Overcoming Recurrent Pregnancy Loss with Advanced Fertility Techniques: A Case Report on Combined Stem Cell Therapy, In-vitro Activation and Lymphocyte immunotherapy

doi:10.21203/rs.3.rs-5403424/v1

Infertility continues to be one of the most challenging issues that affects a significant number of people worldwide and is often accompanied by advanced maternal age and poor ovarian reserve. These factors also contribute significantly to recurrent miscarriage and secondary infertility. The traditional fertility treatment proves to be a failure in cases where the women have clinically low AMH (anti-Müllerian hormone) levels and multiple miscarriages. Novel therapeutic approaches are required to address such complex case scenarios. A 46-year-old woman with a history of secondary infertility and four miscarriages presented with an AMH level of 0.88. Traditional fertility treatments had previously failed to yield successful outcomes. the patient underwent a combined therapy involving Bone marrow-derived stem cell therapy, laparoscopy and hysteroscopy, lymphocyte immunotherapy (LIT) and in vitro activation. Post-treatment, the woman conceived and delivered a normal baby who previously faced infertility and Recurrent Pregnancy Loss. This case indicates the potential of an innovative combined approach using Bone marrow-derived stem cell therapy, In-vitro Activation and LIT in the treatment of complex fertility problems, especially in women with diminished ovarian reserve and Recurrent Pregnancy Loss.

Recurrent Pregnancy Loss

anti-Müllerian hormone

lymphocyte immunotherapy

Bone marrow-derived stem cell therapy

Miscarriage

In-Vitro Activation

diminished ovarian reserve

The World Health Organisation (WHO) defines infertility as the inability to conceive or maintain a pregnancy to the point of live birth[1]. It strongly contributes to recurrent miscarriages and secondary infertility, often coexisting with advanced maternal age and low ovarian reserve [2]. Traditional reproductive therapies usually fail in women who have had multiple miscarriages and have low anti-Müllerian hormone (AMH) levels, necessitating alternative treatments[3].

Age-related fertility decline, caused by the limited amount of oocytes in females, has become a concern [4]. The aim of regenerative medicine in this field is to reignite the remaining ovarian 'reproductive gold reserve' in patients with diminished ovarian reserve (DOR) [5].

Stem cell biology, specifically bone marrow-derived stem cells, has been identified as a possible strategy to improve ovarian tissue quality and the environment needed for the formation of new follicles, thereby promoting conception and pregnancy[6].

Several strategies for stimulating the growth of dormant follicles have recently been developed, as well as bone marrow-derived stem cell therapy to rejuvenate ovarian tissue and improve the ovarian microenvironment[7].

Lymphocyte Immunisation Therapy (LIT) has emerged as a novel intervention addressing immune dysregulation, one of the underlying causes of recurrent pregnancy loss (RPL) [8].

We present a case of a 46-year-old patient with poor ovarian reserve and RPL who achieved pregnancy using a combined treatment of bone marrow-derived stem cell therapy, in-vitro activation, and lymphocyte immunotherapy.

Patient Information-

On November 9, 2022, a 46-year-old woman arrived at our center. Her husband was 47 and they'd been married for eight years. The patient has secondary infertility and a bad obstetric history. Her menstrual cycle was normal, and her last menstrual period (LMP) was on October 23rd, 2022. Her menstrual period lasts for 4–5 days, and her cycle is 28–30 days long.

She has a history of 4 miscarriages. The first miscarriage occurred in October 2016 at 11 weeks gestation, conceived naturally. The second miscarriage was in July 2017 at 10 weeks gestation, through D&C, where the fetus had Down syndrome. They consulted a genetic counsellor, but everything seemed normal. The third miscarriage occurred in December 2017 at 8 weeks gestation, naturally conceived, with a blighted ovum. Following these miscarriages, they changed their gynaecologist. In May 2019, they underwent 1 IVF cycle, which was unsuccessful, where 13 follicles were seen, 9 mature eggs were retrieved out of which 5 fertilized, and 2 reached the blastocyst stage, but both embryos had genetic abnormalities. In December 2019, they opted for Ayurvedic treatment for 6 months and conceived in July 2020 with no cardiac activity.

Initial Assessment:

Her AMH was 0.88 ng/ml. Her husband’s semen analysis was found to be normal with 82 million counts, and 71% motility. Transvaginal sonography was performed, which revealed that the uterus was anteverted and of normal size, with no malformations. It also revealed two anterior wall subserous/intramural fibroids, with diameters measuring D1 11.9 mm and D2 9.9 mm, as seen in Fig. 1, late secretory phase endometrium, and homogenous myometrium. Both ovaries appear to have a poor reserve. There was no free fluid in the pouch for Douglas.

