We would like to thank the healthcare professionals who helped us with participant recruitment and tissue collection and all the patients who donated their ovarian tissue. We also acknowledge the Childhood Cancer Fund (PR2020-0096) and Karolinska Institutet PhD funding KID, whose funding made this work possible. We would also like to thank the Morphological Phenotype Analysis facility for the preparation of the histological slides. We acknowledge Nicola Byers for language editing this manuscript.

All of the procedures conducted were approved by the Swedish Ethical Review Authority (Sveafertil patients, Dnr: 2019-03802) and by the Ethics Committee of Helsinki University Hospital (HCH patients, Dnr 340/13/03/03/2015). Written and oral information about the study were provided by the research nurses, and informed consent was obtained in accordance with the Declaration of Helsinki. For patients under 18 years old, informed consent was obtained from the legal guardians. All samples were pseudonymized and processed acc...

Assessing Ovarian Reserve in Young Patients Using Z-Scores for Follicle Density

<ol>
	<li>Richards, J. A. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+ovarian+cycle.">The ovarian cycle.</a> Vitam Horm. 107, 1-25 (2018).</li><li>Wallace, W. H. B., Kelsey, T. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+ovarian+reserve+from+conception+to+the+menopause.">Human ovarian reserve from conception to the menopause.</a> PLoS One. 5 (1), 1-9 (2010).</li><li>Monniaux, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+ovarian+reserve+of+primordial+follicles+and+the+dynamic+reserve+of+antral+growing+follicles:+What+is+the+link.">The ovarian reserve of primordial follicles and the dynamic reserve of antral growing follicles: What is the link.</a> Biol Reprod. 90 (4), 1-11 (2014).</li><li>Gougeon, A., Trounson, A., Gosden, R., Eichenlaub-Ritter, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The early stages of follicular growth in biology and pathology of the oocyte. , 50-61 (2013).</li><li>Gougeon, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+ovarian+follicular+development:+From+activation+of+resting+follicles+to+preovulatory+maturation.">Human ovarian follicular development: From activation of resting follicles to preovulatory maturation.</a> Ann Endocrinol. 71 (3), 132-143 (2010).</li><li>Hansen, K. R., Craig, L. B., Zavy, M. T., Klein, N. A., Soules, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ovarian+primordial+and+non-growing+follicle+counts+according+to+the+stages+of+reproductive+aging+workshop+(STRAW)+staging+system.">Ovarian primordial and non-growing follicle counts according to the stages of reproductive aging workshop (STRAW) staging system.</a> Menopause. 19 (2), 164-171 (2012).</li><li>Gold, E. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+timing+of+the+age+at+which+natural+menopause+occurs.">The timing of the age at which natural menopause occurs.</a> Obstet Gynecol Clin North Am. 38 (3), 425-440 (2011).</li><li>Alberico, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Workflow+optimization+for+identification+of+female+germline+or+oogonial+stem+cells+in+human+ovarian+cortex+using+single-cell+RNA+sequence+analysis.">Workflow optimization for identification of female germline or oogonial stem cells in human ovarian cortex using single-cell RNA sequence analysis.</a> Stem Cells. 40, 523-536 (2022).</li><li>White, Y. A. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oocyte+formation+by+mitotically+active+germ+cells+purified+from+ovaries+of+reproductive-age+women.">Oocyte formation by mitotically active germ cells purified from ovaries of reproductive-age women.</a> Nat Med. 18 (3), 413-422 (2012).</li><li>Wagner, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-cell+analysis+of+human+ovarian+cortex+identifies+distinct+cell+populations+but+no+oogonial+stem+cells.">Single-cell analysis of human ovarian cortex identifies distinct cell populations but no oogonial stem cells.</a> Nat Commun. 11, 1147 (2020).</li><li>Yoshihara, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+reply:+Revisiting+claims+of+the+continued+absence+of+functional+germline+stem+cells+in+adult+ovaries.">In reply: Revisiting claims of the continued absence of functional germline stem cells in adult ovaries.</a> Stem Cells. 41 (2), 205-206 (2023).</li><li>Yoshihara, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+continued+absence+of+functional+germline+stem+cells+in+adult+ovaries.">The continued absence of functional germline stem cells in adult ovaries.</a> Stem Cells. 41, 105-110 (2023).