Effect of ovarian stimulation on developmental speed of preimplantation embryo in a mouse model.

Ovarian stimulation protocols are widely used to collect oocytes in assisted reproductive technologies (ARTs). Although the influence of ovarian stimulation on embryo quality has been described, this issue remains controversial. Here, we analyzed the influence of ovarian stimulation on developmental speed and chromosome segregation using live cell imaging. Female mice at the proestrus stage were separated by the appearance of the vagina as the non-stimulation (-) group, and other mice were administered pregnant mare serum gonadotropin (PMSG) and human chorionic gonadotropin (hCG) as the stimulation (+) groups. The cumulus-oocyte complexes from both groups were inseminated with sperm suspensions from the same male mice. Fertilization rates and developmental capacities were examined, and the developmental speed and frequency of chromosome segregation errors were measured by live-cell imaging using a Histone H2B-mCherry probe. The number of fertilized oocytes obtained was 1.4-fold more frequent in the Stimulation (+) group. The developmental rate and chromosome stability did not differ between the groups. Image analysis showed that the mean speed of development in the stimulation (+) group was slightly higher than that in the non-stimulation (-) group. This increase in speed seemed to arise from the slight shortening of the 2- and 4-cell stages and third division lengths and consequent synchronization of cleavage timing in each embryo, not from the emergence of an extremely rapidly developing subpopulation of embryos. In conclusion, ovarian stimulation does not necessarily affect embryo quality but rather increases the chances of obtaining high-quality oocytes in mice.

Beyond appearance: Can morphologically low-grade euploid blastocysts yield successful pregnancies?

Purpose: The primary objective of this investigation is to evaluate how morphological quality affects the pregnancy outcomes in euploid embryos determined by preimplantation genetic testing for aneuploidies (PGT-A). Concurrently, as a secondary objective, we aim to identify which specific aspects of morphological evaluation exert the most significant impact on these outcomes. Methods: A retrospective analysis of 451 single euploid embryo transfer cycles at our clinic was conducted. Embryos were evaluated based on the degree of blastocyst expansion, inner cell mass (ICM), trophectoderm (TE) morphology, and the day of blastocyst vitrification. Outcomes between morphologically low-grade and high-grade embryos were compared. Additionally, the study analyzed which morphological factors most influenced pregnancy outcomes. Results: Pregnancy outcomes were significantly lower in morphologically low-grade blastocysts compared to high-grade ones. Among the morphological evaluations, the ICM assessment was significantly associated with the live birth rate. Conclusion: Our study indicates that the morphological quality of euploid embryos, particularly the evaluation of the ICM, plays a crucial role in IVF-ET success.

Anti-Müllerian hormone for screening, diagnosis, evaluation, and prediction: A systematic review and expert opinions.

AIM: To present evidence-based recommendations for anti-Müllerian hormone (AMH) measurement as an ovarian reserve test. METHODS: A systematic literature search for the clinical utility of AMH was conducted in PubMed from its inception to August 2022 to identify studies, including meta-analyses, reviews, randomized controlled trials, and clinical trials, followed by an additional systematic search using keywords. Based on this evidence, an expert panel developed clinical questions (CQs). RESULTS: A total of 1895 studies were identified and 95 articles were included to establish expert opinions subdivided into general population, infertility treatment, primary ovarian insufficiency, polycystic ovary syndrome, surgery, and oncofertility. We developed 13 CQs and 1 future research question with levels of evidence and recommendations. CONCLUSION: The findings of the current systematic review covered the clinical utility of AMH including its screening, diagnosis, evaluation, and prediction. Although some clinical implications of AMH remain debatable, these expert opinions may help promote a better understanding of AMH and establish its clinical significance.

Risk factors for empty follicle syndrome in assisted reproductive technology with gonadotropin-releasing hormone agonist trigger.

