Conventional multi-step warming protocols (MWP) for vitrified blastocysts are labor-intensive and susceptible to handling variability. This study evaluated whether a shortened warming protocol (SWP) could support post-warming recovery and developmental competence comparable to those of conventional MWP. A retrospective propensity score-matched cohort study was conducted using 546 single vitrified-warmed blastocyst transfer cycles (271 SWP and 275 MWP). Post-warming developmental behavior was assessed using time-lapse imaging, including the start of re-expansion (tSRE), completion of re-expansion (tCRE), and hatching start point (tHSP). Morphological recovery, blastocyst quality score (BQS), and inner cell mass (ICM) and trophectoderm (TE) grades were also evaluated. Blastocysts warmed using SWP demonstrated significantly faster post-warming recovery, with shorter tSRE (0.148 vs. 0.179 h, P = 0.009), tCRE (2.16 vs. 2.91 h, P < 0.001), and tHSP (3.08 vs. 5.24 h, P < 0.001). The SWP group showed a significantly higher proportion of blastocysts with improved post-warming morphology and grade A ICM and TE than the MWP group (P < 0.05). The distribution of hatching stages and clinical pregnancy outcomes did not differ significantly between the groups. These findings suggest that SWP provides efficient and biologically stable post-warming recovery without compromising embryonic quality or clinical outcomes.

Objective:To evaluate the impact of blastulation speed and blastocyst quality on pregnancy outcomes in single vitrified-warmed blastocyst transfer (SVBT) cycles. Methods:A total of 736 SVBT cycles conducted between 2019 and 2021 were analyzed. Blastocysts were classified according to blastulation speed (day 5 vs. day 6) and morphological quality (top, good, average, and poor). A supplementary analysis included 395 blastocysts from 171 cycles that underwent pre-implantation genetic testing for aneuploidy between 2022 and 2023. Ongoing pregnancy rates and euploidy rates were compared across blastulation speed and quality categories. Results:In SVBT cycles, the ongoing pregnancy rate was significantly higher in the day 5 group than in the day 6 group. However, no significant difference in ongoing pregnancy rates was observed between top-quality and good-quality blastocysts prior to vitrification, irrespective of blastulation speed. Average- and poor-quality blastocysts were more frequently observed in the day 6 group than in the day 5 group. In addition, euploidy rates were significantly higher in the day 5 group than in the day 6 group. However, no significant difference in euploidy rates was detected between top- or good-grade blastocysts derived from day 5 and day 6 embryos. By contrast, significant differences in euploidy rates were observed between average- and poor-quality blastocysts from day 5 and day 6. Conclusion:Blastocyst quality has a significant influence on clinical outcomes, whereas blastulation speed appears to be comparatively less critical. For slow-developing embryos, vitrification of high-quality day 6 blastocysts may be preferable to the transfer of low-grade day 5 blastocysts, thereby providing practical clinical options for patients experiencing developmental delays.