</li><li>Van Kasteren, Y. M., Schoemaker, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Premature+ovarian+failure:+A+systematic+review+on+therapeutic+interventions+to+restore+ovarian+function+and+achieve+pregnancy.">Premature ovarian failure: A systematic review on therapeutic interventions to restore ovarian function and achieve pregnancy.</a> Hum Reprod Update. 5 (5), 483-492 (1999).</li><li>McGlacken-Byrne, S. M., Conway, G. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Premature+ovarian+insufficiency.">Premature ovarian insufficiency.</a> Best Pract Res Clin Obstet Gynaecol. 81, 98-110 (2022).</li><li>Viuff, M., Gravholt, C. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Turner+syndrome+and+fertility.">Turner syndrome and fertility.</a> Ann Endocrinol. 83 (4), 244-249 (2022).</li><li>Poirot, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ovarian+tissue+cryopreservation+for+fertility+preservation+in+418+girls+and+adolescents+up+to+15+years+of+age+facing+highly+gonadotoxic+treatment:+Twenty+years+of+experience+at+a+single+center.">Ovarian tissue cryopreservation for fertility preservation in 418 girls and adolescents up to 15 years of age facing highly gonadotoxic treatment: Twenty years of experience at a single center.</a> Acta Obstet Gynecol Scand. 98 (5), 630-637 (2019).</li><li>Jensen, A. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cryopreservation+of+ovarian+tissue+for+fertility+preservation+in+a+large+cohort+of+young+girls:+Focus+on+pubertal+development.">Cryopreservation of ovarian tissue for fertility preservation in a large cohort of young girls: Focus on pubertal development.</a> Hum Reprod. 32 (1), 154-164 (2017).</li><li>The Practice Committee of the American Society for Reproductive Medicine. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fertility+preservation+in+patients+undergoing+gonadotoxic+therapy+or+gonadectomy:+A+committee+opinion.">Fertility preservation in patients undergoing gonadotoxic therapy or gonadectomy: A committee opinion.</a> Fertil Steril. 112 (6), 1022-1033 (2019).</li><li>. ART Fact Sheet Available from: <a target="_blank" href="https://www.eshre.eu/Press-Room/Resources">https://www.eshre.eu/Press-Room/Resources</a> (2020)</li><li>Antonouli, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+comprehensive+review+and+update+on+human+fertility+cryopreservation+methods+and+tools.">A comprehensive review and update on human fertility cryopreservation methods and tools.</a> Front Vet Sci. 10, 1151254 (2023).</li><li>Bahroudi, Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Review+of+ovarian+tissue+cryopreservation+techniques+for+fertility+preservation.">Review of ovarian tissue cryopreservation techniques for fertility preservation.</a> J Gynecol Obstet Hum Reprod. 51 (2), 102290 (2022).</li><li>Jensen, A. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=86+successful+births+and+9+ongoing+pregnancies+worldwide+in+women+transplanted+with+frozen-thawed+ovarian+tissue:+Focus+on+birth+and+perinatal+outcome+in+40+of+these+children.">86 successful births and 9 ongoing pregnancies worldwide in women transplanted with frozen-thawed ovarian tissue: Focus on birth and perinatal outcome in 40 of these children.</a> J Assist Reprod Genet. 34 (3), 325-336 (2017).</li><li>Donnez, J., Dolmans, M. -. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fertility+preservation+in+women.">Fertility preservation in women.</a> N Engl J Med. 377 (17), 1657-1665 (2017).</li><li>Dolmans, M. M., Falcone, T., Patrizio, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Importance+of+patient+selection+to+analyze+in+vitro+fertilization+outcome+with+transplanted+cryopreserved+ovarian+tissue.">Importance of patient selection to analyze in vitro fertilization outcome with transplanted cryopreserved ovarian tissue.</a> Fertil Steril. 114 (2), 279-280 (2020).</li><li>Demeestere, I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Live+birth+after+autograft+of+ovarian+tissue+cryopreserved+during+childhood.">Live birth after autograft of ovarian tissue cryopreserved during childhood.</a> Hum Reprod. 30 (9), 2107-2109 (2015).</li><li>Ernst, E., Kj&#230;rsgaard, M., Birkeb&#230;k, N. H., Clausen, N., Andersen, C. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Case+report:+Stimulation+of+puberty+in+a+girl+with+chemo-+and+radiation+therapy-induced+ovarian+failure+by+transplantation+of+a+small+part+of+her+frozen/thawed+ovarian+tissue.">Case report: Stimulation of puberty in a girl with chemo- and radiation therapy-induced ovarian failure by transplantation of a small part of her frozen/thawed ovarian tissue.