Purpose: To analyze whether response to the GnRH test is a predictor of empty follicle syndrome (EFS) and to analyze independent risk factors for EFS. Methods: The GnRH test results of 3765 patients from 2016 to 2018 were used to define the reference range of the GnRH test. Risk factors for EFS were estimated by multivariate logistic analysis of 5282 cycles (5247 oocyte-retrieved cycles with GnRH agonist trigger and 35 cycles of EFS) conducted from 2016 to 2019. Results: GnRH testing showed basal hormone values as follows: median LH 5.2 (95 percentile; 1.3-12.6) mIU/mL, LH 30 min 22.0 (6.8-57.1), basal FSH 7.3 (3.0-20.5), FSH 30 min 11.5 (5.1-30.4) and FSH/LH ratio 1.5 (0.6-4.1). Independent risk factors for EFS were antral follicle count (adjusted odds ratio; 0.94, 95% CI; 0.89-0.99), basal LH (0.78, 0.66-0.90), and days duration of ovarian stimulation (1.41, 1.21-1. 60). The respective thresholds were 8 for AFC, 5.0 for basal LH, and 16 days for duration. Conclusions: LH 30 min values of the GnRH test did not predict EFS. Independent risk factors for EFS were AFC, basal LH and days duration of ovarian stimulation.

Clinical outcomes of personalized blastocyst embryo transfer after endometrial receptivity analysis: A multicenter, retrospective cohort study.

Purpose: To evaluate clinical outcomes after endometrial receptivity analysis (ERA). Methods: This was a multicenter, retrospective cohort study involving 861 women who underwent ERA testing at certified fertility clinics in Japan, and who received subsequent personalized blastocyst embryo transfers (ET) between 2018 and 2020. Clinical outcomes, including pregnancies, miscarriages, and live births, were evaluated according to receptivity status for ERA. Results: Mean patient age was 37.7 years (SD = 4.0), and the median number of previous ETs was 2 (interquartile range, 2-3). 41.0% (353/861) of patients were non-receptive for ERA testing. Clinical pregnancy, miscarriage, and live birth rates for personalized blastocyst ET were 44.5% (226/508), 26.1% (59/226), and 26.8% (136/508) for receptive patients, and 43.1% (152/353), 28.3% (43/152), and 28.9% (102/353) for non-receptive patients, all statistically nonsignificant. Multiple logistic regression demonstrated similar nonsignificant associations between receptivity and clinical outcomes. Greater patient age, smoking, and longer duration of infertility were significantly and negatively associated with receptivity, whereas a history of delivery was positively associated and statistically significant. Conclusions: Clinical outcomes after ERA testing were similar between receptive and non-receptive patients. Further prospective study including an appropriate comparison group are warranted to evaluate the efficacy of ERA testing.

Cryptozoospermia: Should we use ejaculated sperm or surgically retrieved sperm for assisted reproductive technology?

Purpose: In cryptozoospermic subjects, it may often may be difficult to secure motile sperm for assisted reproductive technology (ART). We examined the results of ART with frozen thawed ejaculated sperm in cryptozoospermic subjects and evaluated whether sperm retrieval surgery is necessary for such patients in our clinic. Methods: Between 2013 and 2021, we evaluated 197 cryptozoospermic patients. Age, endocrine panel at the time of the initial semen analysis, and anti-müllerian hormone levels at the time of the spouse's first egg retrieval were examined. Cryopreservation of ejaculated motile sperm collected essentially weekly over a 3-month period was carried out. ART data recorded was the number of egg retrieval cycles, normal fertilization rate, and clinical pregnancy rate. Results: ART using frozen sperm as well as sperm ejaculated on the day of egg retrieval was possible in all cases. The normal fertilization rate was 70.4%, the clinical pregnancy rate per embryo transferred was achieved in 31.5% (870 cycles), and the live birth rate per case was 73.8%. Conclusions: Intracytoplasmic sperm injection (ICSI) was possible without sperm retrieval surgery in cryptozoospermia, resulting in 73.8% of live births per patient. Sperm identification, sperm processing, and ICSI technique are especially important in cryptozoospermia. Sperm retrieval surgery can be avoided in cryptozoospermic patients.

