WO2021038543A1 - Treatment of diminished ovarian reserve using menstrual blood stromal cells - Google Patents
Treatment of diminished ovarian reserve using menstrual blood stromal cells Download PDFInfo
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Abstract
The method presented in this invention, the first and only method based on cell therapy using stem cells from menstrual blood to treat infertility caused by decreased ovarian reserve and poor response to ovarian is that ovarian failure to reform, the number and quality of eggs, fertilization rate, embryo quality, pregnancy rate and live birth rate significantly compared with the control group increases. In fact, the natural pregnancy after treatment of cells can be used as the most significant data on the treatment to be considered This invention is the first clinical trial study to demonstrate the safety and efficacy of menstrual blood stem cells in the treatment of infertility, especially the reduction of ovarian reserve.
Description
The method presented in this invention, the first and only method based on cell therapy using stem cells from menstrual blood to treat infertility caused by decreased ovarian reserve and poor response to ovarian is that ovarian failure to reform, the number and quality of eggs, fertilization rate, embryo quality, pregnancy rate and live birth rate significantly compared with the control group increases
In fact, the natural pregnancy after treatment of cells can be used as the most significant data on the treatment to be considered This invention is the first clinical trial study to demonstrate the safety and efficacy of menstrual blood stem cells in the treatment of infertility, especially the reduction of ovarian reserve
Use in medical treatment or therapy (B42D 25/28)
The method presented in this invention is the first and only method based on cell therapy using menstrual blood stem cells to treat infertility due to decreased ovarian reserve (POR). However, there are studies on the effectiveness of stem cells in the treatment of infertility caused by another common problem in women called premature ovarian failure or premature menopause (PREATURE OVARIAN FAILURE; POF), which is as follows:
In one study, amniotic membrane-derived epithelial and mesenchymal cells were used to improve mouse ovarian function and showed that injected stem cells increased the proliferative power of granulosa cells. However, the function of amniotic membrane-derived mesenchymal cells in improving ovarian repair due to telomerase activity and high expression level of potent markers was much higher than that of amniotic membrane-derived epithelial cells (Ding et al., 2017).
In another study, it was reported that the number of follicles as well as the quality of ovulation in ovaries with cyclophosphamide-induced premature insufficiency in the mouse model increased after adipose-derived stem cell transplantation (Sun et al., 2013).
Liu and colleagues transplanted granulosa-like cells derived from pluripotent induced stem cells in the POF mouse model and observed that compared to the control animal group, the growth of granulosa-like cells improved and mature follicles were seen in ovarian tissue. Be. Also, the level of estradiol in peripheral blood increased and the number of atrial fibrils significantly decreased compared to the control group of animals. The researchers concluded that granulosa-like cells derived from pluripotent induced stem cells played a vital role in maintaining ovarian niche, stimulating follicle development, and maturation (Liu et al., 2016).
In 2016, a team of researchers in Egypt studied the effect of injecting bone marrow-derived stem cells into chemically damaged ovaries in an animal model of rats and found that levels of FSH and estradiol, as well as follicogenesis, were treated in treated animals. Improves. They concluded that stem cells can be easily located in the ovarian stroma and are effective in improving ovarian niche and increasing sex hormones and ovarian function (Gabr et al., 2016).
Su and colleagues used labeled adipose tissue-derived stem cell transplantation with collagen scaffold to improve the model of premature ovarian failure in mice. They found that estradiol levels and granulosa cell proliferation were significantly increased in animals receiving stem cells with collagen digestion compared with controls receiving PBS. Also, the number of antral follicles in the stem cell group with collagen scaffold and the stem cell group alone increased significantly compared to the control group. In addition, mating and pregnancy rates increased in the two treated groups compared to the control group. The researchers concluded that collagen scaffolds could be effective in transplanting stem cells to treat POF (Su et al., 2016).
In a clinical trial in humans, Edessy et al. Reported autologous MSCs from the iliac bone marrow being injected laparoscopically into the ovary, and even pregnancy and the birth of a healthy baby were reported (Edessy et al., 2014).
Due to the ability of menstrual blood-derived stem cells to repair various tissues, in a study these cells were used to treat ovarian failure in a mouse model. Intravenous injection of cells improves the microenvironment of the ovary by reducing apoptosis in granulosa cells and fibrous tissue in the interstitial tissue of the ovary. In addition, these cells play an important role in repairing damaged ovaries by increasing the number of follicles and returning sex hormone levels to normal levels (Liu et al, 2014).
In another study, after injecting menstrual blood-derived stem cells into a mouse model of premature ovarian failure (POF), researchers found that levels of ovarian markers such as antimullerian hormone, inhibin and ovarian stimulating hormone receptor, as well as Ki67 proliferative marker, increased. In addition, ovarian weight, normal follicle count, and plasma estradiol levels were higher in the stem cell group compared with the control group. Microarray analysis of cDNA expression pattern also showed that after menstrual blood-derived stem cell transplantation, the gene expression pattern in ovarian cells following host ovarian niche stimulation is similar to human ovarian tissue. The researchers concluded that the characteristics of mesenchymal stem cells derived from Menstrual blood makes them ideal cells for treating premature ovarian failure (Zhen et al, 2017).
Also in a recent study it was shown that injecting these cells into the ovaries of rats with premature ovarian failure causes follicle enlargement ovulation and the secretion of estrogen and progesterone
In 2018, an article was published that used bone marrow stem cells to treat infertility with poor ovarian response (Herraiz, et al. Fertility Sterility, 2018). In this invention, we used menstrual blood stem cells to treat infertility of weak ovarian response, which compared to bone marrow stem cells has advantages such as very easy access and reproducibility (monthly) without any invasion.
Prior to this invention, the repair capacity of menstrual blood stem cells in animal models of premature ovarian failure (different from poor ovarian response) was reported in several articles (Manshadi, et al., Microsc Res Tec.2019, Liu et al. Stem cell and development. 2014, Feng et al. Stem Cell Reviews and Repots, 2019). Although previous studies have shown a significant effect of stem cell therapy on resumption of ovarian function in animal models of premature ovarian failure, no article has been reported to evaluate the efficacy and effectiveness of menstrual blood stem cell administration in poor ovarian response. Also, among the inventions, there have been cases that have used menstrual blood cells, but none of them have been mentioned in the treatment of human infertility. The safety and efficacy of these cells in the treatment of premature ovarian failure in humans have not been reported. This invention is the first clinical trial study to demonstrate the safety and efficacy of menstrual blood stem cells in the treatment of infertility, especially the reduction of ovarian reserve.
