We thank Tatsuji Ihana, Sachiyo Kurimoto, Kazuko Takahasih, Yuki Yoshizawa, Maho Arashi, Kentaro Fujita, Erina Kudo, Yuka Kurimoto and Mayuko Wakatsuki for supporting the drug-free IVA procedure and Prof. Aaron J.W. Hsueh (Stanford University School of Medicine, Stanford, CA) for critical reading and editing of the manuscript. We also thank Rebecca Truman and Gregory Truman for inserting English narration. This study was supported by The Japan Society for the Promotion of Science (JSPS), Scientific Research B (19H03801), and Challenging Exploratory Research (18K19624).

Written informed consent was obtained from each POR patient with diminishing ovarian reserve who enrolled in the drug-free IVA treatment. This study was approved by ethical committee of International University of Health and Welfare (No. 17-S-21). Clinical trial was registered under number UMIN000034464 and carried out in accordance with the Code of Ethics of the World Medical Association (Declaration of Helsinki).
1. Ovarian cortex extraction
<ol>
	<li>Remove part of ovarian cortex (10 x 10 mm, 2-3 mm thickness) from one or both ovaries under general anesthesia through laparoscopic surgery as previous described19,20.</li>
	<li>Place the collected ovarian cortexes in a sterile container containing 10 mL of modified HTF (mHTF) at 37 &#176;C.</li>
</ol>
2. Ovarian cortex fragmentation
NOTE: Prior to dissection of the ovarian cortex, warm up mHTF to 37 &#176;C. Maintain sterile conditions throughout the procedure. All tools for this procedure are listed in Table of Materials.
<ol>
	<li>Place the collected ovarian cortexes in a plastic dish containing 5-10 mL of mHTF at 37 &#176;C and place them on the heat plate to maintain the tissue at 37 &#176;C (Figure 2A).</li>
	<li>Take a picture of each cortex using a digital camera with patient&#39;s name before starting next procedure to link patient background data.</li>
	<li>Place an individual cortex on a sterile gauze moistened with mHTF (Figure 2B). 
	NOTE: Apply additional mHTF with a disposable pipette on the gauze to prevent the surface of cortex being dried out during this procedure.</li>
	<li>Remove residual medulla tissue where looks pink using a micro-scissors so that thickness of tissue reaches 1-2 mm (Figure 2C and D). 
	NOTE: To avoid the loss of follicles at early stage, do not prepare the cortical strips less than 1 mm in thickness, because the early-stage follicles are located within 1-2 mm from the surface of ovarian cortex21.</li>
	<li>Dissect a 1 mm x 1 mm x 5 mm of tissue piece from each ovarian cortical strip using a fine scalpel and subject it to histological analysis to determine the presence of residual follicles as previous described18 (Figure 2E). 
	NOTE: The dissected tissues for histology are immersed in mHTF and keep at 4 &#176;C until fixing (Figure 2F, see Step 4).</li>
	<li>Cut the cortex into 1mm x 1 mm x 10 mm strips using a fine scalpel (Figure 2G). 
	NOTE: It is easier to cut by pressing down on the scalpel than by pulling on it.</li>
	<li>Cut these tissue strips into 1 mm x 1 mm x 1 mm small cubes (Figure 2H). 
	NOTE: To avoid changes of osmotic pressure in the medium, add mHTF in moderation before tissue autografting.</li>
</ol>
3.&#160;Auto-grafting of ovarian cortical fragments
<ol>
	<li>Open the package of the IVA cannula and put it on a sterile drape.</li>
	<li>Rinse inside of the IVA cannula with mHTF by aspirating and discharging mHTF several times.</li>
	<li>Aspirate 100-200 &#181;L of mHTF to fill the tip of IVA cannula to avoid the cortical cubes from drying out during loading (Figure 3A).</li>
	<li>Load the cortical cubes into the tip of IVA cannula using a fine tweezer (Figure 3B-E). 
	NOTE: The number of cortical cubes for loading depends on the grafting site. Generally, 20-30 cubes can be transplanted into an orthotropic remaining ovary, whereas 10-15 cubes can be transplanted into a pouch beneath the serosa of the Fallopian tube.</li>
	<li>After loading the cortical cubes, hold the tip of the cannula containing the cortical cubes by the fingers until transferring the IVA cannula to a surgeon. This will avoid the cube temperature to decreases. 
	&#8203;NOTE: The details of autografting procedure under laparoscopic surgery have been reported previously19. For grafting ovarian cortical cubes, a cannula-shaped device (IVA cannula) was developed by our collaborator, the Kitazato Corporation.</li>
</ol>
4.&#160;Histological analysis of ovarian cortex
NOTE: To count the number of residual follicles, perform histological analysis as previous described15.
<ol>
	<li>Mark the cortical strip surface with a tissue marking dye before sample fixation. This will indicate the side of cortex in strip for histology.</li>
	<li>Fix the tissue strips overnight at 4 &#176;C using Bouin&#39;s solution. 
	NOTE: To detect atretic follicles, we do not recommend using paraformaldehyde for fixing to avoid shrinkage of oocyte cytoplasm.</li>
	<li>Dehydrate and embed the tissue strips in paraffin.</li>
	<li>Serially section the paraffin block containing the tissue sample.</li>
	<li>Stain each section using hematoxylin and eosin to visualize the follicles.</li>
	<li>Count the follicles as previously described17.</li>
</ol>