Infertility treatment for patients having a microdeletion of azoospermic factor (AZF).

In genetic causes of male infertility, Y chromosome microdeletions are the second most common after Klinefelter's syndrome. Although sperm recovery rate is relatively high for subjects with azoospermic factor (AZF) c chromosome microdeletion, intracytoplasmic sperm injection (ICSI) results using retrieved sperm has been reported to be poor. We retrospectively examined the infertility treatment for subjects with AZF microdeletion. From October 2017 to September 2020, chromosomal examination of 67 azoospermic subjects and 12 cryptozoospermia were performed. Of these, twenty-three subjects (29.1%) had AZF microdeletion. Twelve subjects with AZFc microdeletion and one subtype with unknown classification (Ym-9; P3 deletion) received sperm retrieval surgery due to azoospermia. Two subjects obtained motile sperm by microscopic epididymal sperm aspiration (MESA) and four subjects by microscopic testicular sperm extraction (micro-TESE). Pregnancy and healthy delivery were achieved in 6 of 14 subject (42.9%; including one twin) using ICSI. This was comparable with previous reports. Since there were two cases of obstructive azoospermia, we employed MESA to avoid testicular damage. Following observation of the testis and epididymis under operative microscope, a decision was made to perform sperm retrieval surgery to avoid unnecessary testicular damage. Furthermore, since AZFc microdeletion is passed to the next generation, long term follow-up is necessary.

Microscopic Epididymal Sperm Aspiration (MESA) Should be Employed Over Testicular Sperm Extraction (TESE) Sperm Retrieval Surgery for Obstructive Azoospermia (OA).

INTRODUCTION:  Testicular sperm extraction (TESE) has been widely used as a sperm extraction surgery for azoospermia even for obstructive azoospermia (OA) because it does not require surgical skill. However, there are postoperative pain issues, and subsequent testicular atrophy and decreased testosterone levels may occur with TESE. This study examines the usefulness of microscopic epididymal sperm aspiration (MESA) for OA. METHODS:  We studied 108 patients diagnosed with OA and treated with MESA at our institute between April 2004 and December 2021. The MESA was performed using a micropipette with a micropuncture technique under an operative microscope. When no sperm were present or motility was not observed, additional punctures to the epididymal tubule were performed. RESULTS:  Motile sperm were recovered in all cases (108 cases). Of these, intracytoplasmic sperm injection (ICSI) using frozen-thawed sperm was performed in 101 cases and the normal fertilization rate was 76.2%. A total of 436 embryo transfer (ET) cycles were performed. The implantation rate per transfer cycle was 47.9%, the clinical pregnancy rate was 41.0%, and the live birth rate was 23.7%. The per-case live birth rate was 84.8%. CONCLUSIONS:  MESA-ICSI has a very good fertilization rate, clinical pregnancy rate, and delivery rate. Furthermore, the patient's postoperative pain is less, the number of sperm collected is larger, the burden on the embryologist who processes the collected sperm is less, and ICSI can be easily attempted after frozen-thawed sperm. MESA rather than TESE should be employed for the OA subjects.

Factors affecting the distribution of serum anti-müllerian hormone levels among infertile Asian women: a multi-nation, multi-centre, and multi-ethnicity prospective cohort study.