</a> Eur J Cancer. 49 (4), 911-914 (2013).</li><li>Rodriguez-Wallberg, K. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Successful+pregnancies+after+transplantation+of+ovarian+tissue+retrieved+and+cryopreserved+at+time+of+childhood+acute+lymphoblastic+leukemia+-+a+case+report.">Successful pregnancies after transplantation of ovarian tissue retrieved and cryopreserved at time of childhood acute lymphoblastic leukemia - a case report.</a> Haematologica. 106 (10), 2783-2787 (2021).</li><li>Matthews, S. J., Picton, H., Ernst, E., Andersen, C. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Successful+pregnancy+in+a+woman+previously+suffering+from+&#946;-thalassemia+following+transplantation+of+ovarian+tissue+cryopreserved+before+puberty.">Successful pregnancy in a woman previously suffering from &#946;-thalassemia following transplantation of ovarian tissue cryopreserved before puberty.</a> Minerva Ginecol. 70 (4), 432-435 (2018).</li><li>Kristensen, S. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+cryopreserved+ovarian+tissue+in+the+Danish+fertility+preservation+cohort.">Use of cryopreserved ovarian tissue in the Danish fertility preservation cohort.</a> Fertil Steril. 116 (4), 1098-1106 (2021).</li><li>Donfack, N. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expectations+and+limitations+of+ovarian+tissue+transplantation.">Expectations and limitations of ovarian tissue transplantation.</a> Zygote. 25 (4), 391-403 (2017).</li><li>Schmidt, K. L. T., Byskov, A. G., Andersen, A. N., M&#252;ller, J., Andersen, C. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Density+and+distribution+of+primordial+follicles+in+single+pieces+of+cortex+from+21+patients+and+in+individual+pieces+of+cortex+from+three+entire+human+ovaries.">Density and distribution of primordial follicles in single pieces of cortex from 21 patients and in individual pieces of cortex from three entire human ovaries.</a> Hum Reprod. 18 (6), 1158-1164 (2003).</li><li>Hassan, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reference+standards+for+follicular+density+in+ovarian+cortex+from+birth+to+sexual+maturity.">Reference standards for follicular density in ovarian cortex from birth to sexual maturity.</a> Reprod Biomed Online. 47, 103287 (2023).</li><li>Vilela, J. d. e. M. V., Dolmans, M. M., Amorim, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ovarian+tissue+transportation:+A+systematic+review.">Ovarian tissue transportation: A systematic review.</a> Reprod Biomed Online. 42 (2), 351-365 (2021).</li><li>Gibson, E., Mahdy, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Anatomy, abdomen and pelvis, ovary. , (2023).</li><li>Nagaraj, A. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Establishment+and+analysis+of+tumor+slice+explants+as+a+prerequisite+for+diagnostic+testing.">Establishment and analysis of tumor slice explants as a prerequisite for diagnostic testing.</a> J Vis Exp. (141), e58569 (2018).</li><li>Adeniran, B. V., Bjarkadottir, B. D., Appeltant, R., Lane, S., Williams, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improved+preservation+of+ovarian+tissue+morphology+that+is+compatible+with+antigen+detection+using+a+fixative+mixture+of+formalin+and+acetic+acid.">Improved preservation of ovarian tissue morphology that is compatible with antigen detection using a fixative mixture of formalin and acetic acid.</a> Hum Reprod. 36 (7), 1871-1890 (2021).</li><li>Bankhead, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=QuPath:+Open-source+software+for+digital+pathology+image+analysis.">QuPath: Open-source software for digital pathology image analysis.</a> Sci Rep. 7 (1), 1-7 (2017).</li><li>Bj&#246;rvang, R. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Persistent+organic+pollutants+and+the+size+of+ovarian+reserve+in+reproductive-aged+women.">Persistent organic pollutants and the size of ovarian reserve in reproductive-aged women.</a> Environ Int. 155, 106589 (2021).</li><li>Kwok, R., Johnson, N. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ovarian+biopsy+has+no+role+as+a+routine+diagnostic+test+of+ovarian+reserve:+A+systematic+review.">Ovarian biopsy has no role as a routine diagnostic test of ovarian reserve: A systematic review.</a> Reprod Biomed Online. 24, 492-495 (2012).</li><li>Ahmad, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-throughput+spatial+sensitive,+quantitative+phase+microscopy+using+low+spatial+and+high+temporal+coherent+illumination.">High-throughput spatial sensitive, quantitative phase microscopy using low spatial and high temporal coherent illumination.</a> Sci Rep. 11 (1), 1-13 (2021).</li></ol>

