A practical survey of fertility-preservation treatments in the startup phase in Japan.

AIM: The actual status of fertility preservation treatments in the startup phase in Japan was investigated as a basis for discussing future directions. METHODS: This study was conducted as "Research project to promote support of children and parenting 2016" which was supported by Ministry of Health in Japan with the approval of the institutional review board at St. Marianna University. Subjects of the survey were facilities registered with the Japan Society of Obstetrics and Gynecology as fertility preservation facilities, and facilities belonging to the Japan Association of Private Assisted Reproductive Technology Clinics and Laboratories. We provided questionnaires to survey both the medical care system and cases for which fertility preservation was implemented between 2006 and 2016. RESULTS: Responses were obtained from 68 facilities (of the 64, 59 [92.2%] responded to the questionnaire and 9 clinics cooperated). Many facilities limited the cryopreservation of oocytes and ovaries to patients 40-41 years old and the use of eggs to patients 44-45 years old. In the patient survey, 812 cases of oocyte cryopreservation and 201 cases of ovarian tissue cryopreservation were performed during study period. Breast cancer was the most indicated disease, with oocyte cryopreservation in the late 30s and ovarian tissue cryopreservation in the early 30s. Very few babies were born from fertility preservation, and no live birth cases of ovarian tissue cryopreservation were identified. CONCLUSIONS: Even from the early days, fertility preservation was implemented according to certain standards in Japan, but was characterized by a large variety of facilities.

Characteristics of the cytoplasmic halo during fertilisation correlate with the live birth rate after fresh cleaved embryo transfer on day 2 in minimal ovarian stimulation cycles: a retrospective observational study.

BACKGROUND: Information regarding the influence of cytoplasmic events during fertilisation on the clinical outcome remains limited. The cytoplasmic halo is one of these events. A previous study that used time-lapse technology found an association of the presence and morphokinetics of the cytoplasmic halo with cleavage patterns, development to the blastocyst stage, and the ongoing pregnancy rate after blastocyst transfer. Therefore, the cytoplasmic halo may be a useful predictor of the pregnancy outcome after cleaved embryo transfer. This study evaluated the ability of the cytoplasmic halo to predict a live birth after fresh cleaved embryo transfer on day 2, and sought to identify factors potentially influencing the presence and morphokinetics of the halo. METHODS: A total of 902 embryos cultured in the EmbryoScope+® time-lapse system and subjected to single fresh cleaved embryo transfer were retrospectively analysed. The presence and duration of a cytoplasmic halo were annotated. The initial positions of the pronuclei were also observed. The correlation between the cytoplasmic halo and live birth was evaluated and the association of the cytoplasmic halo with patient, cycle, and embryonic characteristics was determined. RESULTS: Absence of a cytoplasmic halo was associated with a significant decrease in the likelihood of a live birth after fresh cleaved embryo transfer. Prolongation of the halo, especially the duration of central repositioning of cytoplasmic granules, had an adverse impact on the live birth rate. The characteristics of the cytoplasmic halo were not affected by the ovarian stimulation method used, female age, the serum steroid hormone level on the day of trigger, or semen quality. However, the cytoplasmic halo was significantly affected by male age, oocyte diameter, and the initial position of the male pronucleus. CONCLUSIONS: Absence or prolongation of the cytoplasmic halo was negatively correlated with the live birth rate after fresh cleaved embryo transfer. The characteristics of the cytoplasmic halo were strongly associated with oocyte diameter, male age, and the initial position of the male pronucleus. These findings indicate that the characteristics of the cytoplasmic halo can be used to select more competent embryos for transfer at the cleavage stage.

Pregnancy prediction performance of an annotation-free embryo scoring system on the basis of deep learning after single vitrified-warmed blastocyst transfer: a single-center large cohort retrospective study.