STUDY QUESTION: How do age, ethnicity, and other characteristics affect serum anti-mullerian hormone (AMH) levels in Asian women undergoing fertility treatment? SUMMARY ANSWER: Age, ethnicity, obesity (BMI ≥ 30 kg/m2), and polycystic ovarian syndrome (PCOS) significantly impacted serum AMH levels, with the rate of decrease accelerating as age increased; a concentration of 4.0 ng/ml was the optimal cut-off for diagnosis of PCOS. WHAT IS KNOWN ALREADY: There are significant differences in ovarian reserve among women from different races and ethnicities, and Asian women often have poorer reproductive outcomes during assisted reproductive treatment cycles. STUDY DESIGN, SIZE, DURATION: A population-based multi-nation, multi-centre, multi-ethnicity prospective cohort study of 4613 women was conducted from January 2020 to May 2021. Infertile women of 20-43 years of age were enrolled. The exclusion criteria included: age <20 or >43, non-Asian ethnicity, and missing critical data. PARTICIPANTS/MATERIALS, SETTING, METHODS: Participants were Asian women of Chinese, Japanese, Korean, Thai, Vietnamese, Malay, Indian, and Indonesian ethnicities from 12 IVF centres across Asia. These women were all naïve to ovarian stimulation cycles and attended IVF centres for fertility assessment. The AMH measurement was performed using an AMH automated assay on a clinically validated platform. MAIN RESULTS AND THE ROLE OF CHANCE: A total of 4556 infertile Asian women were included in the final analyses. The mean ± SD for serum AMH concentrations (ng/ml) across specific age groups were: overall, 3.44 ± 2.93; age <30, 4.58 ± 3.16; 30-31, 4.23 ± 3.23; 32-33, 3.90 ± 3.06; 34-35, 3.21 ± 2.65; 36-37, 2.74 ± 2.44; 38-39, 2.30 ± 1.91; 40 and above, 1.67 ± 2.00. The rate of AMH decrease was ∼0.13 ng/ml/year in patients aged 25-33 and 0.31 ng/ml/year in women aged 33-43. The highest rates of PCOS were found in Indians (18.6%), Malays (18.9%), and Vietnamese (17.7%). Age (P < 0.001), ethnicity (P < 0.001), obesity (P = 0.007), PCOS (P < 0.001), and a history of endometrioma cystectomy (P = 0.01) were significantly associated with serum AMH values. Smoking status, pretreatment with GnRH agonist (GnRHa) or the oral contraceptive pill (OCP), freezing-thawing of blood samples, and sampling on Day 2 to Day 5 of the menstrual cycle or randomly did not appear to affect serum AMH levels. An AMH concentration of 4.0 ng/ml was the optimal cut-off for PCOS diagnosis with a sensitivity of 71.7% and specificity of 75.8% (AUC = 0.81, CI 95%: 0.79-0.83; P < 0.001). LIMITATIONS, REASONS FOR CAUTION: The incidence of PCOS was supposedly high in this cohort as some IVF clinics were tertiary referral centres for managing specific fertility issues encountered by women with PCOS. Treatment with GnRHa or OCP before AMH testing was regionally and ethnically confined, mostly in Hong Kong SAR and Japan. Moreover, this reference for serum AMH value is limited to Asian women of the ethnicities examined and may not apply to other ethnicities not included in the study. WIDER IMPLICATIONS OF THE FINDINGS: This is the first study to collate and construct age-specific reference ranges for serum AMH levels using the same bioassay on Asian women of different ethnicities. The findings of this investigation can assist clinicians to counsel and prognosticate about Asian women's ovarian reserve and reproductive potential, thus providing better strategies for personalized fertility interventions. STUDY FUNDING/COMPETING INTEREST(S): This study was technically supported by Ferring Pharmaceuticals and received no specific grant from any funding agency. All authors have no competing interests to disclose. TRIAL REGISTRATION NUMBER: NCT04203355.

Evaluation of the serum zinc concentration in male infertility patients: an analysis of 2010 cases.

Zinc is an important trace element, and its importance for male infertility has been reported. The aim of the study was to assess whether the serum zinc concentrations were related to semen quality in male infertility patients. In 2010 subjects who consulted at our male infertility clinic between November 2018 and May 2021, serum zinc concentrations were assessed along with age, sperm concentration, sperm motility, endocrine panel, and body mass index (BMI). A normal zinc concentration was observed in 1069 (53.2%), subclinical deficiency in 845 (42.0%), and deficiency in 79 subjects (3.9%). On the other hand, high a zinc level was observed in only 17 subjects (0.9%). The serum zinc concentration did not relate with age, sperm concentration, sperm motility, luteinizing hormone (LH), follicle stimulating hormone (FSH), testosterone, and body mass index (BMI). However, normozoospermic subjects showed significantly higher zinc concentrations than among azoospermic (included non-obstructive; NOA and obstructive; OA) and cryptozoospermic patients. Furthermore, the zinc concentration was lower in NOA subjects when comparing to oligozoospermia and/or asthenozoospermia. An association between zinc concentration and semen analysis remained unclear. This study was cross-sectional and retrospective, however, this is a largest investigation of the zinc concentration during reproductive life span in Japan. Further accumulation of cases are required to further examine the potential relationship between zinc concentration and semen quality.