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The authors confirm that they have no conflict of interest.

In this manuscript, we showed a detailed laboratory protocol for drug-free IVA. The drug-free IVA is a new approach of infertility treatment for POR patient with diminishing ovarian reserve to promote secondary follicles growth, resulting in yielding more mature oocytes after ovarian stimulation and increasing in successful pregnancy20. In 15 POR patients with diminishing ovarian reserve, this approach achieved one spontaneous pregnancy, in vitro fertilization-embryo transfer allowed four live births, together with one ongoing pregnancy19.
For preparation of grafting ovarian cortex, medulla tissue was removed using a micro-scissors with 1-2 mm of thickness. Previously, we demonstrated that primordial, primary and majority of secondary follicles were located within 1 mm thickness from the surface of ovarian cortex in the patients with normal ovarian reserve21. In POI patients, we found that secondary follicles were located deeper than 1 mm but within 2 mm from the surface of ovarian cortex21. Antral follicles are known to locate in medulla tissues21, but no follicle was found in medulla tissues in POI patients21. Although POR patients with diminishing ovarian reserve could have antral follicles in medulla tissue, antral follicles are difficult to survive after grafting due to lack of blood vessel communication22. Therefore, we determined the thickness of ovarian cortex to be 1-2 mm and the critical step within the protocol is not making the cortical strips &#60; 1-2 mm to avoid loss of precious residual follicles. During preparation of cortical strips and cubes, we used mHTF, but one can use similar medium which include 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffer.
In this procedure, we transplanted ovarian cortex after cutting it into 1 mm3 cubes15,20. Due to the difficulty in handling such small fragments for grafting, previous grafting ovarian cubes one by one under the laparoscopic surgery took 3-4 hours with some cubes occasionally got lost during surgery. After development of the IVA cannula, we could shorten the operation time to 1-2 hours, resulting in minimizing the invasiveness of the surgery to allow the drug-free IVA becoming a single day-surgery. Moreover, we could eliminate the loss of ovarian cortical cubes during surgery. Because of limited number of follicles in ovarian cortex in POR patients with diminishing ovarian reserve, it is important to avoid loss of ovarian cortical cubes. If the IVA cannula is not available, you may try to find a similar cannula to be able to use under laparoscopic surgery.
The decline of ovarian function is progressively caused by aging and some pathophysiological conditions 23,24,25. Thus, in unmarried women with severe ovarian dysfunction, fertility preservation is important to allow future pregnancy. For these patients, the methods of oocytes or ovarian tissue cryopreservation were reported26,27,28,29. Although oocyte cryopreservation is an established method for fertility preservation1,2, these patients could have very limited number of retrievable oocytes even after ovarian hyperstimulation due to low ovarian reserve. Furthermore, in aging patients with ovarian dysfunction, high incidence of chromosome abnormalities was reported in ovulated oocytes26,30. This leads to the requirement of multiple expensive procedures to ensure sufficient number of oocytes to guarantee pregnancy. To solve these problems, we have developed the drug-free IVA approach for increasing the number of mature oocytes for oocyte retrieval. Because oocyte cryopreservation is no longer an experimental method31,32, the drug-free IVA followed by oocyte cryopreservation is expected to be a promising method for fertility preservation in unmarried women with severe ovarian dysfunction. However, future clinical studies using the freeze-thawed oocytes obtained from the drug-free IVA will be required to determine the effectiveness of this approach for fertility preservation in unmarried women with severe ovarian dysfunction.
In conclusion, we developed the drug-free IVA as a new approach of infertility treatment for POR patient with diminishing ovarian reserve and showed the detail laboratory protocol to be able to repeat our procedures. The methodological limitations of drug-free IVA are difficulty in prediction of residual follicle number before the surgery and also inability to evaluate the graft survival. The laboratory technique of drug-free IVA could become the basis of future application of ovarian tissue culture to obtain mature oocytes without grafting.

