Name: op-ivm: combining in vitro maturation after oocyte retrieval with gynecological surgery
Uploaded: 2021-05-09T14:00:00.000+00:00
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Description: Watch this Scientific Journal Video about OP-IVM: Combining In vitro Maturation after Oocyte Retrieval with Gynecological Surgery at JoVE.com

This work was supported by grants from the China National Key R&#38;D Program (no. 2017YFC1002000, 2018YFC1004001, 2019YFA0801400), the National Science Foundation of China (no. 81571386, 81730038), the CAMS Innovation Fund for Medical Sciences (2019-I2M-5-001), and the Special Research Project of Chinese Capital Health Development (2018-2-4095).

NOTE: Studies related to the OP-IVM method have been approved by the institutional review board (IRB) of Peking University Third Hospital and the Ethics Committee of Peking University (2014S2004). A summary of OP-IVM is shown in Figure 1. The step-by-step procedure will be introduced in the following section.
1. Introduction of OP-IVM to appropriate patients
<ol>
	<li>Identify potential patients who may benefit from OP-IVM such as those described in steps 1.1.1-1.1.3.:
	<ol>
		<li>Polycystic ovarian syndrome (PCOS) patients with clomiphene resistance who need laparoscopic ovarian drilling surgery.</li>
		<li>Infertile patients who need benign gynecological surgeries, such as hysteroscopic myomectomy, polypectomy, transcervical resection of septum and laparoscopic tubal surgery, and oophorocystectomy, before ART treatment.</li>
		<li>Patients with cancer or hematological disease who are receiving chemoradiotherapy or radiotherapy.</li>
	</ol>
	</li>
</ol>
2. Informed consent
<ol>
	<li>Provide full information to patients, including why OP-IVM may be beneficial, its procedure, uses of IVM oocytes (IVF-ET or cryopreservation), estimated CPRs and LBRs, and possible complications. Ask patients to give informed consent by signing the consent form.</li>
</ol>
3. Prepare labels and IVM oocyte medium
<ol>
	<li>Print identification (ID) labels with the patient&#39;s name and dates for culture dishes and tubes.</li>
	<li>Add 0.5 mL of IVM&#160;medium supplemented with 0.75 IU/mL of follicle-stimulating hormone (FSH) and 0.75 IU/mL of luteinizing hormone (LH) to each well of a 4-well plate. Cover the medium with oil. 
	NOTE: Perform all these procedures in laminar flow clean benches.</li>
	<li>Prewarm the 4-well plate with IVM oocyte medium at 37 &#176;C in humidified air containing 5% CO2 and 5% O2 at least 6 h before use.</li>
</ol>
4. Administer anesthesia in an operating room
<ol>
	<li>Check the name of the patient before administering anesthesia.</li>
	<li>Intravenously anesthetize the patient by anesthesiologist.</li>
</ol>
5. Perform oocyte retrieval
<ol>
	<li>Tag the labels with name, date, and ID on the culture dishes and tubes.</li>
	<li>Place the patient in the bladder lithotomy position; constantly disinfect, drape, and scrub the vagina with warm saline.</li>
	<li>Place the ultrasound probe inside the vagina; scan and record the number of follicles in both&#160;ovaries. Find the location closest to the ovary as the puncture site, and avoid the intestine, bladder, and large blood vessels.</li>
	<li>Aspirate follicular fluid.
	<ol>
		<li>Wash the needle with&#160;pH-stable handling medium before puncturing (see Table of Materials).</li>
		<li>Inject the 19 G, single-lumen aspiration needle into the ovaries under the guidance of ultrasound.</li>
		<li>Puncture larger follicles with clear boundaries closest to the probe. At a low position, quickly inject the rinse solution supplemented with 25 U/mL of heparin. Aspirate follicular fluid with the needle under a pressure of 80-90 mmHg, rotating the needle slightly to aspirate as much follicular fluid as possible. Puncture other follicles from the near to far side on this plane. 
		NOTE: During aspiration, follicular fluid containing oocytes will flow into a sterile 10 mL test tube under negative pressure. Heparin can reduce the follicular fluid viscosity to facilitate the aspiration process.</li>
		<li>Remove the needle from the ovary (keep the needle in the vaginal wall). Adjust the direction of the ultrasound probe, and puncture the remaining follicles on other planes. Try to aspirate all the follicles with a diameter of ~5-9 mm. 
		&#8203;NOTE: Adjust the probe&#39;s position to keep it closest to the ovaries at all times. Press the vaginal fornix with appropriate force to reduce injury and bleeding.</li>