OBJECTIVE: To analyze the performance of an annotation-free embryo scoring system on the basis of deep learning for pregnancy prediction after single vitrified blastocyst transfer (SVBT) compared with the performance of other blastocyst grading systems dependent on annotation or morphology scores. DESIGN: A single-center large cohort retrospective study from an independent validation test. SETTING: Infertility clinic. PATIENT(S): Patients who underwent SVBT cycles (3,018 cycles, mean ± SD patient age 39.3 ± 4.0 years). INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): The pregnancy prediction performances of each embryo scoring model were compared using the area under curve (AUC) for predicting the fetal heartbeat status for each maternal age group. RESULT(S): The AUCs of the <35 years age group (n = 389) for pregnancy prediction were 0.72 for iDAScore, 0.66 for KIDScore, and 0.64 for the Gardner criteria. The AUC of iDAScore was significantly greater than those of the other two models. For the 35-37 years age group (n = 514), the AUCs were 0.68, 0.68, and 0.65 for iDAScore, KIDScore, and the Gardner criteria, respectively, and were not significantly different. The AUCs of the 38-40 years age group (n = 796) were 0.67 for iDAScore, 0.65 for KIDScore, and 0.64 for the Gardner criteria, and there were no significant differences. The AUCs of the 41-42 years age group (n = 636) were 0.66, 0.66, and 0.63 for iDAScore, KIDScore, and the Gardner criteria, respectively, and there were no significant differences among the pregnancy prediction models. For the >42 years age group (n = 389), the AUCs were 0.76 for iDAScore, 0.75 for KIDScore, and 0.75 for the Gardner criteria, and there were no significant differences. Thus, iDAScore AUC was either the highest or equal to the highest AUC for all age groups, although a significant difference was observed only in the youngest age group. CONCLUSION(S): Our results showed that objective embryo assessment by a completely automatic and annotation-free model, iDAScore, performed as well as or even better than more traditional embryo assessment or annotation-dependent ranking tools. iDAScore could be an optimal pregnancy prediction model after SVBT, especially in young patients.

Effects of fatty acid supplementation during vitrification and warming on the developmental competence of mouse, bovine and human oocytes and embryos.

RESEARCH QUESTION: Does fatty acid supplementation in vitrification and warming media influence developmental competence in oocytes after vitrification and warming? DESIGN: Mouse oocytes and four-cell embryos were vitrified and warmed with solutions supplemented with fatty acid and cultured to the blastocyst stage. To study lipid metabolism after vitrification, quantitative real-time polymerase chain reaction was used to analyse the expression of genes related to beta oxidation in mouse embryos vitrified and warmed with or without fatty acids. The effects of fatty acid supplementation in the warming solutions on the developmental competence of bovine and human embryos were analysed. Blastocyst outgrowth assay was used to evaluate the potential of human blastocysts for adhesion to fibronectin. RESULTS: The neutral lipid content of mouse oocytes in the fatty acid 1% supplementation group was significantly higher than in the fatty acid 0% group (P = 0.0032). The developmental rate to the blastocyst stage was significantly higher in the fatty acid 1% group than in the fatty acid 0% group in mice (P = 0.0345). Fatty acid supplementation in warming solution upregulated Acaa2 and Hadha in mouse embryos. Fatty acids significantly improved the developmental ability of bovine embryos to the blastocyst stage (P = 0.0048). Warming with 1% fatty acid supplementation significantly increased the proportion of human blastocysts with morphological grade A inner cell mass (P = 0.0074) and trophectoderm (P = 0.0323). CONCLUSIONS: Fatty acid supplementation in the warming solutions improved the developmental competence of vitrified-warmed mouse oocytes by activating the beta-oxidation pathway. Fatty acid supplementation enhanced the developmental rate of bovine embryos to the blastocyst stage and improved morphological characteristics of human embryos vitrified at the cleavage stage.

Comparison of Embryo and Clinical Outcomes in Different Types of Incubator Between Two Different Embryo Culture Systems.