Ovarian function declines progressively during aging and some pathophysiological conditions including karyotype abnormality, autoimmune diseases, chemo- and radiation-therapies, and ovarian surgeries. Fertility preservation is one of the best options for unmarried women with severe ovarian dysfunction to preserve their potential for future pregnancy. For fertility preservation, currently two methods are available mostly in the onco-fertility field. Oocyte cryopreservation is a well-established procedure for fertility preservation and many successful cases have been reported1,2. On the other hand, ovarian tissue cryopreservation was also established for fertility preservation in cancer patients but it is still an experimental strategy3,4. In both methods, multiple numbers of mature oocytes are needed for pregnancy to occur. Generally, patients with premature ovarian insufficiency (POI), who become amenorrhea before 40 years of age, and middle-aged women with low ovarian reserve showed poor ovarian response (POR) to ovarian stimulation for yielding mature oocytes5,6,7. Furthermore, young patients with a low number of antral follicles also showed POR to the ovarian stimulation7. These patients have very limited number of retrievable oocytes even after proper ovarian hyperstimulation, thus requiring multiple expensive procedures to ensure a sufficient number of oocytes for pregnancy.
Oocyte donation followed by in vitro fertilization (IVF) with husband&#39;s sperm and embryo transfer (ET) is the only option for these POI and POR patients who have difficulties obtaining their own oocytes8,9,10. However, oocyte donation is complicated by ethical issues as well as autoimmune and pregnancy complications11,12,13,14. To solve these issues, the establishment of infertility treatment using patients&#39; own oocytes is desired. For POI patients, we have developed the in vitro activation (IVA) approach to allow successful follicle growth and the generation of mature oocytes, leading a number of pregnancies and deliveries15. In IVA, we fragmented ovarian cortexes after removal of ovaries under laparoscopic surgery and cultured them for two days to activate follicles by Akt-stimulating drugs and then perform heterotopic grafting back into artificial pouches made beneath the serosa of Fallopian tubes under second laparoscopic surgery15. This procedure promoted growth of primordial, primary and secondary follicles after ovarian cortex fragmentation to promote Hippo signaling disruption16, followed by two days culture with Akt signaling stimulators17.
In contrast to severe POI cases, POR patients with decreased ovarian reserve have multiple secondary follicles. Because Hippo signaling disruption alone is effective in promoting secondary follicle growth16, we recently demonstrated successful pregnancies and deliveries for POR patients using the drug-free IVA procedure involving cortical fragmentation and orthotopic grafting without treatment of the Akt stimulating drugs. Drug-free IVA stimulates early stage of ovarian follicles to develop to the preantral follicle stage after only one surgery and increased the number of retrieved oocytes for IVF-embryo transfer15,18. The drug-free IVA approach has several advantages as compared with our original IVA by 1) avoiding potential follicle loss during culture, 2) minimizing invasiveness and costs of the second surgery, 3) involving only short-term post-surgery bed rest and 4) potential of spontaneous pregnancy due to orthotopic grafting. We recently published a video article showing the surgical procedures of drug-free IVA19 and detailed protocols of ovarian stimulation after the surgery20. Here, we present details of laboratory methods required for drug-free IVA.