		<li>Pull out the needle after finishing in one ovary, wash the needle with the handling medium, and puncture the other side using the same method. 
		NOTE: Complete follicle aspiration within 25-30 min.</li>
	</ol>
	</li>
	<li>Transfer the follicular fluid from the operation room to the IVF laboratory within a few minutes after confirming the patient&#39;s name and ID. 
	NOTE: Transfer aspirated follicular fluid to the IVF laboratory bench as soon as possible to prevent coagulation.</li>
	<li>Detect active hemorrhage in the pelvic cavity with B-mode ultrasound after puncturing all the follicles. Insert a speculum, and point the tip at the posterior fornix to detect active bleeding at the puncture site. 
	&#8203;NOTE: No active bleeding should be observed at the vaginal puncture site if the ovary position is normal&#160;and oocyte retrieval is performed carefully. For light bleeding that continues even after compression, keep a sterile gauze compress in the vagina for 2-4 h. Control excessive bleeding from small arteries using clamps with vascular forceps for 2-4 h. Bleeding in the pelvic cavity or ovary, which seldom happens, should be controlled by electrocoagulation by laparoscopic surgery.</li>
</ol>
6. Gynecological surgery
<ol>
	<li>Based on the need and condition of the patient, perform appropriate gynecological surgery after oocyte retrieval.
	<ol>
		<li>Perform laparoscopic ovarian drilling surgery for polycystic ovarian syndrome (PCOS) patients with clomiphene resistance.</li>
		<li>Perform benign gynecological surgery for infertile patients before ART treatment, such as hysteroscopic myomectomy, polypectomy, transcervical resection of septum and laparoscopic tubal surgery, and oophorocystectomy.</li>
		<li>Perform ovariectomy for fertility cryopreservation of patients with cancer or hematological disease who need to receive chemoradiotherapy. 
		&#8203;NOTE: These gynecological surgeries are basic and standardized clinical operations. Operation guidelines in various countries and hospitals should be relatively similar.</li>
	</ol>
	</li>
</ol>
7. Perform IVM
NOTE: Perform the whole process of IVM on a 37 &#176;C homothermal flat.
<ol>
	<li>Filter the aspirated follicle fluid with a 70 &#181;m nylon cell strainer. Repeatedly rinse the culture tube and strainer with pre-warmed pH-stable handling medium. Ensure all the immature COCs are completely transferred to the culture dish. Collect the filtered fluid , rinse, and culture in a 100 x 15 mm Petri dish.</li>
	<li>Examine the COCs (Figure 2) under the stereoscope with 40x magnification. Quickly transfer the immatures into a pre-warmed IVM oocyte medium. 
	&#8203;NOTE: Choose an appropriate magnification depending on the operator&#39;s habit.</li>
	<li>Record the number of cultured immature COCs.</li>
	<li>Inform the patient about the number of cultured COCs. Discuss the collection of semen with the patients and their partners. Perform sperm extraction according to the normal procedure.</li>
	<li>Culture the immature COCs at 37 &#176;C in humidified air containing 5% CO2 and 5% O2 for 28-32 h.</li>
	<li>Assess the oocyte maturity.
	<ol>
		<li>Denude the cumulus cells by repeated pipetting using a glass Pasteur pipette under the stereoscope with 40x magnification. Examine the extrusion of the polar body (PB) to identify the developmental stage of the oocytes. See Figure 2 for representative images of oocytes in COCs, metaphase II (MII), metaphase I (MI), and germinal vesicles (GVs) with clear morphological characteristics. 
		NOTE: Use a vertical flame (e.g., of a Bunsen burner) to adjust the diameter of the glass Pasteur pipette to the size of an oocyte.</li>
		<li>Count MII oocytes and record the number.</li>
		<li>Choose mature oocytes for IVF or vitrification. 
		&#8203;NOTE: Collect sperm on the day the oocytes mature for IVF, but not for oocyte vitrification.</li>
		<li>Culture the immature oocytes in GV and MI stage in the original IVM culture medium with cumulus cells for another 10-14 h. Repeat steps 7.6.2-7.6.3.</li>
	</ol>
	</li>
</ol>
8. Perform ICSI or oocyte vitrification
<ol>
	<li>Follow the standard procedure of the reproductive center8.</li>
</ol>
9. Culture embryos and perform embryo cryopreservation
<ol>
	<li>Follow the standard procedure of the reproductive center8.</li>
</ol>

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The authors have nothing to disclose.

