Use of marmoset extra oocytes retrieved at follicle isolation without in vivo gonadotropin pre-treatment

( Seung-yup Ku ),( Yoon Young Kim ),( Byeong Cheol Kang ),( Zev Rosenwaks ),( Hoon Kim ),( Chang Suk Suh ) 대한산부인과학회 2019 대한산부인과학회 학술대회 Vol.105 No.-

Objective: Oocyte in vitro maturation is frequently used for assisted reproduction since established in human and rodent. To improve the current infertility treatment, such procedures as in vitro maturation of early follicles are popularly attempted. At follicular isolation stage, unexpected rupture of follicles yields extra oocytes. Those oocytes obtained during follicle isolation from primate ovary can be used for early follicle maturation studies. Methods: Marmoset (Callithrix jacchus) is a good model used for reproductive biomedical investigation, since its reproductive physiology is very similar to that of human. After the ovaries of female marmosets were surgically retrieved, and the excess oocytes released during follicle isolation were collected without gonadotropin induction. Oocytes were matured in vitro for 72 h in the presence of various concentrations of epidermal growth factor and human chorionic gonadotropin (hCG), and the optimal maturation conditions were assessed. Each oocyte was individually prepared for RT-PCR and the expression of mRNAs and microRNAs (miRs) were evaluated. Results: hCG concentrations significantly influenced on the maturation rate of oocytes. Expression of Figla, Cja-let-7b, and Cja-mir-27a, was highest when the oocytes were matured using 300 mIU/mL hCG. From these results, these up-regulated miRs were considered to regulate the maturation of oocytes. Protein network analysis suggested an association of Figla with Nobox, GDF9 and DAZL. Conclusion: To summarize, these results suggested optimal maturation conditions of marmoset extra oocytes without using in vivo gonadotropin treatment, and showed the specific roles of miRs during early primate oocyte maturation (2016R1E1A1A01943455).

Gonadotropin ratio affects the <i>in vitro</i> growth of rhesus ovarian preantral follicles

Kim, Yoon Young,Yun, Jun-Won,Kim, Jong Min,Park, Chung Gyu,Rosenwaks, Zev,Liu, Hung Ching,Kang, Byeong-Cheol,Ku, Seung-Yup BMJ Publishing Group 2016 Journal of investigative medicine Vol.64 No.4

<P><I>In vitro</I> follicle growth (IVFG) strategy is critical in the fertility preservation of cancer survivors; however, its optimal protocol needs to be developed using primate models since the availability of human samples is limited. Only a few previous studies have reported the successful IVFG of rhesus monkey ovaries using low-dose follicle-stimulating hormone (FSH) (0.3 or 3 ng/mL) and long-term culture (up to 5 weeks) and it is still uncertain in regard to the optimal culture duration and effective dose of treated gonadotropins applicable to the IVFG of rhesus preantral follicles. Recently, we have reported that the FSH to luteinizing hormone (LH) ratio affects the <I>in vitro</I> growth of murine ovarian follicles. We aimed to investigate whether gonadotropin ratios affect the efficiency of rhesus follicular growth <I>in vitro</I>. Ovaries were collected from six necropsied rhesus macaques (4–9 years) and preantral follicles were retrieved and cultured for 14 days using 200 mIU/mL FSH. The characteristics of follicular growth were compared between the FSH:LH=1:1 (n=24) and FSH:LH=2:1 (n=24) groups. High concentration gonadotropin treatment shortened the duration required for <I>in vitro</I> maturation of rhesus preantral follicles. The FSH:LH=2:1 group showed a faster follicular growth and enabled the acquisition of mature oocytes, although the expression of growth differentiation factor (GDF)-9 and anti-Müllerian hormone (AMH) did not differ significantly between the two groups. Taken together, high dose gonadotropin treatment can shorten the duration of IVFG and the gonadotropin ratio is important in the IVFG of rhesus monkey ovaries.</P>

Expression of Transcripts in Marmoset Oocytes Retrieved during Follicle Isolation Without Gonadotropin Induction

Kim, Yoon Young,Kang, Byeong-Cheol,Yun, Jun Won,Ahn, Jae Hun,Kim, Yong Jin,Kim, Hoon,Rosenwaks, Zev,Ku, Seung-Yup MDPI 2019 INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES Vol.20 No.5

<P>The in vitro maturation of oocytes is frequently used as an assisted reproductive technique (ART), and has been successfully established in humans and rodents. To overcome the limitations of ART, novel procedures for the in vitro maturation of early follicles are emerging. During the follicle isolation procedure, the unintended rupture of each follicle leads to a release of extra oocytes. Such oocytes, which are obtained during follicle isolation from marmosets, can be used for early maturation studies. Marmoset (<I>Callithrix jacchus</I>), which is classified as a new-world monkey, is a novel model that has been employed in reproductive biomedical research, as its reproductive physiology is similar to that of humans in several aspects. The ovaries of female marmosets were collected, and the excess oocytes present during follicle isolation were retrieved without pre-gonadotropin induction. Each oocyte was matured in vitro for 48 h in the presence of various concentrations of human chorionic gonadotropin (hCG) and epidermal growth factor (EGF), and the maturity of oocytes and optimal maturation conditions were evaluated. Each oocyte was individually reverse-transcribed, and the expression of mRNAs and microRNAs (miRs) were analyzed. Concentrations of hCG significantly affected the maturation rate of oocytes [the number of metaphase II (MII) oocytes]. The expression of <I>BMP15</I> and <I>ZP1</I> was highest when the oocytes were matured using 100 IU/L of hCG without pre-treatment with gonadotropins, and that of <I>Cja-mir-27a</I> was highest when cultured with follicle stimulating hormone. These results suggest that these up-regulated miRs affect the maturation of oocytes. Interactions with other protein networks were analyzed, and a strong association of BMP15 and ZP1 with sperm binding receptor (ACR), anti-Müllerian hormone (AMH), and AMH receptor was demonstrated, which is related to the proliferation of granulosa cells. Collectively, on the basis of these results, the authors propose optimal maturation conditions of excess oocytes of marmoset without in vivo gonadotropin treatment, and demonstrated the roles of miRs in early oocyte maturation at the single-cell level in marmosets.</P>

Synergistic regenerative effects of functionalized endometrial stromal cells with hyaluronic acid hydrogel in a murine model of uterine damage

Kim, Yoon Young,Park, Kyu-Hyung,Kim, Yong Jin,Kim, Moon Suk,Liu, Hung Ching,Rosenwaks, Zev,Ku, Seung-Yup Elsevier 2019 ACTA BIOMATERIALIA Vol. No.