The authors have no conflicts of interest.

Women are born with a finite pool of oocytes, which cannot be replenished. Ovarian reserve is a key determinant of a woman&#39;s reproductive potential. This article outlines a method for the histological quantification of follicle density and its comparison to reference standards using Z-scores of ovarian cortical follicle densities in young patients and women up to 25 years of age.
In this protocol, ovarian tissue is obtained from girls and women at high or very high risk of infertility unde...

The ovary is a dynamic, endocrinologically active organ, consisting of an outer cortex and an inner medulla. Heterogeneously distributed within the ovaries are follicles, which contain the gonadal oocyte surrounded by stromal granulosa cells1. All follicles are already formed during fetal development, peaking at around 20 weeks of gestational age, followed by an exponential decline. At birth, 1-2 million primordial follicles remain within the cortex, and only a few hundred thousand follicles survive until puberty2. The non-growing follicle pool, also referred to as the ovarian reserve, influences a woman&#39;s fertility, among other factors. The ovarian reserve resides within the cortex and moves into the medulla as the follicles grow and mature. Some researchers recognize the ovarian reserve as being comprised of primordial follicles only, while others include all unilaminar follicles (i.e., primordial, intermediary, and primary)3,4,5,6. During a woman&#39;s lifetime, only 300-400 follicles mature and survive to eventually undergo ovulation. Menopause commences when the follicle number drops to around a thousand, which typically occurs at around 50 years of age2,7. There is currently an ongoing debate regarding the presence of oogonial stem cells (OSCs) in adult human ovaries that can form oocytes after xenotransplantation. Some studies suggest isolating cells that express a gene profile of primitive germ cells and differentiate into oocytes8,9, while others indicate that the alleged OSCs are perivascular cells of the blood vessels10,11,12.
Premature ovarian insufficiency (POI) is defined by the discontinuation of the menstrual cycle with raised FSH levels before the age of 40 years13. The causes of POI are multifactorial and can occur idiopathically; however, it often arises as a result of medical conditions14. For example, certain congenital genetic conditions are associated with POI due to a reduced ovarian reserve already at birth, such as Turner&#39;s syndrome15. POI can also occur as a side effect of gonadotoxic treatments, which is particularly problematic in very young patients as it may lead to an inability to undergo normal puberty and drastically impair the ovarian reserve. Depending on the risk of POI and the patient&#39;s age, several fertility preservation options are available, such as cryopreservation of mature oocytes, cryopreservation of embryos after in vitro fertilization, and ovarian tissue cryopreservation (OTC). For pre-pubertal patients, OTC is the only feasible option due to their sexual immaturity16,17. In the United States, fertility preservation through OTC has recently been considered an acceptable clinical routine18. However, OTC is still considered experimental in many European countries, and ethical approval is required, particularly for auto-transplantation19,20. Therefore, inclusion criteria for OTC protocols must be carefully considered, and a risk-benefit analysis should be performed for each patient. Not all children receiving gonadotoxic therapies are eligible for fertility preservation. In Sweden, only patients