This study aimed to compare the clinical outcomes of an oxidative stress-reducing embryo culture system (ORES) containing compounds that minimize intercellular oxidative stress, with those of a standard embryo culture system (StES). Furthermore, we investigated the efficiency of the ORES regarding the type of incubator used (time-lapse incubator [TLI] or non-time-lapse dry incubator [non-TLI]) and maternal age. In this retrospective study, we analyzed 3610 oocyte retrieval cycles (in 2537 patients) and 1726 single vitrified-warmed blastocyst transfer (SVBT) cycles (in 1726 patients) performed in a single center between April 2018 and July 2019. Transfers of single vitrified-warmed blastocysts, confirmed by fetal heartbeat, were used to assess clinical outcomes. The clinical outcomes of ORES and StES were compared in both TLI and non-TLI. Groups were stratified according to maternal age (≤39 years old, young age group; ≥40 years old, advanced age group). A significant difference in ongoing pregnancy rates was observed between the ORES and StES groups when non-TLI was used (34.9 vs. 27.0%, respectively; p < 0.05), unlike when TLI was used. Furthermore, ongoing pregnancy rates were significantly higher in the ORES group (24.8%) than in the StES group (14.9%) in the advanced age group, unlike in the young age group when non-TLI was used. In conclusion, compared to StEs, the ORES during all in vitro fertilization procedures improved ongoing pregnancy rates in the advanced age group using the non-TLI.

Prolactin receptor expression and its role in trophoblast outgrowth in human embryos.

RESEARCH QUESTION: What is the gene expression pattern of prolactin receptor (PRLR) in human pre-implantation embryos and what are its functions during the embryonic development and adhesion process? DESIGN: A total of 405 discarded human vitrified oocytes and embryos donated for research by consenting couples were used in this study. The oocytes and embryos were used to analyse PRLR expression and to evaluate the influence of prolactin (PRL) supplementation in the embryo culture medium on embryo developmental competence and viability. The rates of blastocyst development and adhesion, outgrowth area, cytoskeletal reorganization and nascent adhesion formation were compared between groups. RESULTS: PRLR expression increased significantly after embryo compaction (P < 0.0001) and blastulation (P < 0.0001). Supplementation of the embryo culture medium with PRL did not improve the developmental rate and morphological grade. In contrast, blastocyst outgrowth was significantly increased in embryos cultured with PRL (P = 0.0004). Phosphorylation of JAK2, downstream of the prolactin receptor family, was markedly higher in the PRL-treated embryos than in embryos cultured without PRL. Furthermore, the expression of mRNAs encoding ezrin-radixin-moesin proteins and epithelial-mesenchymal transition-related genes was stimulated by the activation of PRL-JAK2 signalling. The PRL-treated embryos had higher mRNA expression of integrins than non-treated embryos, and transcriptional repression of cadherin 1 was observed after PRL treatment. More nascent adherent cells expressed focal adhesion kinase and paxillin in PRL-treated embryos than in non-treated embryos. CONCLUSIONS: Human embryos express PRLR at the morula and blastocyst stages, and PRLR signalling stimulates blastocyst adhesion by promoting integrin-based focal adhesions and cytoskeletal organization during trophoblast outgrowth.

Establishment of day 7 blastocyst freezing criteria using blastocyst diameter for single vitrified-warmed blastocyst transfer from live birth outcomes: a single-center, large cohort, retrospectively matched study.

PURPOSE: To establish blastocyst freezing criteria for day 7 blastocyst (day 7 BL) for single vitrified-warmed blastocyst transfer (SVBT) by examining the diameter of blastocysts. METHODS: Patients who underwent day 7 BL transfer cycles (1143 cycles, mean age: 38.5 ± 3.5) and randomly selected patients after 1:1 matching who underwent day 6 BL transfer cycles and day 2-single-embryo transfer (SET) cycles were used for analysis. Comparison of the miscarriage (per clinical pregnancy) and live birth rates were made among day 2-SET, day 7 BL, and day 6 BL. These blastocyst groups were stratified into six groups based on blastocyst diameter, namely, 180 µm, 190 µm, 200 µm, 210 µm, over 220 µm, and hatched, for making the freezing criteria. RESULTS: For each diameter, 180 µm, 190 µm, 200 µm, 210 µm, over 220 µm, and hatched, the live birth rates of day 7 BL after SVBT were 9.0%, 11.9%, 11.5%, 15.6%, 20.0%, and 19.9%, respectively. Compared with the 14.6% live birth rate of the day 2-SET group, the live birth rate of 220 µm day 7 BL was significantly higher (P < 0.05) and was around the same in other diameter groups. CONCLUSION: Our study demonstrates that sufficient live birth rates can be obtained after SVBT even from blastocysts on day 7 when blastocysts were vitrified at expanded blastocyst stage of over 180 µm of diameter or at hatched blastocyst stage and were transferred at the optimal time. This is the first study to establish a day 7 blastocyst freezing criteria using blastocyst diameter, which is an objective assessment way.