<table><tbody><tr><td>4.5 onz specimen container</td><td>FALCON</td><td>354013</td><td>Other products may also be suitable</td></tr><tr><td>60mm dish</td><td>FALCON</td><td>351007</td><td>Other products may also be suitable</td></tr><tr><td>50 x 50 cm sterile drape</td><td>HOGY Medical</td><td>SR-823</td><td>Any type of sterile produsts may also be suitable</td></tr><tr><td>Disposable pippete</td><td>FALCON</td><td>357575</td><td>Other products may also be suitable</td></tr><tr><td>Fine scissors, Curved</td><td>WPI</td><td>#14224-G</td><td>Although other products may also be suitable, we strongly recommend use this products</td></tr><tr><td>Hot plate</td><td>TOKAI HIT</td><td>TPiE-SP</td><td>Use at operation room to maintain the temperature of dishes containing ovarian tissue before transplantation</td></tr><tr><td>Human Serum Albumin Solution</td><td>Irvine Scientific</td><td>9988</td><td>Medium for handling ovarian tissue</td></tr><tr><td>IVA cannule</td><td>KITAZATO</td><td>446030 IVA-6030E</td><td>Specific cannula for tissue autografting</td></tr><tr><td>KAI medical Disposable scalpel</td><td>WPI</td><td>#5 10-A</td><td>Although other products may also be suitable, we strongly recommend use this products</td></tr><tr><td>Micro scissors, Curved</td><td>WPI</td><td>#503364</td><td>Although other products may also be suitable, we strongly recommend use this products</td></tr><tr><td>Modified HTF Medium-HEPES</td><td>Irvine Scientific</td><td>90126</td><td>Medium for handling ovarian tissue</td></tr><tr><td>Sterile gauze</td><td>Osaki Medical</td><td>15004</td><td>Any type of sterile produsts may also be suitable</td></tr><tr><td>Swiss Tweezers, Curved Tips</td><td>KAI</td><td>#504505</td><td>Although other products may also be suitable, we strongly recommend use this products</td></tr></tbody></table>

fertility preservation in patients with severe ovarian dysfunction

Ovarian function progressively declines during aging and in some pathophysiological conditions including karyotype abnormality, autoimmune diseases, chemo- and radiation-therapies, as well as ovarian surgeries. In unmarried women with severe ovarian dysfunction, fertility preservation is important for future pregnancies. Although oocyte cryopreservation is an established method for fertility preservation, these patients could only preserve a limited number of oocytes even after ovarian hyperstimulation, leading to repeated stimulations to ensure sufficient oocytes to guarantee future pregnancy. To solve this issue, we have recently developed a drug-free in vitro activation (IVA) procedure, which enable us to stimulate early stages of ovarian follicles to develop to the preantral follicle stage. These preantral follicles can respond to the unique protocol of gonadotropin stimulation, resulting in increased number of retrieved oocytes per ovarian stimulation for cryopreservation. The drug-free IVA comprised from the surgical approach and ovarian stimulation. We removed a part of cortex from one or both ovaries from patients under laparoscopic surgery. The ovarian cortical tissues were cut into small cubes to disrupt the Hippo signaling pathway and stimulate the development of early stage follicles. These cubes were grafted orthotropically into remaining ovaries as well as beneath the serosa of both Fallopian tubes. We have already published the surgical procedure of the drug-free IVA and the protocol of subsequent ovarian stimulation, but herein we present the details of laboratory methods required for drug-free IVA.

In the first publication of the IVA approach15, we transplanted the ovarian cortical cubes one by one into the grafting sites under laparoscopic surgery (Figure 1A). Because 100-150 ovarian cubes were used, it took 3-4 hours for tissue grafting under laparoscopic surgery. Also, some ovarian cubes were lost before grafting. Because the IVA cannula could transfer 20-30 cubes to a grafting site at one time (Figure 1B), we could shorten the operation time to 1-2 hours. Furthermore, the IVA cannula could eliminate the loss of ovarian cortical cubes during surgery.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/62098/62098fig01.jpg" /> 
Figure 1: The effectiveness of the IVA cannula. (A) Previous method with transplanting ovarian cortical cubes one by one using a forceps. (B) The current method with transplanting 20-30 cubes at one time using the IVA cannula. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62098/62098fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/62098/62098fig02.jpg" /> 
Figure 2: Ovarian tissue dissection for drug-free IVA. (A) A piece of ovarian cortex is placed in a plastic dish containing mHTF at 37 &#176;C. (B) To remove residual medulla tissues, the ovarian cortex is placed on a moistened gauze. (C) Medulla tissue is removed using a fine micro-scissor. (D) The thickness of cortex to be prepared is between 1 to 2 mm. (E) After removal of medulla tissue, a 1 mm x 1 mm x 5 mm of tissue piece from each ovarian cortical strip is dissected for histological analysis. (F) The dissected tissue is stored in 1.5 mL tube containing mHTF at 4 &#176;C until fixing for histology. (G and H) The ovarian cortex is cut into 1 mm x 1 mm x 10 mm strips, and then further cut into 1 mm x 1 mm x 1 mm small cubes using a fine scalpel. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62098/62098fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/62098/62098fig03.jpg" /> 
Figure 3: Auto-grafting of ovarian cortical fragments. (A) Before loading cubes, 100-200 &#181;L of mHTF is aspirated to fill the tip of the IVA cannula to avoid the cortical cubes from drying out during loading (B-E) Loading ovarian cortical cubes into the tip of IVA cannula. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62098/62098fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Fertility Preservation in Patients with Severe Ovarian Dysfunction at JoVE.com

