Cryopreservation and Thawing of Human Ovarian Cortex Tissue Slices.

Cryopreserved ovarian cortex tissue can be used to improve or restore female fertility. It can be used for cancer patients to restore fertility after chemotherapy treatment or for social reasons for women who want to postpone their pregnancy wish. In order to preserve ovarian tissue viability in these cases, the tissue needs to be stored by cryopreservation. In this chapter we describe the entire process chain needed to prepare, transport, and cryopreserve human ovarian cortex tissues as well as to subsequently thaw and implant it.

In vitro maturation of immature oocytes from ovarian tissue prior to shipment to a cryobank.

PURPOSE: Female fertility preservation prior to gonadotoxic therapies can be achieved by the cryopreservation of ovarian cortical tissue. Immature oocytes may be recovered during the preparation, matured in vitro and lead to live births, thereby providing an additional option for fertility preservation. The purpose of this study was to test the feasibility of this approach in a setting with unilateral biopsy of a small piece of ovarian tissue and minimal tissue preparation prior to shipment to an external cryobank. METHODS: A prospective observational clinical study in an academic center was performed from January 2018 through December 2019. Ovarian tissue was obtained laparoscopically. Immature oocytes were recovered by minimal preparation of the tissue before shipment to an external cryobank for cryopreservation. In vitro maturation was performed on recovered immature oocytes. RESULTS: Twelve patients were enrolled. Immature oocytes could be recovered for all. The maturation rate was 38.9% (n = 14/36). Metaphase II (MII) were either directly used for intracytoplasmic sperm injection (ICSI) with a fertilization rate of 66.6% (n = 4/6) or vitrified (n = 8). PNs were cryopreserved (n = 4). Vitrified MII were warmed with a post-warming vitality rate of 75.0% (n = 3/4) and used for ICSI with a fertilization rate of 33.3% (n = 1/3). CONCLUSIONS: Immature oocytes can be successfully retrieved from ovarian tissue through minimal tissue preparation prior to shipment to a cryobank, matured in vitro, fertilized and cryopreserved for potential future fertility treatments. The total number of oocytes available for fertility preservation can be increased even without controlled ovarian stimulation in a situation where only ovarian biopsy for cryopreservation is performed. TRIAL REGISTRATION: German Clinical Trials Register (DRKS), DRKS00013170. Registered 11 December 2017, https://www.drks.de/drks_web/navigate.do?navigationId=trial.HTML&TRIAL_ID=DRKS00013170 .

Serum anti-Mullerian hormone (AMH) concentration has limited prognostic value for density of primordial and primary follicles, questioning it as an accurate parameter for the ovarian reserve.

OBJECTIVES: To evaluate the prognostic value of anti-Mullerian hormone (AMH) levels in estimating the ovarian density of primordial and primary follicles, which can be assumed to reflect the real ovarian reserve. STUDY DESIGN: A total of 537 women, average age 30.4 years (range 8.0-43.7 years), underwent ovarian tissue cryopreservation prior to gonadotoxic therapies due to malignant diseases which do not affect ovarian reserve parameters. Standardized ovarian biopsies were obtained, and follicular density was analysed. The prognostic accuracy of serum AMH in estimating ovarian follicle density was evaluated. MAIN OUTCOME MEASURES: Histologically determined follicle density, AMH serum concentration and their correlation. RESULTS: In children, follicle density was high but AMH concentration was low. AMH concentration was predicted to be maximum at the age of 15.5 years. In women aged over 15.5 years, the relationship between AMH concentration and follicle density was evaluated. Crude analysis revealed that serum AMH levels and follicular density were moderately correlated (r = 0.34, p < 0.001). From the adjusted regression model the predicted value of follicle density of women aged 20, 30 and 40 years as well as the associated 50 % and 95 % prediction intervals (50 % PI and 95 % PI, respectively) were calculated. For example, for women aged 40 years with a serum AMH level of 1 ng/ml, a follicle density of 2.3/mm3 (50 %PI: [1.1, 4.6]; 95 %PI: [0.3, 18]) was predicted. These large prediction intervals demonstrate the low predictive value of serum AMH for the ovarian follicle density. CONCLUSIONS: Serum AMH levels have limited prognostic value for the follicle density and therefore for the real ovarian reserve.