Cytoplasmic halo characteristics during fertilization and their implications for human preimplantation embryo development and pregnancy outcome.

RESEARCH QUESTION: Is the spatiotemporal phenomenology of the cytoplasmic halo during fertilization related to embryonic competence? DESIGN: Time-lapse images from 1009 zygotes were retrospectively analysed from 560 patients who underwent IVF with minimal stimulation and single vitrified-warmed blastocyst transfer between April 2017 and March 2018. Halo presence and morphokinetics were monitored and compared relative to embryo quality, blastocyst expansion and ongoing pregnancy. RESULTS: Halo was observed in 88% of fertilized oocytes. Embryos derived from zygotes without halo had significantly higher rates of rapid cleavage (P = 0.0004), cell fusion (P = 0.0028) and asymmetrical division (P = 0.0002) compared with those derived from zygotes with halo. Multivariate logistic regression analysis had significantly higher developmental rates compared with the expanded blastocyst stage in embryos displaying a halo, regardless of its distribution (adjusted odds ratio 0.435; P = 0.0004). Prolonged halo time intervals were significantly correlated with increased asymmetrical division at first cell division (P = 0.0412, P = 0.0088, respectively) and decreased developmental rates to expanded blastocyst stage (P = 0.0062, P = 0.0020, respectively). Additionally, prolonged presence of the cytoplasmic halo was associated with a decreased ongoing pregnancy rate (adjusted odds ratio 0.871; P = 0.006). Poor sperm quality and decreased oocyte diameter were correlated with absence of the cytoplasmic halo (P = 0.0477, P < 0.0001, respectively) or prolonged halo presence (P = 0.0139, P = 0.0002, respectively). CONCLUSIONS: Halo presence and morphokinetics are associated with cleavage patterns, development to blastocyst stage and ongoing pregnancy rate after single blastocyst transfer. Halo morphokinetics seems to reflect sperm and oocyte quality. Cytoplasmic halo might be valuable predictor for refining selection of more developmentally competent blastocysts.

Effects of gonadotropin administration on clinical outcomes in clomiphene citrate-based minimal stimulation cycle IVF.

PURPOSE: Exogenous gonadotropins (EGn) have been used occasionally in clomiphene citrate (CC)-based minimal stimulation cycles to compensate insufficient secretion of endogenous gonadotropin; however, the effectiveness of EGn supplementation remains unknown. In the present study, we assessed whether EGn improved pregnancy outcomes in CC-based minimal stimulation cycles. METHODS: A total of 223 patients treated with CC and EGn (CC-EGn group) were matched one to one to patients treated with CC only (CC group) by propensity score matching. Embryonic and pregnancy outcomes were retrospectively compared between the groups. RESULTS: The numbers of retrieved oocytes, fertilized oocytes, cleaved embryos, and cryopreserved blastocysts were increased in the CC-EGn group compared with the CC group. However, the cumulative live birthrate was comparable between the two groups. Although the increased number of retrieved oocytes was correlated significantly with improvement of the cumulative live birthrate in both groups, the correlation tended to be lower in the CC-EGn group than in the CC group (odds ratio, 1.193 vs 1.553). CONCLUSIONS: In CC-based minimal stimulation cycles, the stimulation should be started with CC only, and EGn administration should be scheduled only if insufficient secretion of endogenous gonadotropin is observed in the late follicular phase.

Success rates in minimal stimulation cycle IVF with clomiphene citrate only.