Abstract Study question What is the mechanism of ovarian damage after doxorubicin (Dox) treatment and how recombinant human anti-Müllerian hormone (rhAMH) suppresses ovarian aging and prevents chemotherapy-induced damage? Summary answer Administration of rhAMH attenuates Dox-induced follicle depletion and protects the ovarian reserve in mice, primarily by reducing DNA damage via Id3 upregulation. What is known already Treatment with rhAMH results in reversible ovarian quiescence that can reduce primordial follicle depletion induced by Dox when it is used as an adjuvant. However, the exact mechanism of Dox-induced ovarian toxicity and the mechanisms by which rhAMH rescue this damage were unknown. Study design, size, duration For the in vivo experiments: 25-day-old mice were randomized into four treatment groups (Control, Dox, Dox+rhAMH, and rhAMH) for three different timepoints (n = 5 per group, total n = 60). Ovaries were removed for analysis at 4, 24 hours and and 1 week after treatment. For the in vitro studies: 21-day-old mice (n = 30) were treated with subcutaneous injection of diethylstilbestrol (DES) to enhance preantral follicle growth, and primary granulosa cells were collected by mechanical dissociation. Participants/materials, setting, methods Immunohistochemistry with DDX4 and counter staining with hematoxylin was used for differential follicle counts. DNA damage and apoptosis were measured by quantification of phosphorylated form of histone 2AX (γH2AX) and by TUNEL assay. MTS assay, clonogenic assay and siRNA Id3 RNA interference knockdown experiment were carried out to reveal the effects of doxorubicin and rhAMH on primary granulosa cell survival. Main results and the role of chance Treatment with Dox alone caused significant loss of PMF reserve (59% reduction, 5.25 versus 12.76 in control, P<0.0001) and this was significantly attenuated by coadministration with rhAMH (33% rescue, 9.47 P<0.001). The DNA damage marker γH2AX was elevated in primordial follicle (PMF) and primary follicle (PF) oocytes from Dox-treated ovaries 4 h after treatment, compared with controls (P<0.0001) and was significantly reduced (95% reduction) in rhAMH cotreated ovaries (P<0.001). Oocytes of secondary follicle (SF) and antral follicle (AF) were negative for γH2AX while granulosa cells (GCs) showed increased positivity for γH2AX after Dox treatment with significant reduction after rhAMH cotreatment. (24% reduction, P<0.001). Oocytes and somatic cells of PMF and PF rarely showed positive TUNEL staining at any of the timepoints evaluated.In vitro study showed reduced double stranded breaks (DSBs) in Dox-rhAMH cotreated GC compared to Dox alone indicated by number and signal intensity of γH2AX positive cells. Finally, while rhAMH treatment increased Id3 expression in early stage oocyte and granulosa cells, siRNA Id3 knock-down in granulosa cells significantly reduced the protective effect of rhAMH, as seen by the increased DSBs and pyknotic morphology of cells after Dox suggesting Id3 is downstream of AMH and enhancing Dox-induced DSB DNA repair. Limitations, reasons for caution This study failed to detect fate of PMF after DNA damage. PMF distinguished without apoptotic finding and this might be because experiment was conducted in limited time point or mechanism other than apoptotic cell death are responsible for PMF loss such as shedding, phagocytosis or necrosis. Wider implications of the findings Understanding of the mechanism behind rhAMH prevention of chemotherapy-induced reproductive aging is important for reproductive health and longevity. Possibility of rhAMH as non-invasive ferto-protective agent during chemotherapy may overcome limitation of current fertility preservation method which is limited by time requirement, cost and success rate. Trial registration number ’not applicable’

Objective Dual trigger is used to induce final oocyte maturation during the process of controlled ovarian hyperstimulation, yet yielding controversial results. Also, there are yet no data regarding the effect of the dosage of the dual trigger on clinical outcomes. Based on the Patient-Oriented Strategies Encompassing IndividualizeD Oocyte Number (POSEIDON) criteria, this study aimed to determine the clinical difference of a single bolus versus two boluses of gonadotropin-releasing hormone agonist (GnRHa) in POSEIDON group IV patients using dual trigger. Methods We screened a total of 1,256 patients who underwent in vitro fertilization (IVF) cycles who met the POSEIDON group IV criteria. Six hundred and twenty-nine patients received one bolus of GnRHa, and 627 patients were given two boluses. All patients received the same dose of recombinant human chorionic gonadotropin during the dual trigger cycle. Results Metaphase II oocyte retrieval rate, fertilization rate and clinical pregnancy rate did not differ between the two groups. However, a lower percentage of at least one top-quality embryo transfer (34.3% vs. 26.0%, P=0.001) in the two bolus-GnRHa group was noted. Conclusion A double bolus of GnRHa did not show superior clinical results compared to a single bolus of GnRHa in the dual trigger IVF cycle. Therefore, GnRHa doses for use should be decided based on individual clinical situations considering cost-effectiveness and patient compliance, but further investigation will be needed.