The OP-IVM method described in this article extends to conventional IVM applications and combines IVM after oocyte retrieval with routine gynecological surgery. Oocytes that would have been lost in the gynecological surgery can now be used for IVF-ET or FP without additional surgical risks. OP-IVM was first used to retrieve oocytes before ovarian drilling in PCOS patients. Its application soon expanded to infertile patients who need benign gynecological surgery and cancer or hematological disease patients who need chemor...

An erratum was issued for:&#160;<a href="https://www.jove.com/t/61647">OP-IVM: Combining In vitro Maturation after Oocyte Retrieval with Gynecological Surgery</a>. The Protocol and Representative Results sections were&#160;updated.
Step 3.2 of the Protocol was updated from:
Add 0.5 mL of IVM&#160;medium supplemented with 0.075 IU/mL of follicle-stimulating hormone (FSH) and 0.075 IU/mL of luteinizing hormone (LH) to each well of a 4-well plate. Cover the medium with oil.
to:
Add 0.5 mL of IVM&#160;medium supplemented with 0.75 IU/mL of follicle-stimulating hormone (FSH) and 0.75 IU/mL of luteinizing hormone (LH) to each well of a 4-well plate. Cover the medium with oil.
The legend for Figure 2 was updated from:
Figure 2:&#160;Morphological characteristics of representative images of oocytes. (A) COCs, (B) MII, (C) MI, (D) GV stage. Abbreviations: COCs = cumulus-oocyte complexes; GV = germinal vesicle; MI = metaphase I; MII = metaphase II.
to:
Figure 2:&#160;Morphological characteristics of representative images of oocytes. (A) COCs, (B) GV, (C) MI, (D) MII&#160;stage. Abbreviations: COCs = cumulus-oocyte complexes; GV = germinal vesicle; MI = metaphase I; MII = metaphase II.

An erratum was issued for:&#160;<a href="https://www.jove.com/t/61647">OP-IVM: Combining In vitro Maturation after Oocyte Retrieval with Gynecological Surgery</a>. The Protocol and Representative Results sections were&#160;updated.

Erratum: OP-IVM: Combining In vitro Maturation after Oocyte Retrieval with Gynecological Surgery

IVM is an ART in which human immature oocytes are cultured in vitro to maturation for IVF-ET or FP. In IVM, ovulation induction medications are not used, thus reducing pain, financial burden, and complications such as ovarian hyperstimulation syndrome (OHSS)1,2. In addition, IVM is particularly suitable for the FP of cancer patients and the infertility treatment of hormone-sensitive patients who may be unable to or have no time to receive ovulation induction therapy3. Therefore, although the number of oocytes retrieved, clinical pregnancy rate (CPR), and live birth rate (LBR) are lower than those of IVF4,5, IVM has its own unique advantages.
Infertile patients with endometrial lesions, hydrosalpinx, or ovarian cysts usually have gynecological surgery before ART treatment, and their oocytes are usually immature. OP-IVM uses guided transvaginal ultrasound to retrieve the immature oocytes and grow them in vitro until maturation for IVF-ET or FP. OP-IVM combines IVM after oocyte retrieval and gynecological surgery, thereby reducing complications that are common in&#160;controlled ovarian hyperstimulation cycles and saving time and money. For fertile patients, OP-IVM could serve as a &#34;fertility insurance&#34; while undergoing routine gynecological surgery.
Furthermore, damages caused by gynecological surgeries, such as electrocautery6,7 and ovarian tumor resection, could be reduced through oocyte retrieval before gynecological surgery. Therefore, compared with routine gynecological surgery, OP-IVM could reduce the number of operations during infertility treatment and prevent the loss of functional oocytes during ovarian surgery.
A previous study has shown that the additional procedure of oocyte retrieval would neither increase surgical complications and adverse pregnancy outcomes, nor prolong the hospital stay8. Some patients have given live birth through OP-IVM8, indicating the feasibility of this method. This paper describes characteristics of patients who may benefit from OP-IVM as well as procedures and critical points of OP-IVM and discusses the evaluation of human oocyte maturity.