Fig:1- Two anterior wall subserous/intramural fibroids

Treatment Plan:

On the same day, we counselled them about various treatment options, including IVF with Donor Egg, Ovarian Rejuvenation with Bone Marrow-derived stem cell therapy, In-Vitro Activation, IVF with own egg, LIT therapy, and PGTA.

Then on November 10th, 2022, the patient came for treatment. We performed a Hysteroscopy where the Internal OS was normal, the Bilateral Ostia looked stenosed which was dilated by opening a grasper, the Endometrium was normal, and the uterine cavity was normal. Then Laparoscopy was done, where the uterus was normal, Bilateral Fallopian Tubes on chromopertubation bilateral free spillage of dye was seen, right-sided ovary In vitro Activation was done, and transplantation was done on the left side below the ovarian ligament. Bilateral Intra Ovarian Bone marrow-derived stem cell therapy was done, and a small fimbrial cyst from the right-sided tube was removed. Then on 25th November 2022 patient underwent Lymphocyte Immunotherapy (LIT).

Treatment Rationale:

In vitro Activation (IVA) seeks to activate inactive ovarian follicles, perhaps promoting follicular development and ovulation [9]. The process includes ovarian fragmentation, disruption of ovarian Hippo signalling, and actin polymerization, Implantation of cortical beads, which may improve reproductive results, especially in women with low ovarian reserve and advanced maternal age [10], [11]. (Fig. 2, Fig. 3 and Fig. 4)

Fig:2- Preparation of Ovarian Strips

Fig:3- Transplantation of Cortical beads

Fig:4- Implantation of Cortical beads

Autologous BMDCs demonstrate potential in ovarian rejuvenation through a variety of methods, including differentiation into ovarian cell types, 'homing' to damaged sites for repair, paracrine actions that release growth factors and cytokines, immunological modulation, and angiogenesis enhancement [12]. These effects may improve the ovarian microenvironment and follicular development, especially in cases of diminished ovarian reserve [13].

LIT addresses immune-related causes of recurrent pregnancy loss and implantation failure. Its benefits include dampening inflammatory responses, reducing the risk of immune rejection of the embryo, and fostering a more favourable uterine environment for successful pregnancy [14]. By modulating the maternal immune system, LIT may improve the chances of maintaining a pregnancy in women[15].

When these three techniques are combined, they may address many factors that contribute to infertility and recurrent pregnancy loss, potentially opening up a new therapy option for difficult patients who have not responded to typical fertility therapies.

The patient was prescribed a 6-month course of Letrozole and Dydrogesterone. The patient then went to the USA. She conceived naturally in November 2022.

Follow up and outcome:

At 6 weeks into the pregnancy, on December 30th, 2022, the patient underwent her first sonogram, revealing a robust fetal heartbeat and a baby measuring 3.24 cm.

On January 14th, 2023, during her second visit, the gynecologist noted that the cervix measured 4.3 cm and recorded the baby's heartbeat at 165 bpm. (Fig.-5)

Fig:5- Sonogram of Cervix

Genetic testing for Down syndrome and other chromosomal abnormalities (trisomies 13, 18, and 21) returned negative results. The fetus was determined to be male. (Fig-6)

Fig:6- Sonogram showing the gender of Fetus

The third ultrasound was conducted on February 14th, 2023, revealing a fetal heartbeat of 162 bpm and a cervix length of 4.6 cm. A test for spina bifida was also performed. (Fig.-7)

Fig:7- PCI of Fetal

An anomaly ultrasound scan on March 7th, 2023, showed a fetal heartbeat of 146 bpm, a weight of 11 ounces, and a cervix length of 3.5 cm.

On April 4th, 2023, another scan revealed a fetal heart rate of 147 beats per minute and a weight of 1 pound 11 ounce. (Fig. 8)

Fig:8- Sonogram of Fetus at 2nd Anomaly Ultrasound Scan

On May 4th, 2023, the third anatomy scan was performed, which measured the spine. The infant weighed two pounds fourteen ounces and had a heart rate of 159 beats per minute.

The 31-week scan on June 2nd, 2023, showed the baby weighing 4 pounds 6 ounces with a heartbeat of 138 bpm.

On July 11th, 2023, a healthy baby boy was delivered naturally, weighing 6 pounds 4 ounces. There were no anomalies or defects detected. The Summary of pregnancy follow-up scans and delivery details is given in Table: 1.