with high or very high risk are recommended to undergo fertility preservation in accordance with the guidelines on fertility preservation of the Nordic Society of Paediatric Haematology and Oncology21. This group includes (1) pre- and post-pubertal children requiring stem cell transplantation or receiving radiation therapy with the ovary in the field of range, and (2) post-pubertal children with high cumulative doses of alkylating chemotherapy. For girls before menarche, inclusion criteria for very high risk of infertility include those treated with any of the following: radiation dose &#62;10 Gy to the ovary, or allogeneic or autologous hematopoietic stem cell therapy (HSCT). Exclusion criteria include those who need additional blood products for other anticoagulant preparations before surgery or have a neutrophil count &#60;1. For girls/women after menarche, inclusion criteria for high or very high risk of infertility include those treated with any of the following: radiation dose &#62;10 Gy to the ovary, or allogeneic HSCT, cyclophosphamide &#62;9 g/m&#178;, ifosfamide &#62;60 g/m&#178;, procarbazine, or nitrosourea compounds (BCNU/CCNU) &#62;360 mg/m&#178;. Exclusion criteria include those with an increased risk of bleeding, increased risk of infection, increased risk of surgical complications, or increased risk of pain problems as assessed by the physician in charge.
For OTC, ovarian tissue is collected during abdominal surgeries by laparotomy or laparoscopy through unilateral oophorectomy or biopsy. The medulla is removed from the collected ovarian tissue, and the remaining cortex is then sectioned into smaller fragments for cryopreservation through controlled-rate freezing or vitrification for later use in fertility restoration22. The samples are then stored in liquid nitrogen until the patient recovers from her treatment and wishes to start a family. Depending on the type of cancer and in the absence of contraindications to ovarian tissue transplantation, the tissue is then thawed and autografted to restore hormonal and ovarian function. OTC has been proven successful, with a 50% pregnancy rate and more than 200 live births reported after grafting of cryopreserved ovarian tissue collected during adulthood23,24,25. Reported live births after auto-transplantation of ovarian tissue collected during childhood are few, and none have been reported after auto-transplantation of undoubtedly pre-pubertal ovarian tissue26,27,28,29,30. Tissue quality is a prerequisite for successful fertility restoration31. Therefore, it is essential to study the quality of the collected tissue samples.
Currently, a way of estimating the quality of the ovarian tissue is through histological sections and manual counting of the follicles. However, assessing the normality of follicle density is difficult due to the natural decline of the reserve with increasing age and the heterogeneous distribution of follicles. Additionally, comparison to reference values is challenging as they are not standardized for biopsy size and age. Other assessment options, such as AMH serum levels or staining of digested tissue with calcein or neutral red, are either not well established or come with their own limitations. Wallace and Kelsey previously published a model of normal ovarian reserve over age by collecting data on follicle numbers from whole ovaries of patients with no known ovarian pathologies2. Furthermore, as biopsies are not representative of the whole ovary, Schmidt et al. developed a method for estimating the total number of follicles based on cortical biopsies32. Using a modified version of this method and the model of normal ovarian reserve, a reference dataset for follicle density of women aged 0-25 years was established33.