<table><tbody><tr><td>19 G single-lumen aspiration needles</td><td>Cook, Australia</td><td>K-OPS-7035-REH-ET</td><td></td></tr><tr><td>4-well plate</td><td>Corning</td><td></td><td></td></tr><tr><td>70 &amp;mu;m nylon cell strainer</td><td>Falcon, USA</td><td>352350</td><td></td></tr><tr><td>CO&lt;sub&gt;2&lt;/sub&gt; Incubator</td><td>Thermo</td><td></td><td></td></tr><tr><td>Culture oil</td><td>Vitrolife, Sweden</td><td>10029,OVOIL&amp;nbsp;</td><td>Step 3.2.</td></tr><tr><td>FSH &amp;amp; LH</td><td>Ferring Reproductive Health, Germany</td><td>MENOPUR&amp;reg;</td><td></td></tr><tr><td>Glass Pasteur pipette</td><td>Hilgenberg GmbH, Germany</td><td>3154102-26</td><td></td></tr><tr><td>G-MOPS medium</td><td></td><td></td><td>pH-stable handling medium for washing the needle before puncturing</td></tr><tr><td>IVM medium</td><td>Origio, Denmark</td><td>ART-1600-B</td><td></td></tr><tr><td>Laminar Flow Clean Benches</td><td>ESCO</td><td></td><td></td></tr><tr><td>Petri dish</td><td>Thermo Fisher Scientific, Denmark</td><td>263991</td><td></td></tr><tr><td>pH stable handing media designed to support the handling and manipulation of oocytes and embryos outside the incubator</td><td>Vitrolife, Sweden</td><td>10130, G-MOPS PLUS</td><td>Step 7.1.</td></tr><tr><td>Rinse solution</td><td>Cook, Australia</td><td>K-SIFB-100</td><td></td></tr><tr><td>Stereoscope</td><td>Nikon</td><td></td><td></td></tr></tbody></table>

op-ivm: combining in vitro maturation after oocyte retrieval with gynecological surgery

The use of in vitro maturation (IVM) before gynecological operation (OP-IVM) is an extension of conventional IVM that combines IVM following oocyte retrieval with routine gynecological surgery. OP-IVM is suitable for patients undergoing benign gynecological surgery who have the need for fertility preservation (FP) or infertility treatments such as in vitro fertilization and embryo transfer (IVF-ET). In the operating room, patients undergoing benign gynecological surgery are first anesthetized and receive ultrasound-guided immature follicle aspiration (IMFA) treatment. As the subsequent gynecological surgery is performed, the cumulus-oocyte complexes (COCs) are examined, and the immature COCs are transferred into the IVM medium and cultured for 28-32 hours in the IVF laboratory. After assessment, mature oocytes in the MII stage will be selected and cryopreserved in liquid nitrogen for FP or fertilized by intracytoplasmic sperm injection (ICSI) for IVF-ET. By combining IVM with gynecological surgery, immature oocytes that would have been discarded can be saved and used for assisted reproductive technology (ART). The procedure, significance and critical aspects of OP-IVM are described in this article.

Until December 2019, OP-IVM was used for fertility preservation of 274 patients. Embryological and reproductive outcomes of 158 patients between 2014 to 2016 were published in a previous paper8. The following example discusses the procedure followed for a PCOS patient receiving OP-IVM in 2016. The patient is a 28-year-old diagnosed with primary infertility, left adnexal cyst, and PCOS. She received laparoscopic cystectomy and OP-IVM on September 28th, 20...

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OP-IVM: Combining In vitro Maturation after Oocyte Retrieval with Gynecological Surgery

In vitro maturation (IVM) before gynecological operation (OP-IVM) combines IVM following oocyte retrieval with routine gynecological surgery and serves as an extension of conventional IVM applications for fertility preservation.

The use of in vitro maturation (IVM) before gynecological operation (OP-IVM) is an extension of conventional IVM that combines IVM following oocyte ...

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3.9K Views.  Peking University Third Hospital. In vitro maturation (IVM) before gynecological operation (OP-IVM) combines IVM following oocyte retrieval with routine gynecological surgery and serves as an extension of conventional IVM applications for fertility preservation.