US9044431B2
Methods of treating stroke using stem cell like menstrual blood cells
A cell type that is a complete match of the transplant recipient appears as an optimal scenario to open treatment options to a large patient population with minimal complications. The use of autologous bone marrow or umbilical cord blood has been proposed as a good source of stem cells for cell therapy. Menstrual blood is found to be another important source of stem cells. Assays of cultured menstrual blood reveal that they express embryonic like-stem cell phenotypic markers and neuronal phenotypic markers under appropriate conditioned media. Oxygen glucose deprivation stroke models show that OGD-exposed primary rat neurons, co-cultured with menstrual blood-derived stem cells or exposed to the media from cultured menstrual blood, exhibited significantly reduced cell death. Transplantation of menstrual blood-derived stem cells, either intracerebral or intravenously, after experimentally induced ischemic stroke in adult rats also significantly reduced behavioral and histological impairments compared to vehicle-infused rats.
In this invention, menstrual blood stem cells have been used to treat stroke, but in my invention, menstrual blood stem cells have been used in the treatment of infertility due to reduced ovarian reserve, as well as the safety and effectiveness of these cells in the treatment of premature ovarian failure in humans.
US20170143764A1
Menstrual blood derived stem cells for the treatment of human pancreatic carcinoma
A method and compound product are created from menstrual blood-derived mesenchymal stem cells (MenSCs) with an anti-tumor effect. In particular, the method and compound show the potential anti-tumor effect MenSCs can have on human pancreatic carcinoma, which has been analyzed on the Mia PaCa 2 human pancreatic carcinoma cell line (ATCC #CRL 1420). MenSCs have been proven to have an in vitro anti-tumor effect both in bi-dimensional cultures (monolayer) and in three-dimensional cultures (tumor spheres). Additionally, MenSCs slow down the appearance of pancreatic tumors when they are co-implanted with the pancreatic carcinoma cell line. MenSCs also have an in vivo therapeutic advantage in the treatment of human pancreatic carcinoma via intra-tumor injections.
In this invention, menstrual blood stem cells have been suggested for the treatment of human pancreatic carcinoma, but in my invention, menstrual blood stem cells have been used in the treatment of infertility due to reduced ovarian reserve, as well as the safety and effectiveness of these cells in treating premature ovarian failure in humans.
WO2017064670A2
Treatment for infection composed of menstrual stem cells
The present invention offers a solution to the lack of effective alternative treatments to fight infectious disease, preferably involving sepsis, comprising active ingredients obtained by non- intrusive and efficient methods. In particular, the present invention is the first to show that mesenchymal stem cells obtained from menstrual fluids (MenSCs) have the capacity to control infectious diseases, especially those leading to a reaction of the host body like sepsis. As shown herein, in vivo experiments illustrate that MenSCs have antibacterial activity in vitro, increase the survival rates of a mouse model for sepsis, regulate several parameters that are altered in sepsis patients and that are related with multi-organ dysfunction, such as the levels of Alanine aminotransferase (ALT), Aspartate aminotransferase (AST), alkaline phosphatase (ALP), glucose in blood, serum albumin, lung injury. Results show that in the mouse model for sepsis, MenScs also regulate the pro- and anti-inflammatory cytokine levels, reduce the loss of lymphocytes during sepsis and systemic bacterial proliferation in blood. The conditioned medium of MenSCs also increases the survival rates of mouse animals affected by sepsis. Overall, the invention offers a promising alternative method to treat infectious diseases. Since it is principally composed of stem cells present in menstrual fluid, the invention provides an ease access and repeated sampling in a non-invasive manner. Such attributes allow the rapid production of the treatment.
In this invention, the menstrual blood stem cells to treat infections has been suggested, but my invention, due to reduced ovarian reserve menstrual blood stem cells as well as the safety and efficacy of these cells have been used in the treatment of infertility of premature ovarian failure in humans.
Methods and Compositions for Treating Ovarian Failure
United States Patent Application 20170258842
Methods and compositions for treating ovarian failure are provided. In one embodiment, the method includes administering stem cells into the ovary of a female subject in need of such treatment. The stem cells are preferably bone marrow derived stem cells (BMSC). In other embodiments, the stem cells are embryonic stem cells, adult stem cells, induced stem cells, induced pluripotent stem cells, umbilical cord blood cells, or combinations thereof. The stem cells can be autologous or heterologous. In one embodiment, the stem cells have the following surface marker profile: CD105 positive, CD166 positive, CD90 positive, and CD73 positive as well as CD14 negative, CD34 negative, CD45 negative, HLA-DR negative, and CD 19 negative. The stem cells are administered in an amount effective to restore ovarian hormone production and promote folliculogenesis.
In this invention, umbilical cord and bone marrow stem cells are used to treat ovarian failure. but my invention, due to reduced ovarian reserve menstrual blood stem cells as well as the safety and efficacy of these cells have been used in the treatment of infertility of premature ovarian failure in humans.
We invented an efficient method for treatment of infertility in women with diminished ovarian reserve. This method is cell therapy of sterile poor ovarian responders (POR) using autologous menstrual blood stem cells (MenSCs) that have encouraging characteristics like easy accessibility, high availability, monthly sampling repeatability, no need to collect samples in an invasive manner, less ethical considerations, no tumorigenesis potential, immune privilege property and significant trans-differentiation capability. Intra-ovarian injection of autologous MenSCs amends ovarian insufficiency, considerably increases oocyte number and quality, fertilization rate, embryo quality, pregnancy rate and live birth rate in comparison with control group. Indeed, incidence of natural pregnancies after MenSCs therapy could be considered as the most noticeable data of this invention. This invention is the first report showing safety and significant efficacy of cell therapy using MenSCs to attenuate infertility problem of POR women. The purpose of this invention is to improve ovarian function, increase the incidence of pregnancy and live birth in women with reduced ovarian reserve and poor ovarian response to conventional therapies.
Poor ovarian responder (POR) refers to women in reproductive age with spontaneous menstruation and diminished ovarian reserve resulting in a reduction in follicular response and number of retrieved oocytes. Vibrant definitions are available for POR, however, the most consensus definition belongs to the Bologna criteria established by ESHRE group in 2011. According to this definition, at least two of the following characteristics are needed for ovaries to be considered as a poor responder: 1) advanced maternal age (>40 years); 2) a previous meager response, unerringly three or less oocytes after conventional ovarian stimulation protocol; 3) an abnormal ovarian reserve test such as antral follicle count (AFC) less than five to seven follicles or anti-Mullerian hormone (AMH) below 0.5–1.1 ng/ml. In rife, a patient who experienced two cycles with three oocytes or less after maximum stimulation could be considered as a poor responder, even in absence of other two criteria.