Background: The single vitrified-warmed blastocyst transfer (SVBT) cycle has been increasingly utilized for assisted reproductive technology. Women of advanced maternal age (AMA) comprise a significant portion of patients who have undergone 'freeze-all' cycles. This study investigated the association between the post-warming extended culture duration and pregnancy outcomes in patients of AMA. Methods: This retrospective cohort study analyzed the outcomes of 697 SVBT cycles between January 2016 and December 2017. The cycles were divided into 3 groups based on the age of the female partners: group I: < 35 years (n = 407), group II: 35-37 years (n = 176); and group III, 38-40 years (n = 114). Data are shown as the mean +/- standard error of the mean. Data were analyzed using one-way ANOVA followed by Duncan's multiple range test. Statistical significance was set at P < 0.001. Results: The blastocyst rate, clinical pregnancy rate, and live birth rate (LBR) was significantly lower in the AMA groups. However, there were no significant differences in LBR in the transfer between the AMA and younger groups according to blastocyst morphology and post-warming extended culture duration. Conclusion: Post-warming extended culture of blastocysts is not harmful to patients of AMA. It could be a useful parameter in clinical counseling and decision making for fertility treatments.

Dual trigger is used to induce final oocyte maturation during the process of controlled ovarian hyperstimulation, yet yielding controversial results. Also, there are yet no data regarding the effect of the dosage of the dual trigger on clinical outcomes. Based on the Patient-Oriented Strategies Encompassing IndividualizeD Oocyte Number (POSEIDON) criteria, this study aimed to determine the clinical difference of a single bolus versus two boluses of gonadotropin-releasing hormone agonist (GnRHa) in POSEIDON group IV patients using dual trigger.We screened a total of 1,256 patients who underwent in vitro fertilization (IVF) cycles who met the POSEIDON group IV criteria. Six hundred and twenty-nine patients received one bolus of GnRHa, and 627 patients were given two boluses. All patients received the same dose of recombinant human chorionic gonadotropin during the dual trigger cycle.Metaphase II oocyte retrieval rate, fertilization rate and clinical pregnancy rate did not differ between the two groups. However, a lower percentage of at least one top-quality embryo transfer (34.3% vs. 26.0%, P=0.001) in the two bolus-GnRHa group was noted.A double bolus of GnRHa did not show superior clinical results compared to a single bolus of GnRHa in the dual trigger IVF cycle. Therefore, GnRHa doses for use should be decided based on individual clinical situations considering cost-effectiveness and patient compliance, but further investigation will be needed.

Previous studies have shown that the angle of spindle in MII oocyte is associated with embryo development. The incidence of zygotes with significant pronucleus size asynchrony is considered to be an indication of the poor embryo development. However, the correlation between angle the of spindle and difference in the size of 2PN have not been studied. In the present study, we investigated the correlation between the angle of spindle and the difference in the size of 2PN, and the effect of its correlation on clinical outcomes. Data were retrospectively collected from polarized light microscopy and time-lapse system performed from March 2018 to February 2020 in the Fertility center of CHA Gangnam Medical Center. We analyzed 235 zygotes from 66 patients. We measured the angle of spindle prior to ICSI using a LC-PolscopeTM (CRI). Oocytes were divided into two groups according to the angle of MII spindle (<30° group =189, ≥30° group =46) and were cultured in EmbryoscopeTM (Vitrolife). The size of 2PN were analyzed by measuring the horizontal diameter at the same focal plane before PN fade. Also, an abnormal division (direct division and not available cell number due to poor quality)in 2-cell embryo was checked. The average of difference in the size of 2PN was significantly increased in ≥30° group (<30° group; 1.4 um vs. ≥30° group; 3.5 um, p< 0.05). The rate of abnormal division (8.5% vs.23.9%) was highly increased but the rate of good quality (63.5% vs. 47.8%) was decreased in the ≥30° group compared with <30° group (p <0.05). When only embryos from the <30° group were selected for transfer, the pregnancy and implantation rates were 67.7% and 57.1%. But when only embryos from the ≥30° group were transferred, those rates were 25.0% and 20.0%, respectively (p <0.05). This is the first study that has analyzed the correlation between spindle angle and difference in the size of 2PN. Further studies with larger sample size are needed. This study could provide a new insight for the embryo selection by enabling noninvasive judgement of embryos that are more likely to become pregnant at the earliest time point. Also, it could be especially useful to assess zygote quality in countries with strict policies.