Diagnosis and Therapy Before Assisted Reproductive Treatments. Guideline of the DGGG, OEGGG and SGGG (S2k Level, AWMF Register Number 015-085, February 2019) - Part 1, Basic Assessment of the Woman.

Introduction Supporting and counselling couples with fertility issues prior to starting ART is a multidisciplinary diagnostic and therapeutic challenge. The first German/Austrian/Swiss interdisciplinary S2k guideline on "Diagnosis and Therapy Before Assisted Reproductive Treatments (ART)" was published in February 2019. This guideline was developed in the context of the guidelines program of the German Society of Gynecology and Obstetrics (DGGG) in cooperation with the Swiss Society of Gynecology and Obstetrics (SGGG) and the Austrian Society of Gynecology and Obstetrics (OEGGG). Aims One third of the causes of involuntary childlessness are still unclear, even if the woman or man have numerous possible risk factors. Because the topic is still very much taboo, couples may be socially isolated and often only present quite late to a fertility center. At present, there is no standard treatment concept, as currently no standard multidisciplinary procedures exist for the diagnostic workup and treatment of infertility. The aim of this guideline is to provide physicians with evidence-based recommendations for counselling, diagnostic workup and treatment. Methods This S2k guideline was developed on behalf of the Guidelines Commission of the DGGG by representative members from different professional medical organizations and societies using a structured consensus process. Recommendations The first part of this guideline focuses on the basic assessment of affected women, including standard anatomical and endocrinological diagnostic procedures and examinations into any potential infections. Other areas addressed in this guideline are the immunological workup with an evaluation of the patient's vaccination status, an evaluation of psychological factors, and the collection of data relating to other relevant factors affecting infertility. The second part will focus on explanations of diagnostic procedures compiled in collaboration with specialists from other medical specialties such as andrologists, human geneticists and oncologists.

Diagnosis and Treatment Before Assisted Reproductive Treatments. Guideline of the DGGG, OEGGG and SGGG (S2k Level, AWMF Register Number 015-085, February 2019) - Part 2, Hemostaseology, Andrology, Genetics and History of Malignant Disease.

Introduction Supporting and counselling couples with fertility issues prior to starting ART is a multidisciplinary diagnostic and therapeutic challenge. The first German-language interdisciplinary S2k guideline on "Diagnosis and Therapy Before Assisted Reproductive Treatments (ART)" was published in February 2019. The guideline was developed in the context of the guidelines program of the German Society of Gynecology and Obstetrics (DGGG) in cooperation with the Swiss Society of Gynecology and Obstetrics (SGGG) and the Austrian Society of Gynecology and Obstetrics (OEGGG). Aim In one third of cases, the cause of involuntary childlessness remains unclear, even if the woman or man have numerous possible risk factors. Because the topic is still very much taboo, couples may be socially isolated and often only present quite late to a fertility center. There is no standard treatment concept for these patients at present, as there are currently no standard multidisciplinary procedures for the diagnostic workup and treatment of infertility. The aim of this guideline is to provide physicians with evidence-based recommendations for counselling, diagnosis and treatment. Methods This S2k guideline was developed on behalf of the Guidelines Commission of the DGGG by representative members from different professional medical organizations and societies using a structured consensus process. Recommendations This second part of the guideline describes the hematological workup for women as well as additional diagnostic procedures which can be used to investigate couples and which are carried out in cooperation with physicians working in other medical fields such as andrologists, geneticists and oncologists.

Overnight ovarian tissue transportation for centralized cryobanking: a feasible option.