Asynchronous division at 4-8-cell stage of preimplantation embryos affects live birth through ICM/TE differentiation.

To improve the performance of assisted reproductive technology, it is necessary to find an indicator that can identify and select embryos that will be born or be aborted. We searched for indicators of embryo selection by comparing born/abort mouse embryos. We found that asynchronous embryos during the 4-8-cell stage were predisposed to be aborted. In asynchronous mouse embryos, the nuclear translocation of YAP1 in some blastomeres and compaction were delayed, and the number of ICMs was reduced. Hence, it is possible that asynchronous embryos have abnormal differentiation. When the synchrony of human embryos was observed, it was confirmed that embryos that did not reach clinical pregnancy had asynchrony as in mice. This could make synchrony a universal indicator common to all animal species.

Search for morphological indicators that predict implantation by principal component analysis using images of blastocyst.

Background: Although the current evaluation of human blastocysts is based on the Gardner criteria, there may be other notable parameters. The purpose of our study was to clarify whether the morphology of blastocysts has notable indicators other than the Gardner criteria. Methods: To find such indicators, we compared blastocysts that showed elevated human chorionic gonadotropin (hCG) levels after transplantation (hCG-positive group; n = 129) and those that did not (hCG-negative group; n = 105) using principal component analysis of pixel brightness of the images. Results: The comparison revealed that the hCG-positive group had grainy morphology and the hCG-negative group had non-grainy morphology. Classification of the blastocysts by this indicator did not make a difference in Gardner score. Interestingly, all embryos with ≥20% fragmentation were non-grainy. The visual classification based on this analysis was significantly more accurate than the prediction of implantation using the Gardner score ≥3BB. As graininess can be used in combination with the Gardner score, this indicator will enhance current reproductive technologies.

Oocyte cytoplasmic diameter of ≥130 µm can be used to determine human giant oocytes.

OBJECTIVE: To determine if a cytoplasmic diameter of ≥130 µm can help identify human giant oocytes (GOs) in clinical practice and confirm the presence of genetic abnormalities in GOs by assessing the spindle length and centromere numbers. DESIGN: Case-control study. SETTING: Private in vitro fertilization clinic. PATIENT(S): The subjects were women aged 20-49 years who underwent oocyte retrieval after ovarian stimulation from January 2014 to December 2020. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): The oocyte diameter was measured; immunofluorescent staining was performed to assess the spindle diameter and centromere numbers. RESULT(S): Among the 254,337 oocytes examined, 561 (0.22%) had a diameter of ≥130 µm. The mean diameter ranges in the normal-sized metaphase II (MII) oocytes (MII group) and GO group were 103.0-119.0 and 132.3-175.9 µm. Spindle size could be measured in 6 GOs with 1 spindle (GO1), 10 GOs with 2 spindles (GO2), and 16 MII groups. The equatorial plane and pole-to-pole distance in the GO1 were significantly longer than in the GO2 and MII groups. The median numbers of centromeres were 86 in GOs with 1 spindle and 42 in each spindle for GOs with 2 spindles among 11 GO1s and 5 GO2s. CONCLUSION(S): This study is the first to define GOs as oocytes with a diameter of ≥130 µm and is a large-scale study surveying the incidence of GO. It is also the first study to analyze and elucidate the relationship between spindle numbers within the cytoplasm of GOs and spindle size and centromeres.

Secondary male infertility: the importance of the urological assessment for couples who desire children in later life.