Peroxisome proliferator-activated receptor gamma (PPARγ) is known as a regulator of cellular functions, including adipogenesis and immune cell activation. The objectives of this study were to investigate the expression of PPARγ and identify the mechanism of primordial follicle activation via PPARγ modulators in mouse ovaries. We first measured the gene expression of PPARγ and determined its relationship with phosphatase and tensin homolog (PTEN), protein kinase B (AKT1), and forkhead box O3a (FOXO3a) expression in neonatal mouse ovaries. We then incubated neonatal mouse ovaries with PPARγ modulators, including rosiglitazone (a synthetic agonist of PPARγ), GW9662 (a synthetic antagonist of PPARγ), and cyclic phosphatidic acid (cPA, a physiological inhibitor of PPARγ), followed by transplantation into adult ovariectomized mice. After the maturation of the transplanted ovaries, primordial follicle growth activation, follicle growth, and embryonic development were evaluated. Finally, the delivery of live pups after embryo transfer into recipient mice was assessed. While PPARγ was expressed in ovaries from mice of all ages, its levels were significantly increased in ovaries from 20-day-old mice. In GW9662-treated ovaries in vitro, PTEN levels were decreased, AKT was activated, and FOXO3a was excluded from the nuclei of primordial follicles. After 1 month, cPA-pretreated, transplanted ovaries produced the highest numbers of oocytes and polar bodies, exhibited the most advanced embryonic development, and had the greatest blastocyst formation rate compared to the rosiglitazone- and GW9662-pretreated groups. Additionally, the successful delivery of live pups after embryo transfer into the recipient mice transplanted with cPA-pretreated ovaries was confirmed. Our study demonstrates that PPARγ participates in primordial follicle activation and development, possibly mediated in part by the PI3K/AKT signaling pathway. Although more studies are required, adapting these findings for the activation of human primordial follicles may lead to treatments for infertility that originates from poor ovarian reserves.

The aim of this study was to evaluate the predictive value of the anti-Mullerian hormone (AMH) level for early pregnancy loss and to compare the significance of AMH level to age as prognostic factors of pregnancy loss in subfertile women. The outcome of 848 subfertile patients confirmed with intrauterine pregnancies by ultrasound within 1 year of measuring serum AMH level were retrospectively analyzed. Among 848 patients, 206 women were diagnosed with early pregnancy loss. The mean age of the 848 patients was 35.66 ± 3.61 years (range: 26-46 years), and the mean AMH level was 2.95 ± 1.89 (range: 0.14-8.82 ng/mL). There were no significant differences in gravidity, parity, body mass index (BMI), and previous abortion history depending on early pregnancy loss. However, multivariable logistic regression analysis confirmed that the probability of early pregnancy loss is significantly affected by age (odd ratio, 1.079: 1.025-1.135, P = 0.004) and AMH (odd ratio, 0.885: 0.797-0.982, p = 0.022). According to this study, AMH level and age are both powerful predictors of early pregnancy loss. While chronological age is already well known as a factor related to early pregnancy loss, AMH was also considered when individualizing risk prediction for early pregnancy loss.

Baculoviral inhibitor of apoptosis repeat-containing 5 (Birc5), also known as survivin, is a member of the inhibitor of apoptosis (IAP) family of proteins and regulates the size of tissues through cell division control. The uterus is the most dynamically sized organ among tissues during the estrous cycle. Although Birc5 is expressed in some terminally differentiated cells, the regulation of its expression in the uterus remains unknown. We investigated the regulation of Birc5 expression in the mouse uterus. RT-PCR analysis showed that Birc5 was expressed in various tissues, including the uterus; the expression level of Birc5 was significantly higher at the diestrus stage. Immunohistochemistry and Western blotting analysis revealed that Birc5 was more active in luminal and glandular epithelium than in endometrial stroma. In ovariectomized mice, Birc5 expression in the uterus was gradually increased by estrogen treatment; however, progesterone injection decreased its expression. Estrogen-induced Birc5 expression was blocked by treatment with estrogen receptor antagonist, ICI 182, 780 and progesterone-reduced Birc5 expression was inhibited by the progesterone receptor antagonist RU486. These results suggest that Birc5 expression is dynamically regulated by a combination of estrogen and progesterone via their receptor-mediated signaling.