Table:1 Summary of pregnancy follow-up scans and delivery details

Date	Scan Type	Details
30th Dec 2022	1st Sonogram (6 weeks)	- Fetal heartbeat: strong - Baby size: 3.24 cm
14th Jan 2023	2nd Appointment	- Fetal heartbeat: strong - Baby size: 3.24 cm
14th Feb 2023	3rd Ultrasound	- Baby's heartbeat: 162 bpm - Cervix length: 4.6 cm - Spina bifida test performed
7th Mar 2023	Anomaly Ultrasound Scan	- Baby's heartbeat: 146 bpm - Weight: 11 ounces - Cervix length: 3.5 cm
4th Apr 2023	2nd Anomaly Ultrasound Scan	- Baby's heartbeat: 147 bpm - Weight: 1 pound 11 ounces
4th May 2023	3rd Anomaly Ultrasound Scan	- Baby's heartbeat: 159 bpm - Weight: 2 pounds 14 ounces - Spine measured
2nd Jun 2023	31 Week Scan	- Baby's weight: 4 pounds 6 ounces - Heartbeat: 138 bpm
11th Jul 2023	Delivery	- Healthy baby boy delivered - Weight: 6 pounds 4 ounces - No anomalies or defects detected

This case study shows the potential efficacy of a combination approach that includes intraovarian stem cell therapy, lymphocyte immunotherapy (LIT), and in vitro activation (IVA) for women with diminished ovarian reserve (DOR) and recurrent pregnancy loss (RPL) who have failed conventional fertility treatments. The successful pregnancy and delivery of a healthy baby by a 46-year-old woman with a history of secondary infertility and four losses illustrates the promise of this new combined method.

The patient's case emphasizes the crucial need for alternate treatments in addressing difficult fertility challenges, especially in older women with substantially reduced ovarian function. The combination of intraovarian stem cell therapy, LIT, and IVA may open up new possibilities in reproductive medicine by rejuvenating ovarian tissue, improving the ovarian microenvironment, modifying the immune response, and stimulating dormant follicles.

It is important to recognize the limitations of relying on a single case study, despite the interesting outcomes observed in this particular case. The use of intraovarian stem cell therapy, lymphocyte immunotherapy (LIT), and in vitro activation (IVA) does not definitively establish its absolute safety and effectiveness. Further research and clinical studies are required to thoroughly determine the safety profile, examine efficacy in a larger sample size, develop acceptable patient selection criteria, and analyse long-term outcomes for both mothers and children.

Future study should focus on improving treatment methods, better understanding the particular roles of each component, and assessing the long-term health of infants born after this therapy. While the results are encouraging, they should be interpreted with care until more extensive evidence is available.

AMH - Anti-Müllerian Hormone, BMDC - Bone Marrow-Derived Cells, DOR - Diminished Ovarian Reserve, IVA - In Vitro Activation, IVF - In Vitro Fertilization, LIT - Lymphocyte Immunotherapy, LMP - Last Menstrual Period, OS - Ostium/Ostia, PGTA - Preimplantation Genetic Testing for Aneuploidy, RPL - Recurrent Pregnancy Loss, WHO - World Health Organization

Acknowledgements:

The authors would like to acknowledge the Institution Review Board (IRB) of Sat Kaival Hospital Pvt Ltd, Anand, Gujarat, for their timely approval of this project. They would also like to acknowledge Akanksha Hospital and Research Institute for funding and technical support during the project.

Ethics Approval: Ethics approval was obtained by Institutional Review Board of Sat Kaival Hospital Pvt Ltd, Anand, Gujarat

Consent to Participate: Informed consent was obtained from all individual participants included in the study.

Consent to Publish: Consent to publish was obtained from the participant, allowing their data and relevant information to be included in this case study

Conflict of Interest: On behalf of all authors, the corresponding author states that there is no conflict of interest.

Availability of data and material- All data underlying the results are available as part of the article and no additional source data are required.

Code availability- Not Applicable

Author Contribution

NP: Concept, experimental design, Investigation, analyses and follow up, Manuscript Writing; NP: Investigation, experimental design; MP: Investigation; HB: Investigation; PG: Investigation and follow up; RP: Investigation; SD: Investigation; PC: Investigation, interpretation of data, Manuscript Writing; KV: Data collection, Manuscript Writing.All authors made significant intellectual contributions in reviewing the manuscript and approved the final article.

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No competing interests reported.

Overcoming Recurrent Pregnancy Loss with Advanced Fertility Techniques: A Case Report on Combined Stem Cell Therapy, In-vitro Activation and Lymphocyte immunotherapy

Status:

Version 1

Abstract

Figures

Introduction

Case Presentation

Patient Information-

Initial Assessment:

Treatment Plan:

Treatment Rationale:

Follow up and outcome:

Discussion

Abbreviations

Declarations

References

Additional Declarations

Status:

Version 1