Due to heterogeneous population of POR patients, it is cumbersome to determine the prevalence of this condition. However, it has been estimated to range between 9 and 24% in variant studies. Indeed, with the global raise in population of older women who seek infertility treatment, the demand of efficient strategies to improve ovarian response in in vitro fertilization (IVF) cycles of poor responders is increasing.
Different strategies have been applied to manage POR women so far. Using novel controlled ovarian stimulation protocols and administration of growth hormone are some evaluated strategies that could mildly improve ICSI outcome and live birth rate in POR women who met the Bologna criteria. Another clinical option for improving ICSI aftermath in PORs is using DHEA as an adjuvant for ICSI that resulted in 23.9% clinical pregnancy rate and 16.4% live birth rate.
The efforts related to these recommendations for clinical handling of PORs have made some gains in this regard, but more effective methods need to be followed. Therefore, new strategies should be brought into existence to manage infertility of PORs and rescue them from oocyte/embryo donation.
In recent years, with the emergence of regenerative medicine, some studies have been conducted to evaluate safety and efficacy of stem cell therapy in treatment of female reproductive disorders. These efforts mostly have been implemented using mesenchymal stromal cells (MSCs) derived from various sources such as bone marrow (BM), umbilical cord and amniotic membrane in order to make them feasible to treat female infertility particularly premature ovarian failure (POF). Beside the impressive efficacy of stem cell therapy in resumption of ovarian function, improvement in FSH, E2, ovarian weight, follicle count as well as the number of pregnancies in animal models of POF, birth of one baby consequence to cell therapy of ten POF women using bone marrow derived MSCs (BM-MSCs) was reported. More recently, cell therapy using bone marrow stem cells (BMSCs) was used to treat 15 women, who were poor responders that resulted in five pregnancies (33.3%) and three live births (20%). Considering AFC and serum AMH, they observed an improvement in ovarian function in 81% of patients after intra-arterial delivery of BM stem cells. The cell therapy resulted in an increased number of antral follicles, especially in the infused ovary, and retrieved oocytes. Of note, bone marrow sampling needs an invasive procedure and has no repeatability that limit accessibility of bone marrow stem cells for clinical application.
Menstrual blood stem cells are reckoned as an outstanding type of MSCs that could be obtained from human menstrual blood shedding of endometrium monthly by a non-invasive and easy procedure. These stem cells possess dramatically proliferative, homing and antigenic potency in comparison with other types of adult stem cells, especially bone marrow MSCs (BM-MSCs) and umbilical cord MSCs (UC-MSCs). These properties associated with remarkable regenerative capacity, low-immunogenicity properties and immunomodulatory effects mediate a promising circumstance to fulfill clinical demand. Moreover, absence of any ethical issue, no teratogenic effect, and greater trans-differentiation ability of these cells into various lineages present them as an authentic candidate for cell therapy in various pathological conditions.
Notably, some scholars indicated the efficacy of MenSCs infusion in restoration of ovarian failure in animal models of POF. In this invention, we declared that cell therapy using autologous MenSCs are capable to ameliorate ovarian function and considerably improves pregnancy rate and live birth rate of POR patients.
In infertility treatments with a group of patients are facing that while premenopausal, but a sharp decline in ovarian reserve (diminished ovarian reserve) face and despite receiving high doses of drugs for ovarian stimulation in Cycles, very few eggs (≤ 3) is obtained. These patients, ovarian poor responders (Poor ovarian responder: POR) are called. Currently there is no medical intervention to treat patients POR and finally had to accept many of these patients using donor (zygotes).
So far, various strategies have been used to treat women with POR. Growth hormone administration in IVF treatment cycles is one of the treatment methods for these patients to increase the chances of pregnancy. By giving this hormone, the rate of clinical pregnancy has increased from 15.1% to 22.1% and the rate of live birth has increased from 10.9% to 14.7%.
Another treatment option is to give dehydroepiandrosterone (DHEA) to POR patients before the start of the treatment cycle. In a study of 286 patients who were in this group according to BOLOGNA criteria, DHEA was prescribed at a dose of 75 mg per day for up to three months. Fetal implantation rate increased from 10.1% to 18.7%, success rate in clinical pregnancy reached 23.9% and live birth rate reached 16.4%.
In addition to the ineffectiveness of methods used in pregnancy statistics in this group of patients, the reported pregnancy was due to the use of IVF methods and no cases of spontaneous pregnancy occurred. The purpose of this invention was to improve ovarian function, increase the incidence of pregnancy and live birth. in women with reduced ovarian reserve and ovarian poor responders to conventional therapy.
In a specific embodiment, sterile POR women are fertile using procedures known in the art. Said procedure includes direct administration of autologous cultured menstrual blood stem cells into patient ovary.
It is known in the art that injected MSCs affect ovarian function through two mechanisms: First allegation is ovarian microenvironment could be improved by paracrine effect of MenSCs. Variety of growth factors and cytokines are released by MenSCs that could play key role in ovarian restoration where the ovarian niche is not able to maintain growth of their already confined follicular pool.
To corroborate this assertion, animal studies demonstrated the improvement of ovarian function and angiogenic induction by cytokines secreted of Men-MSCs. Another point to consider is that probably few pre-antral follicles existed in the ovary that their development and maturation could be accelerated and spurred by extrinsic MenSCs. Based on this concept, AFC was not drastically impressed by injected MSCs, and just maturity of existed pre-antral follicles was induced by indirect or direct impact of injected MenSCs. In line with this assumption, we recently indicated that MenSCs could improve indices of follicular growth and maturation
in vitro. Indeed, it is reckoned that dual effect of Men-MSCs in both ovarian niche and follicles maturation and improvement of their dialogue is more involved in this phenomenon.
The isolated stem cell population preferably expresses one or more markers selected from the group consisting of: CD105, CD73, CD90, CD54, CD106, OCT4, HLA-I markers, vimentin, collagen-1, fibronectin, LFA-3, ICAM-1, PECAM-1, P-selectin, L-selectin, CD29, thrombomodulin, telomerase, CD10, CD13, VCAM-1, CD146, and THY-1. According to more specific embodiments, the mesenchymal stem cell population does not express substantial levels of the markers selected from the group consisting of: HLA-DR, CD34, and CD45.
It is possible, other mesenchymal stem cells by the same properties with MenSCs are provided for ovary administration. These stem cells can be derived from sources selected from the group consisting of: adipose tissue, amniotic membrane, umbilical cord blood, placental tissue, peripheral blood mononuclear cells, exfoliated teeth derived stem cells, for example.