Amongst 942 out-patients who consulted our male infertility division between 2016 to 2020, 85 (9.0%) patients suffered from secondary infertility. Of these, in 59 (69.4%) subjects, the first pregnancy was achieved by natural conception. 81 subjects were evaluated for semen quality except for two subjects who at the time were undergoing cancer treatment and another of two ejaculatory dysfunction (EjD). Semen analysis revealed 16 subjects (19.8%) were azoospermic, whereas 9 (11.1%) were cryptozoospermic at median three years of infertility. Left varicocelectomy had been undertaken in a total of 17 oligoasthenozoospermic and cryptozoospermic cases in order to improve semen quality. For achieving natural pregnancy, microscopic vasoepididymostomy was performed in 3 subjects of obstructive azoospemia and patency was achieved in two of three. 11 azoospermic subjects and two of the EjD underwent sperm retrieval surgery for intracytoplasmic sperm injection (ICSI). Motile sperm recovery was obtained by microscopic epididymal sperm aspiration (5/5=100%), microscopic testicular sperm extraction (micro-TESE, 2/6=33.3%), and retrograde vasal sperm aspiration (2/2=100%). Natural pregnancy was obtained in two subjects following varicocelectomy, and in one following vasoepididymostomy. Seven pregnancies were achieved by ICSI using cryopreserved sperm and surgically retrieved sperm. Even if the first pregnancy occurred naturally, 30.9% subjects showed azoospermia or cryptozoospermia at median duration of three years. We would like to emphasize that earlier urological assessment especially semen analysis is necessary if pregnancy later in life is desired.

A case of abdominal migration of the testes presenting with azoospermia.

31-year-old male was referred to our hospital due to azoospermia. Physical examination revealed impalpable testes in the scrotum, and operative scar presented at lower abdominal midline. Magnetic resonance imaging (MRI) revealed that small testes were located subcutaneously in the lower abdominal midline. Since luteinizing hormone (LH) and follicle stimulating hormone (FSH) were elevated, we diagnosed non-obstructive azoospermia (NOA) due to abdominal migration of the testes. Microscopic testicular sperm extraction (micro-TESE) was performed, however, no sperm were recovered. Pathological diagnosis was Sertoli cell only and no malignant cells were observed. Post-operatively, subjects' hormone levels were unchanged, and testicular tumor markers and computed tomography (CT) were normal. However, renal function gradually deteriorated and a renal transplantation from the farther was carried out eight months after micro-TESE. Attention to the possibility of carcinogenesis of the abdominal migrated testes should be maintained.

Successful outcome of a pregnancy derived from premature ovulation in a gonadotropin-releasing hormone antagonist protocol: A case report.

In the gonadotropin-releasing hormone (GnRH) antagonist protocol, it is necessary to reinforce contraceptive guidance assuming that luteinizing hormone surge is not detected by measurement of serum level and ovulation is not suppressed by GnRH antagonist.

Gonadotropin levels at the start of ovarian stimulation predict normal fertilization after hCG re-trigger in GnRH antagonist cycles.

PURPOSE: To assess the appropriateness of human chorionic gonadotropin (hCG) re-trigger in poor responders to gonadotropin-releasing hormone agonist (GnRHa) trigger in controlled ovarian stimulation (COS) cycles. METHODS: The 2251 cycles in 2251 patients triggered with GnRHa for oocyte stimulation, with or without requiring hCG re-trigger between 2013 and 2018, were retrospectively analyzed to compare gonadotropin levels at the start of COS and the rate of normal fertilization between the re-trigger and non-re-trigger group. Furthermore, patients in the re-trigger group were stratified by the rate of normal fertilization (good: ≥60% or poor: <60%) to compare patient demographics, hormone profiles, and clinical outcome between the subgroups. RESULTS: In the re-trigger group, FSH and LH levels at the start of COS were significantly lower in the good fertilization group than in the poor fertilization group (P < .01). Receiver operating characteristic curves identified cutoff values of the FSH and LH levels of 1.30 and 0.35 mIU/mL, respectively, for predicting ≥60% normal fertilization. CONCLUSION: Gonadotropin levels at the start of COS are predictors of response to GnRHa trigger and hCG re-trigger necessity, and may serve as indicators to help clinicians appropriately choose hCG re-trigger rather than abandoning the cycles or continuing the first oocyte aspiration attempt.