Despite the large number of studies on blastocyst transfers, it is unclear whether day 6 blastocysts have similar pregnancy rates and safety with day 5 blastocysts. Thus, this study aimed to compare the obstetric, neonatal, and clinical outcomes of day 5 and day 6 vitrified blastocyst transfers (VBT). In this retrospective cohort study with propensity score matching, we evaluated 1,313 cycles of VBT performed between January 2014 and December 2015 at the Fertility Center of CHA Gangnam Medical Center. All cycles underwent natural endometrial preparation. We used propensity score matching to compare day 5 and day 6 VBTs in a matched comparison. After propensity score matching, there were 465 cycles of day 5 VBT and 155 cycles of day 6 VBT. Implantation rate (IR), clinical pregnancy rate (CPR), and live birth rate (LBR) were significantly lower in day 6 VBTs (44.2 vs. 53.1%,p= 0.023; 48.4 vs. 60.4%,p= 0.009; 33.5 vs. 51.8%,p< 0.001). Miscarriage rate was significantly higher in day 6 VBTs (29.3 vs. 10.7%,p< 0.001). Rate of multiple gestations was similar between the two groups (29.3 vs. 30.2%,p= 0.816). Assessing 241 and 52 babies from day 5 and day 6 VBTs, no differences were found in neonatal outcomes including rates of low birth weight, preterm birth, and congenital malformations. In propensity score-matched analysis, obstetric, and neonatal outcomes between day 5 and day 6 VBTs were similar so that day 6 VBTs are as safe as day 5 VBTs. IR, CPR, and LBR were are all significantly lower in day 6 VBTs. Therefore, if there are no differences in the morphological grade between day 5 and day 6 blastocysts, transfer of day 5 vitrified blastocysts should be considered first.

Objective To evaluate the effects of the duration of cryostorage on clinical outcomes after embryo transfer of vitrified blastocysts stored in an open-device slush-nitrogen (SN2) system. Methods A retrospective cohort study was carried out on 1632 autologous vitrified-warmed blastocyst transfer cycles between January 2013 and June 2014. Duration of cryostorage was divided into four groups: Group I: 0-6 months (n=937); Group II: 7-12 months (n=299); Group III: 13-24 months (n=165); and Group IV: >= 25 months (n=231). The effects of the duration of cryostorage on the survival rate (SR), clinical pregnancy rate (CPR), live birth rate (LBR), and neonatal outcomes of vitrified blastocysts stored in an open-device SN(2)system were evaluated. Results There were no significant differences between groups in SR, CPR, LBR, and neonatal outcomes after autologous vitrified-warmed blastocyst transfer. Multivariate logistic regression analysis showed no effect on LBR from duration of cryostorage. Conclusion Vitrification using SN(2)and long-term cryostorage in an open-device system are safe and effective and do not significantly affect clinical outcomes after embryo transfer.

The uterus is dynamically regulated in response to various signaling triggered by hormones during the estrous cycle. The Hippo signaling pathway is known as an important signaling for regulating cellular processes during development by balancing between cell growth and apoptosis. Serine/threonine protein kinase 3/4 (STK3/4) is a key component of the Hippo signaling network. However, the regulation of STK3/4-Hippo signaling in the uterus is little known. In this study, we investigated the regulation and expression of STK3/4 in the uterine endometrium during the estrous cycle. STK3/4 expression was dynamically regulated in the uterus during the estrous cycle. STK3/4 protein expression was gradually increased from the diestrus stage and reached the highest in the estrus stage. STK3/4 was exclusively localized in the luminal and glandular epithelial cells of the uterus, and phosphorylated STK3/4 was also increased at the estrus stage. Moreover, the increase of STK3/4 expression in uteri was induced by administration of estradiol, but not by progesterone injection in ovariectomized mice. Pretreatment with an estrogen receptor antagonist ICI 182,780 reduced estrogen-induced STK3/4 expression and its phosphorylation. The estrogen-induced STK3/4 expression was related to the increase in phosphorylation of downstream targets including LATS1/2 and YAP. These findings suggest that STK3/4-Hippo signaling acts a novel signaling pathway in the uterine epithelium and STK3/4-Hippo is one of key molecules for connecting between the estrogen downstream signaling pathway and the Hippo signaling pathway leading to regulate dynamic uterine epithelium during the estrous cycle.