Fertility Preservation in Patients with Severe Ovarian Dysfunction

We present details of laboratory procedures for drug-free in vitro activation (IVA) of ovarian follicles for patients with severe ovarian dysfunction. This method could increase the number of retrievable oocytes per ovarian hyperstimulation and benefit fertility preservation for those patients.

Ovarian function progressively declines during aging and in some pathophysiological conditions including karyotype abnormality, autoimmune diseases, ...

fertility-preservation-in-patients-with-severe-ovarian-dysfunction

Nanyang Technological University

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Anatomy and Physiology

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Unit 1

The Reproductive System

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4.5K Views.  International University of Health and Welfare School of Medicine. Het doel van deze film is om de procedure van onze nieuwe benadering van patiënten met eierstokdisfunctie te demonstreren. Ovariële functie neemt geleidelijk af tijdens veroudering en sommige pathofysiologische aandoeningen, waaronder karyotypeafwijkingen, auto-immuunziekten, chemo- en bestralingstherapieën en eierstokoperaties. De meeste vrouwen van middelbare leeftijd met een lage ovariële reserve vertoonden een slechte ovariële respons op ovariële stimulatie voor het leveren van volw...

Video: Fertility Preservation in Patients with Severe Ovarian Dysfunction

A Contusion Model of Severe Spinal Cord Injury in Rats

The translational potential of novel treatments should be investigated in severe spinal cord injury (SCI) contusion models. A detailed methodology is described to obtain a consistent model of severe SCI. Use of a stereotactic frame and computer controlled impactor allows for creation of reproducible injury. Hypothermia and urinary tract infection pose significant challenges in the post-operative period. Careful monitoring of animals with daily weight recording and bladder expression allows for early detection of post-operative complications. The functional results of this contusion model are equivalent to transection models. The contusion model can be utilized to evaluate the efficacy of both neuroprotective and neuroregenerative approaches.

A contusion model of severe spinal cord injury is described. Detailed pre-operative, operative and post-operative steps are described to obtain a consistent model.

The translational potential of novel treatments should be investigated in severe spinal cord injury (SCI) contusion models. A detailed methodology is ...

Computerized Dynamic Posturography for Postural Control Assessment in Patients with Intermittent Claudication

Computerized dynamic posturography with the EquiTest is an objective technique for measuring postural strategies under challenging static and dynamic conditions. As part of a diagnostic assessment, the early detection of postural deficits is important so that appropriate and targeted interventions can be prescribed. The Sensory Organization Test (SOT) on the EquiTest determines an individual&#39;s use of the sensory systems (somatosensory, visual, and vestibular) that are responsible for postural control. Somatosensory and visual input are altered by the calibrated sway-referenced support surface and visual surround, which move in the anterior-posterior direction in response to the individual&#39;s postural sway. This creates a conflicting sensory experience. The Motor Control Test (MCT) challenges postural control by creating unexpected postural disturbances in the form of backwards and forwards translations. The translations are graded in magnitude and the time to recover from the perturbation is computed.
Intermittent claudication, the most common symptom of peripheral arterial disease, is characterized by a cramping pain in the lower limbs and caused by muscle ischemia secondary to reduced blood flow to working muscles during physical exertion. Claudicants often display poor balance, making them susceptible to falls and activity avoidance. The Ankle Brachial Pressure Index (ABPI) is a noninvasive method for indicating the presence of peripheral arterial disease and intermittent claudication, a common symptom in the lower extremities. ABPI is measured as the highest systolic pressure from either the dorsalis pedis or posterior tibial artery divided by the highest brachial artery systolic pressure from either arm. This paper will focus on the use of computerized dynamic posturography in the assessment of balance in claudicants.

