the Endometrium : From the Discovery of Adult Stem Cells to Pre-Clinical Models. Cells 10: 595.
  11. Nazaroff J, Oyadomari S, Brown N, Wang D (2021) Reporting in clinical studies on platelet-rich plasma therapy among all medical specialties: A systematic review of Level i and II studies. PLoS One 16: 0250007.
  12. Zadehmodarres S, Salehpour S, Saharkhiz N, Nazari L (2017) Treatment of thin endometrium with autologous platelet-rich plasma: A pilot study. J Bras Reprod Assist 21: 54-56.
  13. Tandulwadkar SR, Naralkar MV, Surana AD, Selvakarthick M, Kharat AH (2017) Autologous intrauterine platelet-rich plasma instillation for suboptimal endometrium in frozen embryo transfer cycles: A pilot study. J Hum Reprod Sci 10: 208-212.
  14. Samy A, Abbas AM, Elmoursi A, Elsayed M, Hussein RS (2020) Effect of autologous platelet-rich plasma transfusion in the treatment of infertile women with thin endometrium and its implications in IVF cycles: a literature review. Middle East Fertility Society Journal 25: 5.
  15. Everts P, Onishi K, Jayaram P, Lana JF, Mautner K (2020) Platelet-rich plasma: New performance understandings and therapeutic considerations in 2020. Int J Mol Sci 21: 7794.
  16. Vizoso FJ, Eiro N, Cid S, Schneider J, Perez-Fernandez R (2017) Mesenchymal Stem Cell Secretome : Toward Cell-Free Therapeutic Strategies in Regenerative Medicine. Int J Mol Sci 18: 1852.
  17. Gupta S, Lodha P, Karthick MS, Tandulwadkar SR (2018) Role of autologous bone marrow-derived stem cell therapy for follicular recruitment in premature ovarian insufficiency: Review of literature and a case report of world’s first baby with ovarian autologous stem cell therapy in a perimenopausal woman of age 45 year. J Hum Reprod Sci 11: 125-130.
  18. Mansour RN, Soleimanifar F, Abazari MF, Torabinejad S, Ardeshirylajimi A, et al. (2018) Collagen coated electrospun polyethersulfon nanofibers improved insulin producing cells differentiation potential of human induced pluripotent stem cells. Artif Cells Nanomed Biotechnol 46: 734-739.
  19. Herraiz S, Romeu M, Buigues A, Martínez S, Díaz-García C, et al. (2018) Autologous stem cell ovarian transplantation to increase reproductive potential in patients who are poor responders, Fertil Steril 110: 496-505.
  20. Green LJ, Zhou H, Padmanabhan V, Shikanov A (2019) Adipose-derived stem cells promote survival, growth, and maturation of early-stage murine follicles. Stem Cell Res Ther 10: 1-13.
  21. Tomaszewski CE , Constance E , Lemke MM , Zhou H , Padmanabhan V, et al. (2020) Adipose-derived stem cell-secreted factors promote early stage follicle development in a biomimetic matrix. Biomater Sci 7: 571-580.

Citation: Bansode M, Kore SA, Lee Y, Ghodake V, Verma R (2022) Clinical Approach of Novel Autologous Stem Cell Therapy for Primary Ovarian Insufficiency and Endometrial Regeneration. J Stem Cell Res Dev Ther 8: 102.

Copyright: © 2022  Manoj Bansode, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.


Herald Scholarly Open Access is a leading, internationally publishing house in the fields of Sciences. Our mission is to provide an access to knowledge globally.



© 2023, Copyrights Herald Scholarly Open Access. All Rights Reserved!