Aim For in vitro fertilization, the optimal number of blastocysts to transfer according to blastocyst grade has not been well established, especially with respect to vitrified-warmed blastocyst transfer (VBT) in women aged over 35 years. This study aimed to compare the pregnancy and neonatal outcomes for three different VBT methods with variable numbers and qualities of blastocysts in women aged over 35 years. Methods All VBT cycles were categorized into three groups according to blastocyst grade: GG (two good-quality blastocysts transferred), GP (one good-quality blastocyst transferred with one of poor quality) and GS (one good-quality blastocyst transferred). Blastocysts graded greater than or equal to 3BB were considered good quality. We conducted three 1:1 propensity score-matched analyses (GG vs GS, GP vs GS and GG vs GP) to compare the clinical pregnancy rate (CPR), live birth rate (LBR), multiple pregnancy rate (MPR), preterm birth rate and low birthweight rate. Results Compared to GS, GG had higher CPR and LBR; however, MPR was also higher with GG. There were no significant differences, except implantation rate and MPR between GP and GS. Although implantation rate and CPR with GG were higher than those with GP, there were no significant differences in LBR and MPR. Conclusion To reduce high MPR after double blastocyst transfer methods, single good-quality blastocyst transfers are recommended in the VBT of women aged over 35 years. Transferring a good blastocyst with a poor one should be avoided because it confers no advantage.

Objective As paternal age increases, the quality of sperm decreases due to increased DNA fragmentation and aneuploidy. Higher levels of structural chromosomal aberrations in the gametes ultimately decrease both the morphologic quality of embryos and the pregnancy rate. In this study, we investigated whether paternal age affected the euploidy rate. Methods This study was performed using the medical records of patients who underwent in vitro fertilization (IVF) procedures with preimplantation genetic screening (PGS) from January 2016 to August 2017 at a single center. Based on their morphological grade, embryos were categorized as good- or poor-quality blastocysts. The effects of paternal age were elucidated by adjusting for maternal age. Results Among the 571 total blastocysts, 219 euploid blastocysts were analyzed by PGS (38.4%). When the study population was divided into four groups according to both maternal and paternal age, significant differences were only noted between groups that differed by maternal age (group 1 vs. 3, p=0.031; group 2 vs. 4, p=0.027). Further analysis revealed no significant differences in the euploidy rate among the groups according to the morphological grade of the embryos. Conclusion Paternal age did not have a significant impact on euploidy rates when PGS was performed. An additional study with a larger sample size is needed to clarify the effects of advanced paternal age on IVF outcomes.

BACKGROUND:The standard morphological evaluation has been widely used for embryo selection, but it has limitations. This study aimed to investigate the correlation between morphologic grading and euploidy rate of in vitro fertilization (IVF) preimplantation genetic screening (PGS) and compare the pregnancy rates in young and old ages.METHODS:This is a retrospective study using the medical records of patients who underwent IVF procedures with PGS between January 2016 and February 2017 in a single center. The embryo grades were categorized into 4 groups: excellent, good, fair, and poor. Basic characteristics, euploidy rates, clinical pregnancy (CP) rates and ongoing pregnancy rates were analyzed.RESULTS:The excellent group had significantly higher rate of euploid embryos than fair group (47.82% vs. 29.33%; P = 0.023) and poor group (47.82% vs. 29.60%; P = 0.005). When the four groups were recategorized into two groups (excellent and good vs. fair and poor), they also showed significant difference in euploidy rates (44.52% vs. 29.53%; P = 0.002). When the patients were divided into two groups by age 35, the CP rates for those under and over 35 years old were 44.74% and 47.83%, respectively, which showed no significant difference.CONCLUSION:The significant differences among the euploidy rates of different morphologic embryo grades demonstrated the positive correlations between the morphologic grading of the embryo and the euploidy rate of PGS. Additionally, there was no significant difference between the younger and older patients' CP rates. These findings emphasize the fact that old age patients might benefit from PGS whatever the indication of PGS is.