Individuals with intermittent claudication exhibit poor balance compared to healthy controls. Computerized dynamic posturography is an objective method for measuring an individual's postural responses to balance disturbances. This provides an objective reflection of the person&#x2019;s ability to respond to situations under which sensory stimuli are altered and unexpected perturbations occur.

Computerized dynamic posturography with the EquiTest is an objective technique for measuring postural strategies under challenging static and dynamic ...

Assessment of Vascular Function in Patients With Chronic Kidney Disease

Patients with chronic kidney disease (CKD) have significantly increased risk of cardiovascular disease (CVD) compared to the general population, and this is only partially explained by traditional CVD risk factors. Vascular dysfunction is an important non-traditional risk factor, characterized by vascular endothelial dysfunction (most commonly assessed as impaired endothelium-dependent dilation [EDD]) and stiffening of the large elastic arteries. While various techniques exist to assess EDD and large elastic artery stiffness, the most commonly used are brachial artery flow-mediated dilation (FMDBA) and aortic pulse-wave velocity (aPWV), respectively. Both of these noninvasive measures of vascular dysfunction are independent predictors of future cardiovascular events in patients with and without kidney disease. Patients with CKD demonstrate both impaired FMDBA, and increased aPWV. While the exact mechanisms by which vascular dysfunction develops in CKD are incompletely understood, increased oxidative stress and a subsequent reduction in nitric oxide (NO) bioavailability are important contributors. Cellular changes in oxidative stress can be assessed by collecting vascular endothelial cells from the antecubital vein and measuring protein expression of markers of oxidative stress using immunofluorescence. We provide here a discussion of these methods to measure FMDBA, aPWV, and vascular endothelial cell protein expression.

The degree of vascular dysfunction and contributing physiological mechanisms can be assessed in patients with chronic kidney disease by measuring brachial artery flow-mediated dilation, aortic pulse-wave velocity, and vascular endothelial cell protein expression.

Patients with chronic kidney disease (CKD) have significantly increased risk of cardiovascular disease (CVD) compared to the general population, and this ...

Functional Human Liver Preservation and Recovery by Means of Subnormothermic Machine Perfusion

There is currently a severe shortage of liver grafts available for transplantation. Novel organ preservation techniques are needed to expand the pool of donor livers. Machine perfusion of donor liver grafts is an alternative to traditional cold storage of livers and holds much promise as a modality to expand the donor organ pool. We have recently described the potential benefit of subnormothermic machine perfusion of human livers. Machine perfused livers showed improving function and restoration of tissue ATP levels. Additionally, machine perfusion of liver grafts at subnormothermic temperatures allows for objective assessment of the functionality and suitability of a liver for transplantation. In these ways a great many livers that were previously discarded due to their suboptimal quality can be rescued via the restorative effects of machine perfusion and utilized for transplantation. Here we describe this technique of subnormothermic machine perfusion in detail. Human liver grafts allocated for research are perfused via the hepatic artery and portal vein with an acellular oxygenated perfusate at 21 &#176;C.

We describe a method of ex vivo machine perfusion of human liver grafts at subnormothermic temperature (21 &#176;C).

There is currently a severe shortage of liver grafts available for transplantation.  Novel organ preservation techniques are needed to expand the pool of ...

Ovarian Tissue Oocyte-In Vitro Maturation for Fertility Preservation

Mature oocyte vitrification is the standard of care to preserve fertility in women at risk of infertility. However, ovarian tissue cryopreservation (OTC) is still the only option to preserve fertility in women who need to start gonadotoxic treatment urgently or in prepubertal children. During ovarian cortex preparation for cryopreservation, medullar tissue is removed. Growing antral follicles reside at the border of the cortex-medullar interface of the ovary and are broken during this process, releasing their cumulus-oocyte complex (COC). By thoroughly inspecting the medium and fragmented medullar tissue, these immature cumulus-oocyte complexes can be identified without interfering with the OTC procedure. The ovarian tissue-derived immature oocytes can be successfully matured in vitro, creating an additional source of gametes for fertility preservation. If OTC is performed within or near a medical assisted reproduction laboratory, all necessary in vitro maturation (IVM) and oocyte vitrification tools can be at hand. Furthermore, upon remission and child wish, the patient has multiple options for fertility restoration: ovarian tissue transplantation or embryo transfer after the insemination of vitrified/warmed oocytes. Hence, ovarian tissue oocyte-in vitro maturation (OTO-IVM) can be a valuable adjunct fertility preservation technique.