Video: OP-IVM: Combining In vitro Maturation after Oocyte Retrieval with Gynecological Surgery

Functional Manipulation of Maternal Gene Products Using In Vitro Oocyte Maturation in Zebrafish

Cellular events that take place during the earliest stages of animal embryonic development are driven by maternally derived gene products deposited into the developing oocyte. Because these events rely on maternal products which typically act very soon after fertilization-that preexist inside the egg, standard approaches for expression and functional reduction involving the injection of reagents into the fertilized egg are typically ineffective. Instead, such manipulations must be performed during oogenesis, prior to or during the accumulation of maternal products. This article describes in detail a protocol for the in vitro maturation of immature zebrafish oocytes and their subsequent in vitro fertilization, yielding viable embryos that survive to adulthood. This method allows the functional manipulation of maternal products during oogenesis, such as the expression of products for phenotypic rescue and tagged construct visualization, as well as the reduction of gene function through reverse-genetics agents.

Functional Manipulation of Maternal Gene Products Using In Vitro Oocyte Maturation in Zebrafish

An optimized protocol for the in vitro maturation of zebrafish oocytes used for the manipulation of maternal gene products is presented here.

Cellular events that take place during the earliest stages of animal embryonic development are driven by maternally derived gene products deposited into ...

Developmental Biology

Analysis of Chromosome Segregation, Histone Acetylation, and Spindle Morphology in Horse Oocytes

The field of assisted reproduction has been developed to treat infertility in women, companion animals, and endangered species. In the horse, assisted reproduction also allows for the production of embryos from high performers without interrupting their sports career and contributes to an increase in the number of foals from mares of high genetic value. The present manuscript describes the procedures used for collecting immature and mature oocytes from horse ovaries using ovum pick-up (OPU). These oocytes were then used to investigate the incidence of aneuploidy by adapting a protocol previously developed in mice. Specifically, the chromosomes and the centromeres of metaphase II (MII) oocytes were fluorescently labeled and counted on sequential focal plans after confocal laser microscope scanning. This analysis revealed a higher incidence in the aneuploidy rate when immature oocytes were collected from the follicles and matured in vitro compared to in vivo. Immunostaining for tubulin and the acetylated form of histone four at specific lysine residues also revealed differences in the morphology of the meiotic spindle and in the global pattern of histone acetylation. Finally, the expression of mRNAs coding for histone deacetylases (HDACs) and acetyl-transferases (HATs) was investigated by reverse transcription and quantitative-PCR (q-PCR). No differences in the relative expression of transcripts were observed between in vitro and in vivo matured oocytes. In agreement with a general silencing of the transcriptional activity during oocyte maturation, the analysis of the total transcript amount can only reveal mRNA stability or degradation. Therefore, these findings indicate that other translational and post-translational regulations might be affected.
Overall, the present study describes an experimental approach to morphologically and biochemically characterize the horse oocyte, a cell type that is extremely challenging to study due to low sample availability. However, it can expand our knowledge on the reproductive biology and infertility in monovulatory species.

This manuscript describes an experimental approach to morphologically and biochemically characterize horse oocytes. Specifically, the present work illustrates how to collect immature and mature horse oocytes by ultrasound-guided ovum pick-up (OPU) and how to investigate chromosome segregation, spindle morphology, global histone acetylation, and mRNA expression.

The field of assisted reproduction has been developed to treat infertility in women, companion animals, and endangered species. In the horse, assisted ...

In Vitro Culture Strategy for Oocytes from Early Antral Follicle in Cattle

The limited reserve of mature, fertilizable oocytes represents a major barrier for the success of assisted reproduction in mammals. Considering that during the reproductive life span only about 1% of the oocytes in an ovary mature and ovulate, several techniques have been developed to increase the exploitation of the ovarian reserve to the growing population of non-ovulatory follicles. Such technologies have allowed interventions of fertility preservation, selection programs in livestock, and conservation of endangered species. However, the vast potential of the ovarian reserve is still largely unexploited. In cows, for instance, some attempts have been made to support in vitro culture of oocytes at specific developmental stages, but efficient and reliable protocols have not yet been developed. Here we describe a culture system that reproduce the physiological conditions of the corresponding follicular stage, defined to develop in vitro growing oocytes collected from bovine early antral follicles to the fully-grown stage, corresponding to the medium antral follicle in vivo. A combination of hormones and a phosphodiesterase 3 inhibitor was used to prevent untimely meiotic resumption and to guide oocyte&#39;s differentiation.