In one embodiment, depending on female age and previous clinical outcome, stem cells injection to both ovaries or administration of higher doses or multiple cell injection are provided to enhance benefit of stem cell therapy.
In some embodiments the therapy is performed in combination with a growth factor or a plurality of growth factors. This includes, without limitation, angiogenic factors and other molecules competent to induce angiogenesis, including acidic and basic fibroblast growth factors, vascular endothelial growth factor, epidermal growth factor, transforming growth factor, platelet-derived endothelial growth factor, platelet-derived growth factor, hepatocyte growth factor and insulin like growth factor. Dependent on embodiment, MenSCs and/or growth factors may be provided intravascularly, intravenously, intraarterially, intraperitoneally, via intraventricular infusion, via infusion catheter, via balloon catheter, via bolus injection, or via direct application to tissue.
Various female reproductive disorders are amendable to treatment with the current invention. They include premature ovarian failure, thin endometrium, Asherman syndrome, endometriosis, polycystic ovary syndrome and recurrent pregnancy loss.
In recent years, endometrial tissue and high angiogenesis that occurs during the menstrual cycle in this tissue has attracted the attention of researchers. Several studies have implied that the reason for the high proliferation and angiogenesis in the tissue of the menstrual cycle takes place menstrual blood stem cells with features such as a rich source of stem cells that can be easily lost in each cycle is known. Menstrual blood is accessible and available source of stem cells without inflicting pain to donor to achieve source of stem cells. There is no need to prescribe drugs to stimulate the production of more stem cells in the donor, nor to prescribe painkillers, antibiotics, or supplements to improve the donor. Compared to other sources, stem cells extracted from menstrual blood stimulate the recipient's immune system much less, which makes it possible for a larger population of recipients to use this source without imposing immunological restrictions on other sources, and consequently at a cost. It facilitates the selection of the appropriate donor and decreases the costs of the transplant in terms of psychological economics, medicine, manpower, etc., and reduces the costs of treatment due to complications.
The ability to reconstruct and revive the injured tissues using menstrual blood stem cells is the same as that of cord blood and bone marrow stem cells, and allows any woman of childbearing age to maintain these stem cells for herself or other family members without moral problems, and by providing a bank. The menstrual blood stem cells of young women can be used clinically during menopause to treat various diseases, and therefore, while creating more hope for treatment of disorders in the future. Due to the remarkable characteristics of menstrual blood stem cells, we developed studies based on cell therapy in women with poor ovarian response.
This invention can be used as the first report of significant safety and efficacy of cell therapy using stem cells from menstrual blood to reduce the problem of infertility in women with POR. In fact, a new approach in the field of return chances of having a baby with a normal pregnancy experience in patients without hormonal stimulation of the ovaries and challenges of intra-cytoplasmic sperm injection (ICSI) is provided. As well as women who experience pregnancy need to use a protocol routine hormonal stimulation of the ovaries and then ICSI, can utilize the efficacy of intraovarian injection of stem cells from menstrual blood to improve the number and quality of eggs, the fertilized egg and embryo quality. Therefore, due to the easy access, sampling, isolation and high reproducibility of these stem cells as an autologous source, it seems that this treatment in infertile women with POR who have to use egg donation methods to treat infertility are useful.
Steps of cell product preparation and release (Fig.1):
Stem cell isolation from menstrual blood: Menstrual blood was collected from women using sterile Diva cup (Diva International Co., Lunette, Finland) at the second day of menstruation. The specimen was delivered into the collection tube containing GMP-grade Dulbecco’s Modified Eagle’s Medium-F12 (DMEM-F12, Gibco, UK),2.5 μg/mL fungizone (GIBCO, UK), 100 μg/mL streptomycin, 100 U/mL penicillin (Sigma-Aldrich, MO, USA) and 0.5 mM EDTA in GMP-grade phosphate buffered saline (PBS) without Ca2+ or Mg2+(GIBCO, UK) and quickly conveyed to class B cell culture clean room in order to isolate and cultivate MenSCs. The samples were suspended in DMEM-F12 medium, fortified with 5% platelet lysate (Gibco, Fisher Scientific, UK) and then kept in 37 °C CO2 incubator. The cells that adhered to cell culture flask the next day, developed colony formation units and reached 80-90% confluence at day 14. At this time, the cells were passaged using % trypsin-EDTA (Gibco, UK) and divided in multiple cell culture flasks to propagate.
Stem cells cryopreservation and storage in the quarantine cell bank: Upon reaching confluency, cells were detached and then cryopreserved at a density of 1.5×10
6 /1.8 mL cryovials to quarantine in a pre-master cell bank (PMCB). After characterizing this cell bank and confirming all quality control tests, the cells were taken out of quarantine and transferred to master cell bank (MCB) and working cell bank (WCB).
Stem cell quality control: The cells were examined for the following characteristics:
- Identity
- Purity
- Stability
- Karyology
Final product release: For generating the final product, the cryopreserved cells were thawed and expanded to passage 2; cell quality control tests were implemented and then released.
Example: MenSCs isolated from 15 POR participants had spindle-shaped morphology in culture (Fig.2). Immunophenotyping analysis showed that the cultured cells were positive for CD90 (98.5±1.9%), CD73 (99.5±0.5%), CD44 (92±2.35%) and negative for hematopoietic marker CD45 (2±1.02%) as illustrated in figure 3. Moreover, all cultured cells showed normal karyotype pattern. There was no evidence of microbial growth and so the sterility of the cells was confirmed for all cultures. From the results of Gram stains, no colony forming unit (CFU) was observed in the final cellular medicines studied.
In addition, all sample analyzed by DNA amplification were negative for mycoplasma expression. Alongside, endotoxin assay resulted in no LAL clot formation in the assessed samples.
Therapeutic intervention:
Therapeutic interventions were carried out as following steps:
At the time of cell injection (in the follicular phase of the menstrual cycle), on the day of stem cell transfer, AMH levels, number of antral follicles, and ovarian volume were checked again.
Transplantation of menstrual blood-derived stem cells was performed under general anesthesia with a mask (midazolam 1 mg, fentanol 1 μg / kg and induction with propofol 2 mg / kg). After anesthesia, the patient's vagina was washed with sterile normal saline, then 150 μl of cell product with a concentration of 20 million / ml was injected into the left ovary with a vaginal ultrasound guide (due to the ease of access to vaginal ultrasound).
After controlling vaginal bleeding, the patient was under the direct supervision of a gynecologist for 2 hours. If all of the patient's vital signs were normal and no complications were reported from the patient's cell injection, he would be discharged.
For one week after the injection, any side effects from the patient's cell injection were monitored by daily telephone call.