PURPOSE: To determine age-adjusted overall success rates for patients undergoing clomiphene citrate only minimal stimulation cycle (mini) in vitro fertilization (IVF) without any gonadotropin administration. METHODS: Eight hundred thirty-nine women (mean age: 38.4 ± 0.1 years; 2488 cycles) underwent clomiphene citrate only mini-IVF. Their first oocyte retrieval was between January 2009 and December 2009, with follow-up until December 2014. The cumulative live birth rate (CLBR) per oocyte retrieval cycle started and live birth rate per oocyte was retrospectively analyzed. The basic CLBR was calculated as the number of women who achieved a live birth divided by the total number of women who started oocyte retrieval. RESULTS: The mean number of oocytes retrieved was 1.5. The basic CLBRs for all ages after the first and third cycles were 22.6% and 39.2%, respectively. For ≤ 34 years, 35-37 years, 38-40 years, 41-42 years, and ≥ 43 years, CLBRs after the first and third cycles were 42.5% and 70.1%, 32.9% and 49.1%, 20.0% and 38.6%, 12.6% and 25.2%, and 4.4% and 8.8%, respectively. These rates had a significant relationship with age (P < 0.01). The LBR per oocyte for all ages was 9.6%. CONCLUSION: Acceptable overall IVF success rates can be achieved in clomiphene citrate only mini-IVF, as well as acceptable LBR. The CLBRs and LBRs per oocyte are evidently influenced by women's age.

Evaluation of uterine receptivity after gonadotropin releasing hormone agonist administration as an oocyte maturation trigger: a rodent model.

In natural cycle or minimal stimulation cycle IVF, buserelin acetate (buserelin), a gonadotropin-releasing hormone agonist, is often used as a maturation trigger; however, its effect on pregnancy outcomes remains unclear. Therefore, in the present study, we compared uterine receptivity in buserelin-administered mice with that in human chorionic gonadotropin (hCG)-administered mice during the peri-implantation period. Implantation, decidualisation, and term-pregnancy were impaired following hCG, but not buserelin administration. hCG stimulated the synthesis and secretion of progesterone and oestradiol, whereas ovarian steroidogenesis in the buserelin-treated group was comparable with that in the control group. Furthermore, similar to the observation in controls, the buserelin-treated group exhibited activation of progesterone receptor signalling and inhibition of oestrogen receptor signalling in the endometrial epithelium on the day of implantation. However, epithelial progesterone signalling was not detected, and a high expression of genes downstream to oestrogen was observed on day 4 following hCG administration. These results suggest that buserelin administration does not impact uterine receptivity as it did not affect ovarian steroidogenesis and endometrial steroid signalling. Therefore, buserelin is preferred as an oocyte maturation trigger to optimise uterine receptivity during treatments involving timed intercourse, intrauterine insemination, or fresh embryo transfer following in vitro fertilisation.

Blastomere movement post first cell division correlates with embryonic compaction and subsequent blastocyst formation.

BACKGROUND: Blastomere movement (BMov) occurs after the first cell division in human embryos. This movement has been suggested as a prognostic parameter for pregnancy outcome prediction following cleavage-stage embryo transfer. However, the effect of BMov on preimplantation development and pregnancy outcome after blastocyst transfer remains unclear. Therefore, this study aimed to evaluate whether BMov after the first cell division is correlated with blastocyst formation rate and live birth rate after single vitrified-warmed blastocyst transfer (SVBT). METHODS: Nine hundred and sixty-six embryos cultured in the EmbryoScope+® time-lapse system were retrospectively analyzed. The BMov type was categorized into three groups; namely, bouncing, wobbling, and twist-and-crumble. The BMov duration (dBMov) between the first (t2) and second cell division (t3) was monitored, and the ratio of dBMov to the duration of the 2-cell stage was calculated [dBMov/(t3-t2)]. Developmental rates to the 4-cell, 8-cell, morula, blastocyst, and expanded blastocyst stages were assessed, as well as blastocyst morphological grade. The correlations between dBMov and clinical pregnancy, ongoing pregnancy, and live birth rates were evaluated. RESULTS: Increased dBMov/(t3-t2) was significantly correlated with decreased developmental rates to the 8-cell, morula, blastocyst, and expanded blastocyst stages, especially from the 4-cell stage to the morula stage. Analysis of different types of BMov revealed that embryos with bouncing movement exhibited significantly higher developmental rates to the 8-cell, morula, blastocyst, and expanded blastocyst stages compared with embryos with twist-and-crumble movement. The morphological quality of blastocyst-stage embryos with twist-and-crumble movement was significantly lower than that of embryos with bouncing and wobbling movements. The rates of clinical pregnancy, ongoing pregnancy, and live birth after SVBT were not correlated with BMov type or duration. CONCLUSIONS: Embryonic compaction and subsequent blastocyst formation are adversely affected by twist-and-crumble movement and prolonged movement after the first cell division. Our results indicate that the preimplantation developmental competence of human embryos could be predicted by assessing BMov after the first cell division on day 1.