<P><B>Abstract</B></P> <P>Clinically intractable infertility and recurrent miscarriage due to irreversible endometrial damage need to be treated with biomaterial- and cell-based therapies. Some previous studies have reported on the efficacy of a collagen scaffold and/or bone marrow-derived mesenchymal stem cells. However, the functional differentiation of grafted cells was uncertain, and the time required for regeneration was long in these studies.</P> <P>Here, we show the synergistic regenerative effects of hyaluronic acid (HA) hydrogel with <I>in vitro</I> decidualized endometrial stromal cells (EMSCs) in a murine uterine infertility (synechiae) model. Decidualized EMSCs (dEMSCs) were encapsulated with HA hydrogel, combined with three different doses of fibrinogen/thrombin (5, 50, and 500 mIU/mL). The HA/fibrin gel showed biocompatibility when mixed with dEMSCs. The addition of thrombin enhanced gel formation (5 and 50 mIU/mL) and engraftment and enabled the effective release of adhesion molecules.</P> <P>Within two weeks, which is a short duration, treatment with hydrogel decreased the fibrous tissue and increased the thickness of the endometrium. The regenerated endometrium demonstrated functional recovery, as evidenced by the expression and secretion of molecules essential for embryonic implantation, such as Desmin, CD44, PECAM, and IGF-1. Transferred embryos successfully implanted and the normal development of implanted embryos (<I>n</I> = 37) were evaluated by co-localization of distinct markers of the three germ layers (Sox2, Nestin, Brachyury, AFP, and HNF4α). Live birth of offspring was achieved in the regenerated endometrium by HA hydrogel.</P> <P>Therefore, HA hydrogel-mixed dEMSCs can be an innovative treatment strategy with rapid recovery of endometrial damage and may also have therapeutic potential in intractable infertility or recurrent miscarriage.</P> <P><B>Statement of Significance</B></P> <P>Decidualized EMSCs (dEMSCs) encapsulated with HA hydrogel combined with fibrinogen/thrombin (50 mIU/mL) showed injectability and biocompatibility when mixed with dEMSCs.</P> <P>Hydrogel-encapsulated dEMSCs can be a useful treatment for damaged endometrium in short duration, with successful implantation and normal development in a murine model.</P> <P><B>Graphical abstract</B></P> <P>Functional recovery of damaged uteri using HA/fibrin hydrogel. An injectable HA/fibrin hydrogel was manufactured and used to encapsulate <I>in vitro</I> decidualized EMSCs (dEMSCs) retrieved from parous mice. To produce a uterine synechia model, the endometrium underwent chemical damage. HA/fibrin hydrogel with dEMSCs was grafted into the damaged uteri. Subsequent recovery was analyzed using <I>in vitro</I> and <I>in vivo</I> assays. Embryos were transferred into regenerated uteri, and implantation and embryonic development were evaluated.</P> <P>[DISPLAY OMISSION]</P>

Pretreatment of normal responders in fresh in vitro fertilization cycles: A comparison of transdermal estradiol and oral contraceptive pills

Pereira, Nigel,Petrini, Allison C.,Zhou, Zhen N.,Lekovich, Jovana P.,Kligman, Isaac,Rosenwaks, Zev The Korean Society for Reproductive Medicine 2016 Clinical and Experimental Reproductive Medicine Vol.43 No.4

Objective: The aim of this study was to investigate the impact of pretreatment with transdermal estradiol ($E_2$) compared to oral contraceptive pills (OCPs) on controlled ovarian stimulation (COS) response in normal responders undergoing fresh in vitro fertilization (IVF)-embryo transfer (ET) cycles. Methods: A retrospective cohort study was performed of normal responders undergoing fresh IVF-ET cycles who received pretreatment with transdermal $E_2$ versus OCPs prior to fresh IVF-ET. The total days of ovarian stimulation, total dosage of gonadotropins, total number of oocytes, and mature oocytes retrieved were noted. Pregnancy outcomes after ET were also recorded. Results: A total of 2,092 patients met the inclusion criteria: 1,057 and 1,035 patients in the transdermal $E_2$ and OCP groups, respectively. Patients in the OCP group had a longer duration of COS ($10.7{\pm}1.63days$, p< 0.01) than the $E_2$ group ($9.92{\pm}1.94days$). Patients in the OCP group also required higher cumulative doses of gonadotropins ($2,657.3{\pm}1,187.9IU$) than those in the $E_2$ group ($2,550.1{\pm}1,270.2IU$, p= 0.002). No statistically significant differences were found in the total and mature oocytes retrieved or in the rates of biochemical pregnancy, clinical pregnancy, spontaneous miscarriage, and live birth between the groups. Conclusion: Our findings suggest that compared to OCPs, pretreatment with transdermal $E_2$ is associated with a shorter duration of ovarian stimulation and lower gonadotropin utilization, without compromising the oocyte yield or pregnancy outcomes in normal-responder patients undergoing fresh IVF.