After injection for 3 months, the patient was followed up for spontaneous pregnancy and in case of no pregnancy, the patient entered the routine protocol of ovulation stimulation and then ICSI.
In case of pregnancy, the patient underwent routine follow-up of pregnant women and data were recorded until delivery and birth of the baby.
In case of no pregnancy after ovulation induction routine protocol and then ICSI and embryo transfer, the patient was monitored for 14 days for pregnancy.
In case of pregnancy, the patient underwent routine follow-up as a pregnant patient. In case of no pregnancy, patients who wished to receive donated eggs were referred to the donation clinic and patients who did not wish to use donated eggs. In terms of the likelihood of spontaneous pregnancy, AMH levels and the number of antral follicles were followed for at least one year.
Evaluation and follow-up of patients after pregnancy:
In case of pregnancy, maternal and fetal health was assessed according to the following protocol:
- Pregnancy process:
- Visiting patients on a monthly basis for up to 24 weeks
- Refer patients every two weeks between 32-24 weeks of pregnancy
- Visiting patients weekly from week 32 until delivery
- Tests and reviews:
-First trimester: Vaginal ultrasound and cell-free DNA evaluation
-Between 18-22 weeks: Calordapler ultrasound to evaluate fetal heart abnormalities (first screening)
-Between 30-30 weeks: Ultrasound of fetal weight and IUGR examination (second screening)
Assessment of newborns:
Newborns underwent routine national assessments and the results were recorded in their medical records, including:
Assess the baby's height and weight
Baby body temperature
Examination of the baby's scalp and head
Examination of the face and neck
Examination of organs
Heart and chest examination
Joint examination of the hip and spine
Examination of the baby's nervous system (reflexes, muscle tone, crying)
Examination of the genitals and anus
Example: To evaluate therapeutic intervention of intraovarian injection of autologous menstrual blood stem cells, 51 POR participants were studied. Of these, 15 patients were excluded from the study based on the inclusion criteria and thus 36 patients were randomly divided into two main (menstrual blood stem cell therapy group) and control groups (each group consisting of 18 people). The demographic characteristics of participants have been explained in tables and 5.
The reasons for excluding these patients from the plan were:
- Failure to meet the inclusion criteria (9 people)
- Exclusion criteria (6 people)
18 patients in the main group were candidates for cell therapy, 3 of whom were excluded from the study according to the criteria defined for exclusion from the study, and finally 15 of them underwent cell injection. In the control group, 2 people withdrew from the study and finally 16 patients were examined.
Patients in the control group entered the routine ovulation induction protocol after 3 months of opportunity for spontaneous pregnancy and then ICSI was intervened in the same group and the pregnancy results and data obtained from them were recorded. In the absence of pregnancy, the procedure was performed as in the main group.
Results:
Three patients in main group dissuaded from stem cell administration despite of menstrual blood collection and 2 patients in control group changed treatment to egg donation. The situation of participants after stem cell administration in comparison with control group has been indicated in tables 3-8. As shown in these tables, 4 of 15 participants got naturally pregnant just during 3 months after autologous cells administration, in contrast to no natural conception in control group (26.7%: main group vs. 0%: control group). 1 of these naturally pregnancies terminated due to miscarriage, but others (3 pregnant women) gave live birth at due date (20%: main group vs. 0%: control group). Other participants who did not get pregnant after three months (11 in the main group and 16 in the control group) underwent AMH testing and referred to ovary stimulation cycle with the antagonist protocol to implement ICSI plan.
The participants who had satisfying ovum pick up and subsequently mature oocytes continued their therapeutic plan by oocyte insemination through ICSI procedure and embryo transfer. The obtained data showed that mean AMH level of stem cell therapy group did not significantly differ with that of previous cycle and also control group. However, AMH levels significantly decreased in last cycle compared to previous cycle in control group (0.6 (0.7) vs. 0.4 (0.5)). In addition, number of HMG ampoules used by main group was typically less that control group, but this index had no significant difference compared to previous cycle. Although mean AFC count, number of picked up follicles and oocytes number in main group did not indicate considerable difference with those of control group, increase of these parameters in comparison with previous cycle was statistically significant in main group.
Moreover, roughly 94.7 % of retrieved oocytes in main group were in phase II metaphase and had high-quality level. Meanwhile, injection of these oocytes by ICSI resulted in 92% fertilization and development of high-quality embryos, whereas fertilization rate before treatment was 76%. These changes in control group were not significant (76% in previous cycle vs. 67% in last cycle). Furthermore, mean number of high-quality embryos was greater than mean number before the treatment. Instead, these parameters demonstrated significantly worse in control groups. In addition, embryo transfer of 3 of 11 women in main group and 2 of 16 women in control group resulted in paying off pregnancy (27.3% vs. 12.5%). One pregnancy in each group terminated due to abortion, but other conceptions resulted in live births. Therefore, 7 of women enrolled in main group and 2 women in control group got pregnant (46.7% vs. 12.5%). Meanwhile, 5 pregnancies ended in live birth in main group, while only 1 pregnancy in control group resulted in live birth (33.3% vs. 6.3%). All born babies (3 boys and 2 girls in main group and 1 girl in control group) were healthy and were followed up for three months. The mean birth weight of born babies by MenSCs therapy was 3200-3950 g and weight of child in control group was 3320g.
The cells used in this invention are menstrual blood stem cells, which have the following advantages over other stem cell sources:
Due to the fact that it is possible to access this source on a monthly basis, so it does not have the limitations of other sources. It should be emphasized that this cellular source is always available for the person and the person always has stem cells Synthesizes monthly. Therefore, women can use these cells to treat their diseases in the future by storing these cells during their reproductive period, and they may be able to donate these stored cells to other members of their family, away from medical ethics issues.
Menstrual blood can be obtained from sick women themselves and thus reduce the problem of limited donor.
Menstrual blood sampling is a completely non-invasive method and does not cause any side effects from various human, social and economic dimensions.
Sample preparation is monthly and painless.
Sampling is very simple and can be done at no special cost.
By using the patient's own menstrual blood-derived cells, the possibility of rejecting immunological transplantation is reduced, which prolongs the patient's life after transplantation.
Isolation and culture of menstrual blood stem cells can be easily done in the laboratory.
Based on its high self-renewal ability, the use of basic menstrual blood cells is confirmed.
The effects of using these cells in various clinical cases have been proven.
Practically and ethically, the use of menstrual blood stem cells is better than other stem cells such as other adult stem cells or embryonic stem cells.
If the sampling is not successful or it becomes infected due to the contaminated nature of the vaginal canal, it is possible to repeat the sampling in subsequent cycles.
On average, about 400 menstrual blood samples can be taken from each woman during the reproductive period.