RESEARCH QUESTION: Is overnight transportation of ovarian tissue before cryopreservation in a centralized cryobank from the FertiPROTEKT network feasible? DESIGN: Data from 1810 women with cryopreserved ovarian tissue after overnight transportation from December 2000 to December 2017 were analysed with a focus on transportation, tissue activity parameters and pregnancy, and delivery rates after transplantation. RESULTS: A total of 92.4% of tissue samples arrived at ideal temperatures of 2-8°C, 0.4% were transported at temperatures lower than ideal and 6.4% were transported at temperatures that were too high, generally due to mishandling of the inlayed cool packs of the transportation boxes. In 62 women, 78 tissue transplantations were carried out. A subgroup of 30 women who underwent a single orthotopic transplantation with fulfilled criteria of a complete follow-up after transplantation until the end of study, a premature ovarian insufficiency after gonadotoxic therapy as well as the absence of pelvic radiation, was further analysed. In this group, transplantations into a peritoneal pocket accounted for 90%. Transplants were still active at 1 year and above after transplantation in 93.3%. Pregnancy and delivery rates were 46.7% and 43.3%, respectively, with one ongoing pregnancy at the end of the study. CONCLUSIONS: Overnight transportation for central cryobanking is a feasible concept that results in high reproducible success rates through standardized professional tissue freezing and storage.

Concept Paper on the Technique of Cryopreservation, Removal and Transplantation of Ovarian Tissue for Fertility Preservation.

The cryopreservation of ovarian tissue with subsequent transplantation of the tissue represents an established method of fertility protection for female patients who have to undergo gonadotoxic therapy. The procedure can be performed at any point in the cycle and thus generally does not lead to any delay in oncological therapy. With the aid of this procedure, more than 130 births to date worldwide have been able to be recorded. The birth rate is currently approximately 30% and it can be assumed that this will increase through the further optimisation of the cryopreservation and surgical technique. The concept paper presented here is intended to provide guidance for managing cryopreservation and transplantation of ovarian tissue to German-speaking reproductive medicine centres.

Fertility Preservation for Patients with Malignant Disease. Guideline of the DGGG, DGU and DGRM (S2k-Level, AWMF Registry No. 015/082, November 2017) - Recommendations and Statements for Girls and Women.

AIM: The aim of this official guideline published by the German Society of Gynecology and Obstetrics (DGGG) and coordinated with the German Society of Urology (DGU) and the German Society of Reproductive Medicine (DGRM) is to provide consensus-based recommendations, obtained by evaluating the relevant literature, on counseling and fertility preservation for prepubertal girls and boys as well as patients of reproductive age. Statements and recommendations for girls and women are presented below. Statements or recommendations for boys and men are not the focus of this guideline. METHODS: This S2k guideline was developed at the suggestion of the guideline commission of the DGGG, DGU and DGRM and represents the structured consensus of representative members from various professional associations (n = 40). RECOMMENDATIONS: The guideline provides recommendations on counseling and fertility preservation for women and girls which take account of the patient's personal circumstances, the planned oncologic therapy and the individual risk profile as well as the preferred approach for selected tumor entities.

[Fertility Preservation in Prepubertal und Pubertal Children and Adolescents]. / Fertilitätserhalt bei präpubertären und pubertären Kindern und Jugendlichen.

BACKGROUND: Due to rising survival rates in cancer and autoimmune diseases fertility preservation before gonadotoxic therapies has become increasingly important. Although fertility can be significantly affected by gonadotoxic therapies, the possibility of fertility preservation during childhood has not been sufficiently considered so far. METHODS: Selective literature research with presentation of fertility preservation methods, their indications, implementations, risks and efficacy. RESULTS: Measures are indicated in all girls and boys at high risk of gonadal damage. The complexity of the techniques requires special expertise in the counseling and implementation, which is offered to girls in counselling Germany especially in the centers of FertiPROTEKT (www.fertiprotekt.com). In girls, mainly cryopreservation of ovary tissue is considered. In postpubertal girls cryopreservation of oocytes is also possible. In postpubertal boys sperm can be preserved. Freezing of testicular tissue is still experimental in prepubertal boys. Success rates are still difficult to quantify; birth rates of about 50% are discussed. All procedures are not covered by health insurance. CONCLUSION: In children and adolescents, measures of fertility preservation should be considered in cases of highly gonadotoxic therapies, and appropriate advice should be given by specialists.

Fertility protection: complications of surgery and results of removal and transplantation of ovarian tissue.