Ovarian Tissue Oocyte-In Vitro Maturation for Fertility Preservation

Presented here is ovarian tissue oocyte-in vitro maturation (OTO-IVM), an accessible technique within a medical assisted reproduction (MAR) laboratory offering realistic additional fertility preservation options to patients who need ovarian tissue cryopreservation.

Mature oocyte vitrification is the standard of care to preserve fertility in women at risk of infertility. However, ovarian tissue cryopreservation (OTC) ...

Developmental Biology

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

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.

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 ...

Cryopreservation of Oocytes Retrieved from Ovarian Tissue to Optimize Fertility Preservation in Prepubertal Girls and Women

Human ovarian tissue cryopreservation (OTC) is increasingly used worldwide to preserve female fertility in prepubertal girls and women at risk of premature ovarian insufficiency (POI) in the context of urgent gonadotoxic treatments or ovarian surgery. Fertility preservation is challenging because there is no consensus regarding patient management, preservation fertility strategies, or even technical laboratory protocols, which implies that each procedure must be adapted to the characteristics of the patient profile and its own risk-benefit ratio. During OTC, mature/immature oocytes can be aspirated directly from large/small antral follicles within ovarian tissue samples and/or be released into culture media from growing follicles during ovarian tissue dissection in prepubertal girls and women. In this manuscript, we present a protocol that combines ovarian tissue freezing with the cryopreservation of mature/immature oocytes retrieved from ovarian tissue samples, improving the reproductive potential of fertility preservation. Appropriate collection, handling, and storage of ovarian tissue and oocytes before, during, and after the cryopreservation will be described. The subsequent use and safety of cryopreserved/thawed ovarian tissue samples and oocytes will also be discussed, as well as the optimal timing for in vitro maturation of immature oocytes. We recommend the systematic use of this protocol in fertility preservation of prepubertal girls and women as it increases the whole reproductive potential of fertility preservation (i.e., oocyte vitrification in addition of OTC) and also improves the safety and use of fertility preservation (i.e., thawing of oocytes versus ovarian graft), maximizing the chance of successful childbirth for the patients at risk of POI.

We propose a protocol for fertility preservation in prepubertal girls and women at risk of premature ovarian insufficiency. It combines ovarian tissue freezing and cryopreservation of oocytes retrieved from ovarian tissue. This strategy improves the safety and optimizes the reproductive potential of fertility preservation, maximizing the chance of childbirth.

Human ovarian tissue cryopreservation (OTC) is increasingly used worldwide to preserve female fertility in prepubertal girls and women at risk of ...

Fertility Preservation Through Oocyte Vitrification: Clinical and Laboratory Perspectives

Preserving female fertility is crucial in a multifunctional healthcare system that takes care of patients' future quality of life. Oocyte cryopreservation is recognized by several international scientific societies as the gold standard for fertility preservation in postpubertal women, for both medical and non-medical indications. The main medical indications are oncologic diseases, gynecologic diseases such as severe endometriosis, systemic diseases compromising the ovarian reserve, and genetic conditions involving premature menopause. This paper describes the whole clinical and laboratory work-up of a fertility preservation treatment by outlining recommendations for objective and evidence-based counseling. Furthermore, it focuses on the effectiveness of the procedure and describes the most appropriate strategies to fully exploit the ovarian reserve and maximize the number of oocytes retrieved in the shortest possible time. The evaluation of the ovarian reserve, the definition of an ideal stimulation protocol, as well as oocyte retrieval, denudation, and vitrification procedures have been detailed along with approaches to maximize their efficacy, efficiency, and safety.

Oocyte cryopreservation is recognized by several international scientific societies as the gold standard for fertility preservation in postpubertal women. Appropriate clinical and laboratory strategies ensure maximum efficacy, efficiency, and safety of fertility preservation treatments.

Preserving female fertility is crucial in a multifunctional healthcare system that takes care of patients' future quality of life. Oocyte cryopreservation ...