The differentiation of these stem cells into different cell lines and thus their multiplicity has been shown.
Endometrial tissue and menstrual blood can be referred to as "on standby" tissue, which is easily accessible if needed.
Isolation and culture of stem cells from the origin of menstrual blood has an effective role in health economics. Because it is always available and does not include the heavy costs of long-term maintenance of stem cells.
*Stem cells cryopreservation and storage in the quarantine cell bank: Upon reaching confluency, cells were detached and then cryopreserved at a density of 1.5×10
6 /1.8 mL cryovials to quarantine in a pre-master cell bank (PMCB). After characterizing this cell bank and confirming all quality control tests, the cells were taken out of quarantine and transferred to master cell bank (MCB) and working cell bank (WCB).
*Stem cell quality control: The cells were examined for the following characteristics:
- Identity
- Purity
- Stability
- Karyology
*Final product release: For generating the final product, the cryopreserved cells were thawed and expanded to passage 2; cell quality control tests were implemented and then released.
- The results of sterility test, mycoplasma test, endotoxin test was all negative.
- The karyotype of the cultured cells was normal.
Examples
This patented method is used to treat women with reduced ovarian reserve who have at least twice a history of IVF failure. Patients should have two characteristics of the following conditions:
Age equal to or above 40 and under 45 years
Their anti-molar hormone below 1/1 ng / ml
The number of antral follicles is less than 5
Individuals for uterine and ovarian ultrasound, medical diagnostic tests showing thyroid function, prolactin, glucose (FT4, TSH, PRL, FBS), liver enzymes (SGPT-SGOT), renal markers (BUN, Cr), CBC, factors Coagulation (CT-BT-PTT-PT), hypoparathyroidism (Ca, P), Na, K and also in terms of infectious tests (VDRL, HCV, HBS Ag, HIV) should be normal.
Menstrual blood samples according to protocol displayed in the form of a collected from each patient and the isolation, culture, evaluation, freezing, melting and during the injection. Multiple quality control tests including sterility tests, tests for mycoplasma, endotoxin testing and flow cytometry, all cells must be on a stage one stage before freezing and after thawing and before cell injection done.
A patient can use this treatment if he / she meets all of the following conditions:
- Female gender
- Age equal to or under 40 years (40-25 years)
- Confirmed infertility
- Severely reduced ovarian reserve (POR) according to Bologna criteria
- Antimullerian hormone level less than 1/1 ng / ml
- The number of antral follicles is less than 5
- The number of eggs obtained in cycles with a standard drug dose equal to or less than 3
- Normal uterine and ovarian ultrasound
- Analysis of natural sperm of the spouse
- Normal function of thyroid, hypo parathyroid, liver and kidney
- Normal levels of prolactin, sugar, coagulation factors, sodium and potassium
- Negative infectious tests (VDRL, HCV, HBS Ag, HIV, HTLV1,2, CMV)
And If you have any of the following factors, you cannot use this treatment:
- Anatomical defects in the internal genital organs including the uterus, ovaries, and external genitalia and urinary tract
- Having an active genital infection
- Tobacco and alcohol consumption
- History of endometriosis or ovarian cysts
- History of autoimmune disease
- History of malignancy
- History of allergic diseases
- History of mental illness
36 POR women (Based on Bologna criteria) undergoing conventional ICSI-embryo transfer (ET) procedures are entered in a clinical trial. The eligible women (n=36) were divided by block randomization method into main (MSC therapy, n=18) and control (routine ICSI plan, n=18) groups and followed by dedicated code which was defined as POR-Patient number. Three volunteers in MSC group and two participants in control group withdrew from intervention after randomization. Thus, the study was continued with 15 participants in the main group and 16 patients in the control group. The two groups were not significantly different in terms of age, duration of infertility, sperm conditions, previous cycle characteristics, body mass index (BMI), number of oocytes and embryos, AMH and number of antral follicles in the ovaries (Tables 1,2).
Menstrual blood was collected from women using sterile Diva cup (Diva International Co., Lunette, Finland) at the second day of menstruation. The specimen was delivered into the collection tube containing GMP-grade Dulbecco’s Modified Eagle’s Medium-F12 (DMEM-F12, Gibco, UK),2.5 μg/mL fungizone (GIBCO, UK), 100 μg/mL streptomycin, 100 U/mL penicillin (Sigma-Aldrich, MO, USA) and 0.5 mM EDTA in GMP-grade phosphate buffered saline (PBS) without Ca
2+ or Mg
2+ (GIBCO, UK) and quickly conveyed to class B cell culture clean room in order to isolate and cultivate MSCs. The samples were suspended in DMEM-F12 medium, fortified with 5% HyClone™ Serum (U.S.), Standard (Gibco, Fisher Scientific, UK) and then kept in 37 °C CO
2 incubator. The cells that adhered to cell culture flask the next day, developed colony formation units and reached 80-90% confluence at day 14. At this time, the cells were passaged using % trypsin-EDTA (Gibco, UK) and divided in multiple cell culture flasks to propagate. Upon reaching confluency, cells were detached and then cryopreserved at a density of 1.5×10
6 /1.8 mL cryovials to quarantine in a pre-master cell bank. After confirming the quality control tests, cells were transferred to master cell bank and working cell bank. For generating the final product, the cryopreserved cells were thawed and expanded to passage 2 (P2); cell quality control tests were implemented and then released
.
MenSCs isolated from all participants had spindle-shaped morphology in culture (Fig.2). Immunophenotyping analysis showed that the cultured cells were positive for CD90 (98.5±1.9%), CD73 (99.5±0.5%), CD44 (92±2.35%) and negative for hematopoietic marker CD45 (2±1.02%) as illustrated in figure 3, Table 3. Moreover, all cultured cells showed normal karyotype pattern. There was no evidence of microbial growth and so the sterility of the cells was confirmed for all cultures. From the results of Gram stains, no colony forming unit (CFU) was observed in the final cellular medicines studied.
In addition, all sample analyzed by DNA amplification were negative for mycoplasma expression. Alongside, endotoxin assay resulted in no LAL clot formation in the assessed samples.
On the day of cells injection, the cultured and qualified cells were trypsinized, counted and suspended in normal saline included 10% human serum albumin to prepare the density of 20×10
6 cells/ml. Thereafter, 150 μl of prepared suspension was intravaginally by vaginal ultrasonography (Honda 2000-5 MHz, Japan) injected into left ovary of patients after receiving general anesthesia with midazolam and fentanyl. To diminish bias, collection of menstrual blood and injection of MSCs into ovary were performed just by one physician.