Closed embryo culture system improved embryological and clinical outcome for single vitrified-warmed blastocyst transfer: A single-center large cohort study.

While some studies have shown that the closed embryo culture system (CCS) is a possible improvement over standard embryo culture systems (STS) in terms of early embryonic development, information on clinical outcomes of culturing blastocysts following single vitrified-warmed blastocyst transfer (SVBT) in the CCS and STS remains limited. Therefore, the objective of this single-center, large-cohort, retrospective study was to compare embryonic development until the blastocyst stage and clinical outcomes following SVBT between CCS and STS. From May 2017 to October 2018, 2420 oocytes from 1402 patients who underwent in vitro fertilization and blastocyst culture after minimal stimulation were divided into two groups (CCS and STS). The main outcome measures in the two groups were embryological (blastocyst formation rates and utilized blastocyst rates) and clinical outcomes (ongoing pregnancy rates) after a single vitrified-warmed blastocyst transfer (SVBT). There were no significant differences in the blastocyst formation rates between the CCS and STS groups (59.6% versus 59.1%, p = 0.81). However, there were significant differences in utilized blastocyst rates (51.0% versus 46.6%, p < 0.05). Ongoing pregnancy rates per SVBT cycle were significantly higher in the CCS group than in the STS group (41.4% versus 34.4%, p < 0.05). Moreover, after applying multivariable logistic regression analysis, the type of embryo culture system (CCS to STS, adjusted odds ratios: 1.41, 95% CI: 1.04-1.91) was correlated with ongoing pregnancy. Our study suggests that compared to STS, CCS could improve utilized blastocyst rates and ongoing pregnancy rates to a greater extent, following SVBT.

Prolonged blastomere movement induced by the delay of pronuclear fading and first cell division adversely affects pregnancy outcomes after fresh embryo transfer on Day 2: a time-lapse study.

RESEARCH QUESTION: What is the incidence, origin and clinical significance of blastomere movement after the first cell division in the human embryo? DESIGN: A total of 1096 embryos, cultured in the EmbryoScope+ ® time-lapse system and subjected to a single fresh cleaved embryo transfer, were retrospectively analysed. Type and duration of blastomere movement (dBMov) between the first (t2) and second cell division (t3) was monitored, and the ratio of dBMov during the 2-cell stage [dBMov/(t3-t2)] was calculated. Morphological evaluation of embryos was performed by referring to the size of the blastomere and fragmentation after first division in addition to Veeck's criteria on Day 2. The correlation between dBMov and ongoing pregnancy was evaluated and the association of dBMov with patient and embryonic characteristics was determined. RESULTS: Both movement type and the value of dBMov/(t3-t2) were significantly associated with asymmetrical first division, fragment formation and morphological grade on Day 2. Multivariate logistic regression analysis revealed that a higher value of dBMov/(t3-t2) significantly correlated with a decreased ongoing pregnancy rate, even after adjustment for co-founders (odds ratio 0.399, P = 0.0419). The time intervals of pronuclear (PN) alignment and PN fading were significantly correlated with the dBMov/(t3-t2) value. CONCLUSIONS: Embryos with extended blastomere movement after the first cell division, which is associated with the delay of PN fading and first cell division, have a lower competence to initiate an ongoing pregnancy after fresh embryo transfer on Day 2. Thus, blastomere movement could be a useful predictive parameter for selecting embryos at the early cleavage stage.

Cryostorage duration does not affect pregnancy and neonatal outcomes: a retrospective single-centre cohort study of vitrified-warmed blastocysts.