Primordial follicle activation is a process in which individual primordial follicles leave their dormant state and enter a growth phase. While existing hormone stimulation strategies targeted the growing follicles, the remaining dormant primordial follicles were ruled out from clinical use. Recently, in vitro activation (IVA), which is a method for controlling primordial follicle activation, has provided an innovative technology for primary ovarian insufficiency (POI) patients. IVA was developed based on Hippo signaling and phosphatase and tensin homolog (PTEN)/phosphatidylinositol-3-kinase (PI3K)/protein kinase B (AKT)/forkhead box O3 (FOXO3) signaling modulation. With this method, dormant primordial follicles are activated to enter growth phase and developed into competent oocytes. IVA has been successfully applied in POI patients who only have a few remaining remnant primordial follicles in the ovary, and healthy pregnancies and deliveries have been reported. IVA may also provide a promising option for fertility preservation in cancer patients and prepubertal girls whose fertility preservation choices are limited to tissue cryopreservation. Here, we review the basic mechanisms, translational studies, and current clinical results for IVA. Limitations and further study requirements that could potentially optimize IVA for future use will also be discussed.

It has recently been reported that two separate bipolar spindles aligned their poles before anaphase keeping the parental genomes apart during the first cleavage in mammalian zygote including mouse and human. In mouse, the failure of spindle alignment by increasing the distance between the two pronuclei, which led to a larger gap between the spindles, gives rise to multinucleated two-cell embryos phenocopying frequently observed errors in IVF clinics. The purpose of our study is to examine the relationship between pronuclear (PN) proximity in zygote and multinucleation (MN) of early cleavage embryos in human using time-lapse system (TLS). Data were retrospectively collected from TLS performed from May 2018 to February 2019 in the Fertility center of CHA Gangnam Medical Center. We analyzed 220 zygotes from 64 patients. We assessed the PN proximity, distance, MN, and development of early cleavage embryos using EmbryoScopeTM (Vitrolife, Gothenburg, Sweden). Drawing tool was used to measure the distance from a PN center to another PN center in the same focal plane before PN fade. Zygotes were divided into two groups according to the proximity (group Gap [G; n=17] and group Juxtaposition [J; n=203]). The number of MN in 2-cell and 4-cell embryo was checked then the embryos were divided into three groups (No MN, MN, and N/A; not available due to abnormal division). Embryo development was checked up to day 3. The quantitative variables were expressed as mean ± SD and statistically analyzed with Student t test. p <0.05 was considered to be statistically significant. The incidence of gap between PN was considerably low (G; 7.7% vs. J; 92.3%). The average of PN distance was significantly different between two groups (G; 31.2 ± 5.1 μm vs. J; 21 ± 4.9 μm, * p < 0.05). The rate of No MN (23.5%[2C] and 52.9%[4C]) was highly decreased but the rate of MN (41.2%[2C] and 5.9%[4C]) and N/A (35.3%[2C] and 41.2%[4C]) was increased in the Gap group compared with Juxtaposition group (49.8%/72.9%[2C/4C], 38.4%/5.4%[2C/4C], and 11.8%/21.7%[2C/4C]). Also the rate of 3D good quality embryo is slightly decreased in Gap group (G; 82.4% vs. J; 89.7%). This study suggests that the PN proximity is also one of the applicable explanations for the MN in early cleavage embryos of human due to the failure of zygotic spindle alignment as other mammalian embryos such as mouse. As the presence of MN in human embryos, especially during the first and second mitotic divisions, is generally considered to be abnormal, our study on the relationship between PN proximity and MN combined with TLS could be used as a noninvasive technique to enhance selection of competent embryos likely to have the greatest potential of development. This may be of particular benefit to patients desiring elective single embryo transfer without PGS screening.

Supplementation of growth hormone (GH) during controlled ovarian stimulation (COS) has been suggested to improve ovarian response. Despite potential benefits in poor responders, multiple injections of GH during COS are inconvenient. We conducted a randomized controlled study to evaluate the efficacy and safety of sustained-release human GH in poor responders undergoing in vitro fertilization (IVF).