All patients were followed by evaluation of safety and efficacy of treatment up to one year after treatment. To assess the safety, some clinical symptoms including fever, pain, infection, bleeding and allergic reactions were monitored after cell administration. Thereupon, the efficacy of procedure was evaluated by incidence of natural pregnancy in patients during three months. The patients who encountered menstruation arrest were checked by measurement of βHCG. If patients were not conceived after 3 months, they were referred to ICSI. The efficacy of procedure was evaluated by incidence of natural pregnancy in patients during three months. The patients who encountered menstruation arrest were checked by measurement of βHCG. If patients were not conceived after 3 months, they were referred to ICSI.
Followed by confirmation of biochemical pregnancy, clinical pregnancy was approved by detection of fetal heart rate in the 7
th week after last menstrual period and live birth rate by physician report. Pregnancy complications such as miscarriage, ectopic pregnancy, preeclampsia, premature rupture of membranes, intrauterine fetal death, and diabetes were evaluated in each clinical visit during pregnancy.
The first trimester screening test was done between 12-14
th weeks of pregnancy. The levels of free βHCG and PAPP-A hormones were checked and followed by ultrasound to assess the risk of Down syndrome and some other abnormalities caused by chromosomal defects.
Moreover, during 14-16
th weeks of pregnancy, Quadruple marker screening was performed as a blood screening test for AFP, HCG, estriol and Inhibin-A. In 18-20
th weeks, fetal echocardiogram and targeted obstetric ultrasound were accomplished to evaluate heart defects and fetal anatomy, respectively. Complications related to placenta or amniotic fluid at due date were assessed and neonatal information including gender, birth weight, and birth defect was recorded.
Interval duration between two check-ups was 4 weeks until 28
th, 3 weeks until 34
th, 2 weeks until 38
th of gestation week and eventually a week before labor. In each visit, blood pressure, body weight, fetal heart rate and uterine size was checked. Moreover, blood and urine samples were analyzed in each trimester to assess blood glucose level, CBC, TSH, 25-OH-VitD, proteinuria and infection.
For patients with no menstruation arrest up to three months’ post cells administration (MSC group), their AMH and AFC were measured. By reasonable circumstance, ovary stimulation by GnRH antagonist protocol, ovum pick up, ICSI, embryo transfer and clinical pregnancy were followed for the patients.
Patients were recovered post-administration as usual without any sign of pain, nausea, infection, bleeding or fever and so were discharged from hospital after 4 hours. Monitoring of participants by daily phone calls demonstrated no clinical sign. Moreover, no suspicious symptoms were observed by physical exam or sonography done by the experienced clinician during follow up.
Three patients in MSC group dissuaded from MSCs administration despite of menstrual blood collection and 2 patients in control group changed treatment to egg donation. The situation of participants after MSC administration in comparison with control group has been indicated in tables 4-9. As shown in these tables, 4 of 15 participants got naturally pregnant just during 3 months after autologous cells administration, in contrast to no natural conception in control group (26.7%: MSC group
vs. 0%: control group). 1 of these naturally pregnancies terminated due to miscarriage, but others (3 pregnant women) gave live birth at due date (20%: MSC group
vs. 0%: control group). Other participants who did not get pregnant after three months (11 in the MSC group and 16 in the control group) underwent AMH testing and referred to ovary stimulation cycle with the antagonist protocol to implement ICSI plan. The participants who had satisfying ovum pick up and subsequently mature oocytes continued their therapeutic plan by oocyte insemination through ICSI procedure and embryo transfer. The obtained data showed that mean AMH level of MSC group did not significantly differ with that of previous cycle and also control group.
However, AMH levels significantly decreased in last cycle compared to previous cycle in control group (0.6 (0.7)
vs. 0.4 (0.5)). In addition, number of HMG ampoules used by MSC group was typically less that control group, but this index had no significant difference compared to previous cycle. Although mean AFC count, number of picked up follicles and oocytes number in MSC group did not indicate considerable difference with those of control group, increase of these parameters in comparison with previous cycle was statistically significant in MSC group. Moreover, roughly 94.7 % of retrieved oocytes in MSC group were in phase II metaphase and had high-quality level.
Meanwhile, injection of these oocytes by ICSI resulted in 92% fertilization and development of high-quality embryos, whereas fertilization rate before treatment was 76%. These changes in control group were not significant (76% in previous cycle
vs. 67% in last cycle). Furthermore, mean number of high-quality embryos was greater than mean number before the treatment. Instead, these parameters demonstrated significantly worse in control groups. In addition, embryo transfer of 3 of 11 women in MSC group and 2 of 16 women in control group resulted in paying off pregnancy (27.3%
vs. 12.5%). One pregnancy in each group terminated due to abortion, but other conceptions resulted in live births. Therefore, 7 of women enrolled in MSC group and 2 women in control group got pregnant (46.7%
vs. 12.5%). Meanwhile, 5 pregnancies ended in live birth in MSC group, while only 1 pregnancy in control group resulted in live birth (33.3%
vs. 6.3%). All born babies (3 boys and 2 girls in MSC group and 1 girl in control group) were healthy and were followed up for three months. The mean birth weight of born babies by MSC therapy was 3200-3950 g and weight of child in control group was 3320g.
This invention presents a new approach to restore the chance of childbirth in infertile POR women with an increased chance of natural pregnancy that could exempt them of ovarian stimulation and intracytoplasmic sperm injection (ICSI) challenges. Even though, the women who need to refer to routine ovarian stimulation and ICSI plan could benefit from improving effect of MenSCs therapy in oocyte number and quality, oocyte fertilization rate and embryo quality. Therefore, considering easy accessibility, sampling, isolation and high propagation capability of these cells as autologous source, it seems that this therapeutic approach would have a useful utilization in POR women that tackle with infertility and inevitably should be referred to egg donation. About 30% of infertile women who need advanced techniques to treat infertility, decreased ovarian reserve, respectively. Therefore, according to the population of infertile women around 15-20% and 20-25% of women globally women make up the country, the demand is very high in this treatment.
Claims (15)
- What is claimed that cell therapy is a method based on menstrual blood stem cells (MenSCs) to treat infertility due to diminished ovarian reserve or poor ovarian response, which amends ovarian failure and the number and quality of eggs, fertilization rate, embryo quality, pregnancy rate and live birth rate. In fact, the natural pregnancy after treatment of cells can be used as the most significant data on the treatment to be considered.
- According to claim 1, this patented method is used to treat women with diminished ovarian reserve who have had at least twice a history of IVF failure. Patients should have two characteristics of the following conditions:
- Age equal to or above 40 and under 45 years
-Their Anti-Mullerian Hormone is below 1/1 ng /ml
- The number of antral follicles is less than 5 - According to claim 2, the steps of preparing the product are as follows: On the first to second day of menstruation, menstrual blood is collected by the menstrual cup.