This article describes the strategy for assessing the normality of follicle density based on small ovarian&#160;samples by (1) direct quantification of the follicles using histology and (2) comparison of the results to age-normalized standard references via Z-scores. The use of Z-scores describes the relationship of an ovarian biopy&#39;s follicle density to an age- and size-standardized reference group mean. This will be of great value for physicians in counseling their patients for auto-transplantation and further treatment after fertility preservation.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1x Dulbecco&#39;s Phosphate Buffered Saline</td>
			<td>Thermo Fisher</td>
			<td>14190144</td>
			<td></td>
		</tr>
		<tr>
			<td>1x Phosphate Buffered Saline w/ CaCl2, MgCl2, 1 g/L D-glucose, 36 mg/L pyruvate&#160;</td>
			<td>Thermo Fisher</td>
			<td>14287-080</td>
			<td></td>
		</tr>
		<tr>
			<td>37% Hydrochloric acid</td>
			<td>Sigma-Aldrich</td>
			<td>1.09057.1000</td>
			<td></td>
		</tr>
		<tr>
			<td>70% ethanol</td>
			<td>Histolabs</td>
			<td>01370.01L</td>
			<td></td>
		</tr>
		<tr>
			<td>95% ethanol</td>
			<td>Fisher scientific</td>
			<td>10572143</td>
			<td></td>
		</tr>
		<tr>
			<td>Absolute ethanol</td>
			<td>Histolabs</td>
			<td>01399.01L</td>
			<td></td>
		</tr>
		<tr>
			<td>Bouin&#39;s solution</td>
			<td>Sigma-Aldrich</td>
			<td>HT10132-1L</td>
			<td></td>
		</tr>
		<tr>
			<td>Eosin Y Free acid</td>
			<td>Sigma-Aldrich</td>
			<td>E4009-5G&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Formaldehyde</td>
			<td>Thermo Fisher</td>
			<td>28908</td>
			<td></td>
		</tr>
		<tr>
			<td>Haematoxylin solution according to Delafield</td>
			<td>Sigma-Aldrich</td>
			<td>03971-250ML</td>
			<td></td>
		</tr>
		<tr>
			<td>ImageJ</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Microsoft Excel</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Microtome&#160;</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Pertex mounting solution&#160;</td>
			<td>Histolabs</td>
			<td>00840-05</td>
			<td></td>
		</tr>
		<tr>
			<td>Propofol</td>
			<td>Baxter Holding B.V.</td>
			<td>N01AX10</td>
			<td>ATC-code provided instead of catalog number</td>
		</tr>
		<tr>
			<td>QuPath</td>
			<td></td>
			<td></td>
			<td>https://qupath.github.io/</td>
		</tr>
		<tr>
			<td>Rocuronium bromide&#160;</td>
			<td>B. Braun Melsungen AG&#160;</td>
			<td>M03AC09</td>
			<td>ATC-code provided instead of catalog number</td>
		</tr>
		<tr>
			<td>Scott&#39;s tap water substitute concentrate</td>
			<td>Sigma-Aldrich</td>
			<td>S5134-6x100ml</td>
			<td></td>
		</tr>
		<tr>
			<td>Sevoflurane</td>
			<td>Baxter Medical AB&#160;</td>
			<td>N01AB08</td>
			<td>ATC-code provided instead of catalog number</td>
		</tr>
		<tr>
			<td>Slide scanner&#160;</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Sufentanil</td>
			<td>Hameln Pharma gmbh</td>
			<td>N01AH03</td>
			<td>ATC-code provided instead of catalog number</td>
		</tr>
		<tr>
			<td>Sugammadex</td>
			<td>Baxter Holding B.V.</td>
			<td>V03AB35</td>
			<td>ATC-code provided instead of catalog number</td>
		</tr>
		<tr>
			<td>SuperFrost plus white&#160;</td>
			<td>Histolabs</td>
			<td>&#160;06400&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>SuperFrost Plus White microscope slides</td>
			<td>Histolabs</td>
			<td>&#160;06400&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Xylene</td>
			<td>Saveen Werner</td>
			<td>131769.1611</td>
			<td></td>
		</tr>
	</tbody>
</table>