We describe the procedures for isolation of growing oocytes from ovarian follicles at early stages of development, as well as the setup of an in vitro culture system which can support the growth and differentiation up to the fully-grown stage. &#160;

The limited reserve of mature, fertilizable oocytes represents a major barrier for the success of assisted reproduction in mammals. Considering that ...

Defining the Program of Maternal mRNA Translation during In vitro Maturation using a Single Oocyte Reporter Assay

Events associated with oocyte nuclear maturation have been well described. However, much less is known about the molecular pathways and processes that take place in the cytoplasm in preparation for fertilization and acquisition of totipotency. During oocyte maturation, changes in gene expression depend exclusively on the translation and degradation of maternal messenger RNAs (mRNAs) rather than on transcription. Execution of the translational program, therefore, plays a key role in establishing oocyte developmental competence to sustain embryo development. This paper is part of a focus on defining the program of maternal mRNA translation that takes place during meiotic maturation and at the oocyte-to-zygote transition. In this method paper, a strategy is presented to study the regulation of translation of target mRNAs during in vitro oocyte maturation. Here, a Ypet reporter is fused to the 3&#39; untranslated region (UTR) of the gene of interest and then micro-injected into oocytes together with polyadenylated mRNA encoding for mCherry to control for injected volume. By using time-lapse microscopy to measure reporter accumulation, translation rates are calculated at different transitions during oocyte meiotic maturation. Here, the protocols for oocyte isolation and injection, time-lapse recording, and data analysis have been described, using the Ypet/interleukin-7 (IL-7)-3&#39; UTR reporter as an example.

This protocol describes a reporter assay to study the regulation of mRNA translation in single oocytes during in vitro maturation.

Events associated with oocyte nuclear maturation have been well described. However, much less is known about the molecular pathways and processes that ...

Vitrification of In Vitro Matured Oocytes Collected from Adult and Prepubertal Ovaries in Sheep

In livestock, in vitro embryo production systems can be developed and sustained thanks to the large number of ovaries and oocytes that can be easily obtained from a slaughterhouse. Adult ovaries always bear several antral follicles, while in pre-pubertal donors the maximal numbers of oocytes are available at 4 weeks of age, when ovaries bear peak numbers of antral follicles. Thus, 4 weeks old lambs are considered good donors, even if the developmental competence of prepubertal oocytes is lower compared to their adult counterpart. 
Basic research and commercial applications would be boosted by the possibility of successfully cryopreserving vitrified oocytes obtained from both adult and prepubertal donors. The vitrification of oocyte collected from prepubertal donors would also allow shortening the generation interval and thus increasing the genetic gain in breeding programs. However, the loss of developmental potential after cryopreservation makes mammalian oocytes probably one of the most difficult cell types to cryopreserve. Among the available cryopreservation techniques, vitrification is widely applied to animal and human oocytes. Despite recent advancements in the technique, exposures to high concentrations of cryoprotective agents as well as chilling injury and osmotic stress still induce several structural and molecular alterations and reduce the developmental potential of mammalian oocytes. Here, we describe a protocol for the vitrification of sheep oocytes collected from juvenile and adult donors and matured in vitro prior to cryopreservation. The protocol includes all the procedures from oocyte in vitro maturation to vitrification, warming and post-warming incubation period. Oocytes vitrified at the MII stage can indeed be fertilized following warming, but they need extra time prior to fertilization to restore damage due to cryopreservation procedures and to increase their developmental potential. Thus, post-warming culture conditions and timing are crucial steps for the restoration of oocyte developmental potential, especially when oocyte are collected from juvenile donors.

The protocol aims at providing a standard method for the vitrification of adult and juvenile sheep oocytes. It includes all the steps from the preparation of the in vitro maturation media to the post-warming culture. Oocytes are vitrified at the MII stage using Cryotop to ensure the minimum essential volume.

In livestock, in vitro embryo production systems can be developed and sustained thanks to the large number of ovaries and oocytes that can be easily ...

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) ...

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 ...

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.

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, ...

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, ...

OP-IVM: Combining In vitro Maturation after Oocyte Retrieval with Gynecological Surgery

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