Male infertility treatment for cancer survivors: does anticancer treatment affect infertility treatment?

We investigated the impact of prior anticancer treatments such as chemotherapy and radiotherapy on subsequent infertility treatment in cancer survivors who consulted our male infertility division. Of 1,525 male infertility patients who consulted our division between 2008 and 2018, 56 (3.7%) were cancer survivors. Of these, 32 received anticancer treatment (group A) and 24 were treated with surgery alone or were seen before anticancer treatment (group B). Semen analysis revealed that azoospermia in 26 subjects (81.3%) and 14 (58.3%) in groups A and B respectively. Ejaculatory dysfunction was observed 1 in group A and in 2 group B subjects. Sperm cryopreservation before anticancer treatment was performed 4 subjects. Sperm retrieval surgery for intracytoplasmic sperm injection (ICSI) was performed in 13 cases in group A and 10 in group B. Motile sperm were recovered in 7 subjects and in 8 subjects in group A and B respectively. Overall pregnancies and deliveries with ICSI were achieved for 7 subjects (21.9%) in group A, and 9 (37.5%) in group B. Successful sperm retrieval may not be affected by prior anticancer treatment as shown in this study. However, some patients abandoned infertility treatment due to the cost of testing and sperm retrieval surgery. Support for the cost of infertility treatment in cancer survivors is necessary.

Microsurgical seminal reconstruction; our experiences in a single institute.

We assessed the contribution of microsurgical seminal reconstruction to achieving natural conception in conjunction with advanced assisted reproductive technologies. Ninety obstructive azoospermic subjects who underwent microsurgical seminal reconstruction were evaluated. Vasovasostomy (VV) was undertaken in 45 subjects whereas vasoepididymostomy (VE) in 45, respectively. VV was performed by employing a two microlayer anastomotic technique, whilst VE was undertaken using double needle longitudinal vaspepididymostomy (LIVE). Patency was achieved in 41 VV (91.1%), and 25 VE (55.6%) cases. In cases where patency was achieved, pregnancy and healthy delivery were recorded following natural intercourse in 7/41 (17.0%) VV, and in 7/25 (28.0%) VE cases. Where patency was not achieved, the use of cryopreserved sperm for intracytoplasmic sperm injection (ICSI), resulted in a healthy delivery in 4/4 (100%) VV and 14/21 (66.6%) in VE subjects. Although natural pregnancy was achieved only in a limited number of subjects treated (14/90; 15.6%), sperm harvested during surgery and cryopreserved for future ICSI use proved valuable, doubling the overall delivery rate (32/90; 36.6%). Surgical intervention is considered to be a useful technique in order to allow the possibility of a natural conception and by harvesting sperm at the same time contributes to the cost-effectiveness.

Development of an automated two pronuclei detection system on time-lapse embryo images using deep learning techniques.

PURPOSE: To establish an automated pronuclei determination system by analysis using deep learning technology which is able to effectively learn with limited amount of supervised data. METHODS: An algorithm was developed by explicitly incorporating human observation where the outline around pronuclei is being observed in determining the number of pronuclei. Supervised data were selected from the time-lapse images of 300 pronuclear stage embryos per class (total 900 embryos) clearly classified by embryologists as 0PN, 1PN, and 2PN. One-hundred embryos per class (a total of 300 embryos) were used for verification data. The verification data were evaluated for the performance of detection in the number of pronuclei by regarding the results consistent with the judgment of the embryologists as correct answers. RESULTS: The sensitivity rates of 0PN, 1PN, and 2PN were 99%, 82%, and 99%, respectively, and the overlapping 2PN being difficult to determine by microscopic observation alone could also be appropriately assessed. CONCLUSIONS: This study enabled the establishment of the automated pronuclei determination system with the precision almost equivalent to highly skilled embryologists.