- According to claim 3, menstrual blood is transferred to the production line in a GMP grade-clean room by observing the cold transfer chain (temperature 4 ° C) and the sample is taken to a collection tube containing GMP-grade Dulbecco’s Modified Eagle’s Medium-F12, 2.5 μg/mL fungizone, 100 μg/mL streptomycin, 100 U/mL penicillin and 0.5 mM EDTA in GMP-grade phosphate buffered saline without Ca2+ or Mg2+ will be transferred.
- According to Claim 4, in order to isolate and cultivate MenSCs, the sample is suspended in DMEM-F12 medium, fortified with 5% platelet lysate and then kept in 37 °C CO 2 incubator.
- According to claim 5, the cells adhere to cell culture flask the next day, develop colony formation units and reach 80-90% confluence at day 14. At this time, the cells are passaged using % trypsin-EDTA and divided in multiple cell culture flasks to propagate.
- According to claim 6, cryopreservation and storage of cells in the quarantine cell bank are as follows: The cells are stored in the pre-master cell bank until the necessary quality to enter the master cell bank and working cell banks was determined. After characterizing these cells and confirming all quality control tests, the cells were taken out of quarantine and transferred to other cell banks.
- According to claim 7, the cells are examined for the following characteristics:
• Identity
• Purity
• Stability
• Karyology - According to claim 1, the method designed for sampling and injection of menstrual blood stem cells into the ovary is autologous and does not stimulate the immune response in the recipient. Also, for collecting menstrual blood in women by diminished ovarian reserve through menstrual cup and without imposing pain.
- According to claim 9, menstrual blood stem cells prepared from individuals with diminished ovarian reserve do not develop chromosomal abnormalities with the proposed protocol. Menstrual blood stem cells prepared from individuals with diminished ovarian reserve are mesenchymal in nature and express markers related to mesenchymal stem cells as well as cryopreservation. and melting menstrual blood stem cells does not change their characteristics.
- According to claim 10, injection of 150 microliters of autologous menstrual blood stem cell product with a concentration of 20 million per ml using an ultrasound guide and under anesthesia is suitable for direct injection into the one ovary.
- According to claim 11, intra-ovarian injection of menstrual blood stem cells into patients with diminished ovarian reserve with the present protocol is a safe method and does not cause any early complications such as fever, nausea or late complications such as tumor formation in the infertile woman.
- According to claim 12, menstrual blood stem cells after entering the ovary with the present protocol stimulate follicogenesis in the ovary, increase the number of antral follicles, the number of mature eggs and the formation of quality embryos. In addition, menstrual blood stem cells after entering the ovary modulate sex hormones affecting the patient's ovaries. Injection of menstrual blood stem cells into the ovaries of individuals with diminished ovarian reserve with the proposed protocol also increases the incidence of pregnancy and live birth.
- According to claim 1, elements of the invention include:
1. Selection of patients with diminished ovarian reserve
2.Isolation and culture of menstrual blood-derived stem cells
3- Quality control of cells is as follows: Determination of surface markers of cultured cells by flow cytometry, Mycoplasma evaluation, Sterility test, Endotoxin test, Karyotype assessment
4- Freezing of cells
5- Thawing and passage of frozen cells
6- Injection of cells into patients' ovaries - According to claim 14, new techniques and technical design, as elements of the invention:
1- Establishing a method for isolation and culture of menstrual blood-derived stem cells from women with diminished ovarian reserve
2- Establishing a method for quality control of menstrual blood-derived stem cells
3- Stabilization of a suitable method for freezing, thawing and passage of menstrual blood stem cells
4- Stabilization of the appropriate cell dose for injection into the ovary
5- Stabilization of a suitable method for injecting cells into the ovary
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
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| PCT/IB2020/058379 WO2021038543A1 (en) | 2020-09-09 | 2020-09-09 | Treatment of diminished ovarian reserve using menstrual blood stromal cells |
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| Application Number | Priority Date | Filing Date | Title |
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| PCT/IB2020/058379 WO2021038543A1 (en) | 2020-09-09 | 2020-09-09 | Treatment of diminished ovarian reserve using menstrual blood stromal cells |
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| WO2021038543A1 true WO2021038543A1 (en) | 2021-03-04 |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114196619A (en) * | 2021-12-27 | 2022-03-18 | 深圳博雅感知医疗科技有限公司 | Mobilized peripheral blood concentrated cell therapeutic agent for treating premature ovarian failure |
-
2020
- 2020-09-09 WO PCT/IB2020/058379 patent/WO2021038543A1/en active Application Filing
Non-Patent Citations (4)
| Title |
|---|
| CHEN L. ET AL.: "The multi-functional roles of menstrual blood-derived stem cells in regenerative medicine", STEM CELL RESEARCH & THERAPY, vol. 10, no. 1, 3 January 2019 (2019-01-03), pages 1 - 10, XP055794671 * |
| FARAMARZI H. ET AL.: "The Potential of Menstrual Blood-Derived Stem Cells in Differentiation to Epidermal Lineage: A Preliminary Report", WORLD JOURNAL OF PLASTIC SURGERY, vol. 5, no. 1, January 2016 (2016-01-01), pages 26 - 31, XP055794674 * |
| MANLEY H. ET AL.: "Menstrual Blood-Derived Mesenchymal Stem Cells: Women's Attitudes, Willingness, and Barriers to Donation of Menstrual Blood", JOURNAL OF WOMEN'S HEALTH (LARCHMT), vol. 28, no. 12, 1 December 2019 (2019-12-01), pages 1688 - 1697, XP055794673 * |
| ZAFARDOUST S. ET AL.: "Improvement of Pregnancy Rate and Live Birth Rate in Poor Ovarian Responders by Intraovarian Administration of Autologous Menstrual Blood-Derived- Mesenchymal Stromal Cells: Phase I/II Clinical Trial", STEM CELL REVIEW AND REPORTS, vol. 16, no. 4, 20 March 2020 (2020-03-20), pages 755 - 763, XP037207057, DOI: 10.1007/s12015-020-09969-6 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114196619A (en) * | 2021-12-27 | 2022-03-18 | 深圳博雅感知医疗科技有限公司 | Mobilized peripheral blood concentrated cell therapeutic agent for treating premature ovarian failure |
| CN114196619B (en) * | 2021-12-27 | 2023-11-07 | 深圳博雅感知医疗科技有限公司 | Mobilized peripheral blood concentrated cell therapeutic agent for treating premature ovarian failure |
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