z-scores for assessing ovarian reserve in young patients undergoing fertility preservation

Women are born with a non-renewable pool of ovarian follicles, referred to as the ovarian reserve. This reserve consists of primordial follicles in the ovaries and can be affected by many factors, such as genetic and endocrine disorders, medical interventions, and endocrine disruptors. Fertility preservation is recommended when gonadotoxic treatments are necessary. The preferred options for women are oocyte and embryo cryopreservation. However, in very young, sexually immature patients, ovarian tissue cryopreservation is the only option. Knowing the follicular density of cryopreserved tissue samples is essential in fertility counseling for young patients. This protocol demonstrates the use of Z-scores for cortical follicle density as a tool to evaluate the quality of ovarian tissue in girls and young women aged 25 years and below who are undergoing fertility preservation. Follicle density in patient samples is compared to age-normalized reference standards, developed by Hassan et al. to estimate possible deviations from the standard. The follicle density is measured through histological quantification. For this, a small piece of ovarian cortical tissue (~2 mm x 2 mm x 2 mm) is fixed in either Bouin&#39;s or formaldehyde solution, embedded in paraffin, sectioned at 4 &#181;m thickness, stained with hematoxylin and eosin, and digitized using a slide scanner. Follicular stages present in the cortex range from primordial to primary follicles. The cortical area was 1 mm from the surface epithelium on histological sections. Follicle density is calculated using a modified formula presented by Schmidt et al., and the Z-score is determined using the reference mean and standard deviation. The Z-score indicates how much the measured value deviates from the reference mean, determining&#160;reduced (&#60;-1.7 Z-score) ovarian reserve. With this method, follicle densities can be used as a valuable measure of ovarian reserve for young patients requiring fertility preservation.

Author Spotlight: Advancing Fertility Assessment with Z-Score-Based Follicle Density Evaluation in Cryopreserved Ovarian Tissue

Z-Scores for Assessing Ovarian Reserve in Young Patients Undergoing Fertility Preservation

We wanted to assess ovarian reserve based on small ovarian samples in patients aged up to 25 years old that require fertility preservation through ovarian tissue cryopreservation. Using a modified formula that estimates the total number of follicles in ovarian samples and the model for normal ovarian volume, we established a reference dataset for follicle densities in women age zero to 25 years old. Assessing the normality of follicle density is difficult because of, one, the natural decline of ovarian reserve with increasing age.And two, the heterogeneous distribution of follicles. Comparison to reference values is also challenging because they're not standardized to age and biopsy size, so we're trying to bridge this gap. Our protocol describes the strategy for assessing the normality of follicle density based on small ovarian samples.We directly quantify these follicles using histology and compare the results to age-normalized standard references via z-scores. With this tool, meta-analysis become possible because we can compare data from multiple studies. It is important to know if the ovarian reserve is low.We focus on identifying patients with reduced ovarian reserve and suggest a cutoff value for that. This is of great value for physicians in counseling their patients, for autotransplantation of their cryopreserved ovarian tissue, as well as for further treatment after fertility preservation.

Ovarian follicles are quantified after successful sectioning, staining, and scanning of slides. The different stages and health of the follicles are distinguished and further analyzed. In Figure 4A, an HE-stained cross-section of a Bouin-fixed ovary from an adult is displayed, with examples of cortical and medullary tissue. Figure 4B shows a representative picture of a child&#39;s ovary with an example of surface epithelium. The ovarian reserve (i.e., the non-gr...

The present protocol describes a method for assessing ovarian reserve in patients under 25 years old who require fertility preservation through ovarian tissue cryopreservation. This method involves: (1) histological assessment of ovarian reserve in cortical samples, (2) comparison to a reference dataset, and (3) calculation of Z-scores.

Women are born with a non-renewable pool of ovarian follicles, referred to as the ovarian reserve. This reserve consists of primordial follicles in the ...

z-scores-for-assessing-ovarian-reserve-young-patients-undergoing

Research

JoVE Journal

Developmental Biology

309 Görüntüleme.  Karolinska Institutet. Over dokusu kriyoprezervasyonu ile fertilitenin korunması gereken 25 yaşına kadar olan hastalarda küçük yumurtalık örneklerine dayanarak yumurtalık rezervini değerlendirmek istedik. Yumurtalık örneklerindeki toplam folikül sayısını ve normal yumurtalık hacmi modelini tahmin eden modifiye edilmiş bir formül kullanarak, sıfır ila 25 yaş arası kadınlarda folikül yoğunlukları için bir referans veri seti oluşturduk. Folikül yoğunluğ...

Video: Yazar Spotlight: Dondurularak Saklanmış Yumurtalık Dokusunda Z-Skoru Tabanlı Folikül Yoğunluğu Değerlendirmesi ile Doğurganlık Değerlendirmesini Geliştirme