A retrospective cohort study of 8736 autologous single vitrified-warmed blastocyst transfer cycles was conducted in a single centre to investigate the effect of cryostorage on clinical and neonatal outcomes. Cryostorage duration was classified into three groups: (A) 0-2 months (n = 4702); (B) 2-13 months (n = 2853) and (C) 13-97 months (n = 1181). Blastocysts were vitrified using the Cryotop method. No significant differences were observed in live birth rates: (A) 37.3%; (B) 34.9%; (C) (35.2%). Gestational period was significantly shorter in group C: (A) 38.7 ± 1.8; (B) 38.6 ± 1.6; (C) 38.1 ± 1.7; P < 0.05. This was clinically unimportant as the average gestational age was more than 38 weeks. No significant differences between groups were observed in birth weight: (A) 3060 ± 455 g; (B) 3052 ± 449 g; (C) 2992 ± 445 g, or congenital malformation rates: (A) 2.2%; (B) 1.9%; (C) 1.8%. The limitation of this study was that maximum storage duration was 8 years; most blastocysts were in cryostorage for much shorter periods. Long-term storage of blastocysts that are vitrified using an open device vitrification system has no negative effect on pregnancy and neonatal outcomes.

Infertility treatment strategy involving combined freeze-all embryos and single vitrified-warmed embryo transfer during hormonal replacement cycle for in vitro fertilization of women with hypogonadotropic hypogonadism.

AIM: Hypogonadotropic hypogonadism (HH) is a condition caused by the deficient secretion of pituitary gonadotropins, leading to diminished ovarian function. Several studies of in vitro fertilization (IVF) in women with HH revealed acceptable clinical pregnancy outcomes but high multiple pregnancy rates after multiple fresh embryo transfer (ET). The purpose of this study was to analyze the outcomes of combined freeze-all embryos and single vitrified-warmed ET in women with HH. METHODS: Of 91 infertile women with HH (basal luteinizing hormone and follicle-stimulating hormone levels <2.0 mIU/mL), we excluded patients aged ≥40 years (n = 2) and women who preferred fresh ET (n = 10). Seventy-nine women underwent 117 oocyte retrieval cycles and 135 vitrified-warmed ET during hormone replacement (HR) cycles from 2008 to 2014 at the Kato Ladies Clinic and Juntendo University Hospital. RESULTS: In 26 single cleavage ET cycles, the rates of clinical pregnancy and live birth were 34.6% (9/26 ET) and 26.9% (7/26 ET), respectively. Regarding the outcomes after single vitrified-warmed blastocyst transfer, clinical pregnancy and live birth rates were 65.1% (71/109 ET) and 50.5% (55/109 ET), respectively. Multiple conceptions and ovarian hyperstimulation syndrome did not occur in any of the women with HH. CONCLUSION: Our results demonstrated that IVF followed by single vitrified-warmed ET in adjusted endocrine milieu during the HR cycle is an effective fertility treatment for women with HH and decreases the incidence of complications, including multiple conceptions.

Intrinsic fertility of human oocytes.

OBJECTIVE: To study the intrinsic fertility of the human oocyte. DESIGN: A large retrospective study of natural cycle single embryo transfer (ET) IVF cycles. SETTING: Private IVF clinic, university, and private hospital. PATIENT(S): Patients were enrolled consecutively over an 8-year period in a single ET natural cycle protocol. INTERVENTION(S): A total of 13,949 oocyte retrievals with natural IVF single ET. Software package R (version 3.2.5) was used for statistical calculations. MAIN OUTCOME MEASURE(S): Live baby rate per oocyte according to age. RESULT(S): A total of 14,185 natural cycle oocytes resulted in 1,913 live babies from single ET. The number of oocytes required to make one live baby in this large series varied with the age of the female partner. For those under 35, the live baby born per oocyte was 26%. For over age 42 it decreased to 1%. These results fit very robustly with a logistic function curve, which is at first steady (horizontal), followed by a linear decline after age 35 with a 10% loss every year until age 43, and then a flattening out (horizontal) by age 44. CONCLUSION(S): The intrinsic fertility per oocyte in natural cycle is far greater than reported in hyperstimulated cycles, varying robustly from 26% to 4% with age from <35 to 42 years. The curve is relatively flat until age 34, and then declines rapidly 10% per year thereafter.

Complete zona pellucida removal from vitrified-warmed human blastocysts facilitates earlier in-vitro attachment and outgrowth.