Fertility-preserving measures are becoming important for patients receiving oncological treatment. One method involves cryopreservation of ovarian tissue and transplanting it when treatment is completed. We report complications resulting from surgical and fertility medicine, and the results of procedures for the removal and transplantation of ovarian tissue carried out within the FertiProtekt network. A survey using a structured questionnaire was conducted among the FertiProtekt network centres between November 2015 and June 2016. The analysis included surgical techniques used to remove and transplant ovarian tissue, surgical complications and results. Laparoscopic removal and transplantation of ovarian tissue have a low risk of complications. Surgical complications occurred in three of the network's 1373 ovarian tissue removals (n = 1302) and transplantations (n = 71); two complications (0.2%) occurred during removal and one during transplantation. Menstruation resumed in 47 out of 58 women (81%) who underwent ovarian tissue transplantation. Hormonal activity occurred in 63.2% of transplantations with a follow-up of 6 months or over. Sixteen pregnancies occurred in 14 patients, with nine births. The risks and complications of removal and transplantation of ovarian tissue are similar to those of standard laparoscopy. These procedures are becoming standard for fertility protection in cancer patients.

Repetitive Maturation of Oocytes From Non-Stimulated Xenografted Ovarian Tissue From a Prepubertal Patient Indicating the Independence of Human Ovarian Tissue.

INTRODUCTION: Modern anti-cancer strategies have distinctly increased survival rates; nevertheless, often accompanied by sterility. Currently, the only option for preserving fertility in prepubertal females is to cryopreserve ovarian tissue and re-transplant frozen-thawed tissue to restore fertility after treatment. Our aim was to report the occurrence of repetitive antral follicle formation and oocyte maturation in a prepubescent ovarian tissue xenograft without exogenous hormone stimulation. MATERIAL AND METHODS: Frozen-thawed ovarian tissue from a 6-year-old patient suffering from nephroblastoma was xenotransplanted in oophorectomized severe combined immunodeficiency (SCID) mice to evaluate follicle development. ERGEBNISSE: Repetitive follicle development to the antral stage occurred in the same xenograft of prepubertal ovarian tissue without exogenous hormone administration; 37 days after retrieving a maturing oocyte (this first retrieval has been previously published), another, completely mature oocyte was harvested from the xenograft. Subsequent histological evaluation of the grafted tissue showed primordial follicles, nearly all stages of developing follicles, as well as large atretic ones. Many clusters with dormant primordial follicles were also present. CONCLUSION: Xenotransplanted prepubertal ovarian tissue has the potential for repetitive oocyte retrieval cycles without administering exogenous hormones. The results indicate that the human ovarian tissue might be able to synchronize the hypothalamus-hypophysis-axes of the mouse to the physiological human cycle; this should be investigated in future studies.

Chapter 15 Development of a Nationwide Network for Ovarian Tissue Cryopreservation.

Ovarian tissue cryopreservation is gaining much interest since the publication of the first live birth after retransplantation of frozen-thawed tissue in 2004 (Donnez et al., Lancet 364:1405-1410, 2004). In contrast to cryopreservation of gametes and embryos, ovarian tissue freezing is a complex requiring a proper approach in order to make this a viable option for fertility preservation of cancer patients. Due to the need in terms of laboratory space, equipment, personnel, and adequate logistics, an ovarian tissue cryobank is most economic if managed as a centralized service unit that interacts with numerous clinics covering the surgical part. Transportation of ovarian tissue under appropriate conditions from the surgical unit to the cryobank for subsequent preparation and freezing has been shown to have no impact on cryo-survival (Schmidt et al., Hum Reprod 18:2654-2659, 2003; Isachenko et al., Fertil Steril 91:1556-1559, 2009). Several children have been born after retransplantation of such tissue that was derived from the cryobank in Bonn, Germany (Homepage FertiPROTEKT. http://www.fertiprotekt.de ). This cryobank is one of the largest in the world with more than 1300 tissue samples that were frozen from 2003 until today. It is integrated in the network FertiPROTEKT (Homepage FertiPROTEKT. http://www.fertiprotekt.de ) and is served by 108 surgical centers that are located all over Germany. The concept of this cryobank is a blueprint for success and has recently been used for another regionally centralized cryobank in Beijing, China. In this chapter the most important topics that need to be considered while creating a centralized cryobank within a national or regional network are highlighted.