Laparoscopic Oocyte Retrieval and Cryopreservation during Vaginoplasty for Treatment of Mayer-Rokitansky-Kuster-Hauser Syndrome

In Mayer-Rokitansky-Kuster-Hauser Syndrome (MRKHS) patients who are scheduled for laparoscopic vaginoplasty and have a desire for biological motherhood, we propose that a concomitant laparoscopic oocyte retrieval for cryopreservation is performed. Oocyte retrieval is pursued at the beginning of the laparoscopy. Right and left 5 mm trocars are positioned, through which a 17 G ovum aspiration needle is used for puncture of the right and left ovaries, respectively. To facilitate exposure of the follicles, the ovaries are mobilized and held with laparoscopic forceps.
When aspirating multiple follicles near each other, the needle tip is retained in the ovary to reduce the number of times that the ovarian cortex is transfixed and due to the inherent risk of bleeding. Subsequent steps are unchanged compared to the Davydov laparoscopic modified technique for vaginoplasty. Prior to surgery, controlled ovarian stimulation is performed with a gonadotropin hormone-releasing hormone (Gn-RH) antagonist protocol, and the concomitant procedure of oocyte retrieval and vaginoplasty is scheduled 36 h after the final follicular maturation trigger. Follicular fluid is collected in the same 10 mL sterile tubes used during transvaginal oocyte retrieval and transferred in a warming block (37 &#176;C) to the assisted reproduction laboratory, where mature (metaphase II) oocytes are vitrified.
In this case, a series of 23 women with MRKH, oocytes were successfully retrieved and cryopreserved in all patients; vaginoplasty was subsequently conducted without modifications, and the inpatient and outpatient postoperative care (day of urinary catheter removal, day of hospital discharge, dilator use, and comfort at follow-up) remained unaffected. One postoperative complication occurred in one patient (fever developing on day 5 post surgery and intraperitoneal fluid detection on transabdominal ultrasound) and resolved after conservative treatment. Rather than performing surgical vaginoplasty and delaying oocyte retrieval in MRKH patients, this approach combines both procedures in a single laparoscopy, thereby minimizing surgical invasiveness and anesthesiologic risks.

A dedicated team can offer Mayer-Rokitansky-Kuster-Hauser patients the option to perform controlled ovarian stimulation and oocyte cryopreservation at the time of laparoscopic vaginoplasty.

In Mayer-Rokitansky-Kuster-Hauser Syndrome (MRKHS) patients who are scheduled for laparoscopic vaginoplasty and have a desire for biological motherhood, ...

Follicular Viability Assessment in Vitrified Human Ovarian Cortex Tissues

Vitrification of Ovarian Cortex Tissue to Achieve a Glassy State of Aggregation

Ovarian tissue cryopreservation (OTC) is an important option for fertility preservation. For patients whose gonadotoxic treatments cannot be postponed or for pre-pubertal girls, it is often the only option for fertility protection. Cryopreservation can be performed either by vitrification or by slow freezing. Slow freezing is currently the standard approach. An increasing number of studies indicate that vitrification can replace slow freezing in the state-of-the-art in vitro fertilization (IVF) laboratories, significantly improving thawing survival rates and simplifying the technical aspects of cryopreservation. A metal grid-based, high-throughput protocol for rapid vitrification of ovarian cortex tissue, suitable for clinical routine, is described. The sterilization of metal grids and liquid nitrogen ensures high quality, meeting good manufacturing practice (GMP) standards. Vitrification was conducted to ensure ultra-rapid cooling rates. Instead of slowly thawing, samples were rapidly warmed. To assess follicular viability, calcein staining was performed both prior to cryopreservation and after rapid warming. The successful application of vitrification and rapid warming using metal grids is reported. No significant differences in follicular viability were observed prior to vitrification and after rapid warming. These results substantiate the high capacity of tissue vitrification for clinical routine applications as a potential substitute for the widely used slow-freezing method.

Author Spotlight: Advancing Fertility Preservation in Young Female Cancer Patients Through Ovarian Tissue Vitrification and In Vitro Follicular Growth

A protocol for the vitrification of ovarian tissue, as an alternative cryopreservation method to the widely used slow freezing protocol, is presented.

Ovarian tissue cryopreservation (OTC) is an important option for fertility preservation. For patients whose gonadotoxic treatments cannot be postponed or ...

Fertility Preservation in Patients with Severe Ovarian Dysfunction

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