Partial removal of the zona pellucida (ZP) has been performed using a laser system to promote hatching of vitrified-warmed blastocysts. However, low-viability blastocysts cannot hatch even after partial ZP removal. This study examined whether complete removal of the ZP improves embryonic adhesion and outgrowth of vitrified-warmed blastocysts compared with partial removal, using a blastocyst outgrowth model. In all, 217 vitrified human blastocysts, which were discarded and donated for research by consenting couples, were warmed and subjected to assisted hatching to remove the ZP partially or completely, or did not undergo assisted hatching (zona intact controls). Blastocysts were cultured using time-lapse microscopy to monitor hatching, adhesion and outgrowth. Despite partial ZP removal, 36% of blastocysts failed to hatch. Blastocyst outgrowth assays showed improved adhesion rate, shorter time for adhesion and larger outgrowth area in the blastocysts with completely removed ZP compared with those with partially-removed ZP. mRNA expression of integrin α5 and ß1 was upregulated in blastocysts with completely removed ZP compared with those with partially-removed ZP. Study findings reveal the advantages of complete ZP removal for assisted hatching. In conclusion, complete ZP removal increases the chance of blastocyst adhesion and subsequent outgrowth in vitro after the vitrification-warming procedure.

Developmental Competence of Vitrified-Warmed Bovine Oocytes at the Germinal-Vesicle Stage is Improved by Cyclic Adenosine Monophosphate Modulators during In Vitro Maturation.

Cryopreservation of mature oocytes and embryos has provided numerous benefits in reproductive medicine. Although successful cryopreservation of germinal-vesicle stage (GV) oocytes holds promise for further advances in reproductive biology and clinical embryology fields, reports regarding cryopreservation of immature oocytes are limited. Oocyte survival and maturation rates have improved since vitrification is being performed at the GV stage, but the subsequent developmental competence of GV oocytes is still low. The purpose of this study was to evaluate the effects of supplementation of the maturation medium with cyclic adenosine monophosphate (cAMP) modulators on the developmental competence of vitrified-warmed GV bovine oocytes. GV oocytes were vitrified-warmed and cultured to allow for oocyte maturation, and then parthenogenetically activated or fertilized in vitro. Our results indicate that addition of a cAMP modulator forskolin (FSK) or 3-isobutyl-1-methylxanthine (IBMX) to the maturation medium significantly improved the developmental competence of vitrified-warmed GV oocytes. We also demonstrated that vitrification of GV oocytes led to a decline in cAMP levels and maturation-promoting factor (MPF) activity in the oocytes during the initial and final phases of maturation, respectively. Nevertheless, the addition of FSK or IBMX to the maturation medium significantly elevated cAMP levels and MPF activity during IVM. Taken together, our results suggest that the cryopreservation-associated meiotic and developmental abnormalities observed in GV oocytes may be ameliorated by an artificial increase in cAMP levels during maturation culture after warming.

Hydroxypropyl cellulose as an option for supplementation of cryoprotectant solutions for embryo vitrification in human assisted reproductive technologies.

Hydroxypropyl cellulose (HPC) was investigated as a replacement for serum substitute supplement (SSS) for use in cryoprotectant solutions for embryo vitrification. Mouse blastocysts from inbred (n = 1056), hybrid (n = 128) strains, and 121 vitrified blastocysts donated by infertile patients (n = 102) were used. Mouse and human blastocysts, with or without zona pellucida, were vitrified and warmed in either 1% or 5% HPC or in 5% or 20% SSS-supplemented media using the Cryotop (Kitazato BioPharma Co. Ltd, Fuji, Japan) method, and the survival and oxygen consumption rates were assessed. Viscosity of each vitrification solution was compared. Survival rates of mouse hybrid blastocysts and human zona pellucida-intact blastocysts were comparable among the groups. Mouse and human zona pellucida-free blastocysts, which normally exhibit poor cryoresistance, showed significantly higher survival rates in 5% HPC than 5% SSS (P < 0.05). The 5% HPC-supplemented vitrification solution showed a significantly higher viscosity (P < 0.05). The blastocysts were easily detached from the Cryotop strip during warming when HPC-supplemented vitrification solution was used. The oxygen consumption rates were similar between non-vitrified and 5% HPC groups. The results suggest possible use of HPC for supplementation of cryoprotectant solutions and provide useful information to improve vitrification protocols.