Fertility-preservation counselling and treatment for medical reasons: data from a multinational network of over 5000 women.

Fertility-preservation techniques for medical reasons are increasingly offered in national networks. Knowledge of the characteristics of counselled patients and techniques used are essential. The FertiPROTEKT network registry was analysed between 2007 and 2013, and included up to 85 university and non-university centres in Germany, Austria and Switzerland; 5159 women were counselled and 4060 women underwent fertility preservation. In 2013, fertility-preservation counselling for medical reasons increased significantly among nullipara and women aged between 21 and 35 years (n = 1043; P < 0.001). Frequency of GnRH applications slowly decreased, whereas tissue, oocytes and zygote cryopreservation increased. In 2013, women with breast cancer mainly opted for tissue freezing, whereas women with lymphoma opted for GnRH agonist. Women younger than 20 years predominantly opted for GnRH agonists and ovarian tissue cryopreservation; women aged between 20 and 40 years underwent a variety of techniques; and women over 40 years opted for GnRH agonists. The average number of aspirated oocytes per stimulation cycle decreased as age increased (< 30 years: 12.9; 31-35 years: 12.3; 36-46: 9.0; > 41 years: 5.7). For ovarian tissue cryopreservation, removal and cryopreservation of fewer than one ovary was preferred and carried out in 97% of cases in 2013.

Spontaneous antral follicle formation and metaphase II oocyte from a non-stimulated prepubertal ovarian tissue xenotransplant.

BACKGROUND: Current strategies in cancer treatment have markedly increased the rates of remission and survival for cancer patients, but are often associated with subsequent sterility. While there are various options available to an adult female depending on the patient's particular situation, the only realistic option for preserving fertility in prepubertal females is to cryopreserve ovarian tissue. This is the first report of a morphologically mature oocyte collected from non-stimulated prepubertal ovarian tissue xenotransplants. METHODS: Ovarian tissue from a 6 year old patient suffering from nephroblastoma was removed and cryopreserved for fertility preservation. The frozen-thawed ovarian tissue fragments were xenotransplanted to bilaterally oophorectomized severe combined immunodeficiency (SCID) mice to assess follicle development. RESULTS: Antral follicle formation occurred post-xenotransplantation in a single ovarian fragment without exogenous hormone stimulation. A morphologically maturing oocyte was harvested from these follicles. CONCLUSIONS: Prepubertal human ovarian follicles and oocytes can be matured after xenotransplantation even without exogenous hormone stimulation. These results indicate that tissue collected from prepubertal patients can support fertility in cancer survivors.

The impact of culture conditions on early follicle recruitment and growth from human ovarian cortex biopsies in vitro.

OBJECTIVE: To investigate the effects of a dynamic fluidic culture system on early in vitro folliculogenesis in standardized ovarian cortex biopsies. DESIGN: Cortical small strips were cultured for 6 days in a conventional static or in a dynamic fluidic culture system. SETTING: University-affiliated laboratory with an associated cryobank facility. PATIENT(S): Ovarian cortex from postpuberal female cancer patients (26.1 ± 1.3 y) who opted for cryopreservation of their tissue for fertility protection before gonadotoxic cancer therapy. With informed consent of the Institutional Ethics Committee, part of the tissue was available for patient-related research studies. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): The viability and proliferative capacity of the cortex biopsies were evaluated by chemiluminescent microparticle immunoassay for detection of in vitro produced E2 and P in the supernate, by viable follicle counting via calcein staining, by histologic analyses, and by total RNA preparation and reverse transcription for real-time polymerase chain reaction of selected early folliculogenesis genes. RESULT(S): The data support the notion that early follicle development can be better achieved in vitro in a dynamic fluidic culture system. The findings are based on the presence of more viable follicles, higher expression levels of early folliculogenesis genes KIT-L, INHB, and GDF9, and the absence of premature luteinization of follicles. CONCLUSION(S): This study provides evidence that dynamic fluidic culture is a promising approach for investigating early follicular recruitment and growth in cortical biopsies. It may serve as a first step in a multistep culture system to design a complex in vitro system for complete folliculogenesis.