Shape of the first mitotic spindles impacts multinucleation in human embryos.

During human embryonic development, early cleavage-stage embryos are more susceptible to errors. Studies have shown that many problems occur during the first mitosis, such as direct cleavage, chromosome segregation errors, and multinucleation. However, the mechanisms whereby these errors occur during the first mitosis in human embryos remain unknown. To clarify this aspect, in the present study, we image discarded living human two-pronuclear stage zygotes using fluorescent labeling and confocal microscopy without microinjection of DNA or mRNA and investigate the association between spindle shape and nuclear abnormality during the first mitosis. We observe that the first mitotic spindles vary, and low-aspect-ratio-shaped spindles tend to lead to the formation of multiple nuclei at the 2-cell stage. Moreover, we observe defocusing poles in many of the first mitotic spindles, which are strongly associated with multinucleation. Additionally, we show that differences in the positions of the centrosomes cause spindle abnormality in the first mitosis. Furthermore, many multinuclei are modified to form mononuclei after the second mitosis because the occurrence of pole defocusing is firmly reduced. Our study will contribute markedly to research on the occurrence of mitotic errors during the early cleavage of human embryos.

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.

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.

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.

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.

Chromosome segregation error during early cleavage in mouse pre-implantation embryo does not necessarily cause developmental failure after blastocyst stage.

In the pre-implantation embryo, aneuploidy resulting from chromosome segregation error is considered responsible for pregnancy loss. However, only a few studies have examined the relationship between chromosome segregation errors during early cleavage and development. Here, we evaluated this relationship by live-cell imaging using the histone H2B-mCherry probe and subsequent single blastocyst transfer using mouse embryos obtained by in vitro fertilization. We showed that some embryos exhibiting early chromosomal segregation error and formation of micronuclei retained their developmental potential; however, the error affected the blastocyst/arrest ratio. Further, single-cell sequencing after live-cell imaging revealed that all embryos exhibiting micronuclei formation during 1st mitosis showed aneuploidy at the 2-cell stage. These results suggest that early chromosome segregation error causing micronuclei formation affects ploidy and development to blastocyst but does not necessarily cause developmental failure after the blastocyst stage. Our result suggests the importance of the selection of embryos that have reached blastocysts.

Performance of anti-Müllerian hormone (AMH) levels measured by Beckman Coulter Access AMH assay to predict oocyte yield following controlled ovarian stimulation for in vitro fertilization.

PURPOSE: We evaluated the performance of anti-Müllerian hormone (AMH) measured by the Beckman Coulter fully automated Access assay to predict oocyte yield following controlled ovarian stimulation (COS) for in vitro fertilization (IVF). METHODS: The correlation between the Access assay and the pre-mixing method with Generation II ELISA assay (Gen II pre-mix assay) was assessed using 230 blood samples. The relationship of AMH level measured by the Access assay and the actual number of oocytes retrieved following COS was assessed using 3296 IVF cycles. The performances of AMH, follicle stimulating hormone (FSH), and estradiol (E2) in predicting the responses to COS were also evaluated by constructing receiver operating characteristic (ROC) curves. RESULTS: The AMH levels measured just before oocyte retrieval by the Access assay and the number of oocytes retrieved following COS showed a good correlation with R = 0.655. The ROC analysis revealed that the sensitivity of AMH was comparable with or lower than that of E2 but higher than that of FSH. CONCLUSIONS: With the improved Access AMH assays, AMH was as sensitive as E2 and could become an accurate marker of ovarian response to COS in more than 3000 Japanese IVF patients.

Clinical utility of chlormadinone acetate (Lutoral™) in frozen-thawed embryo transfer with hormone replacement.

PURPOSE: The clinical utility of chlormadinone acetate tablets (Lutoral™), an orally active progestin which has been available since June 2007, was compared to an in-house vaginal suppository formulation of progesterone used between 2006 and 2007 for assisted reproductive technology (ART). METHODS: We retrospectively evaluated the efficacy and safety of chlormadinone acetate by comparing the pregnancy rates and the incidences of birth defects and hypospadias in frozen-thawed embryo transfer cycles using the in-house vaginal progesterone and those using chlormadinone acetate for luteal phase support. RESULTS: The pregnancy rates in the frozen-thawed embryo transfer cycles were 31.2% (259/831) with vaginal progesterone for luteal phase support and 31.6% (4228/13 381) with chlormadinone acetate (no significant difference). In the cycles resulting in live birth following administration of chlormadinone acetate between July 2007 and December 2015, the incidence of birth defects was 2.8% (80/2893), and the incidence of hypospadias was 0.03% (1/2893). CONCLUSIONS: These results indicate that the pregnancy rate following frozen-thawed embryo transfer using chlormadinone acetate for luteal phase support was comparable with that using vaginal progesterone, with no increased risk of birth defects, including hypospadias, which has been a concern following the use of progestins.

Long-term outcomes of freeze-all strategy: A retrospective analysis from a single ART center in Japan.

PURPOSE: To demonstrate the benefits of the freeze-all strategy for in vitro fertilization treatment based on retrospective analyses. METHODS: Post-thaw embryo survival rates of slow-frozen embryos in 294 cycles and vitrified embryos in 12 195 cycles were assessed. Progesterone (P4) and estradiol (E2) levels per mature oocyte by age category were assessed in 9081 cycles and pregnancy rates with fresh embryo transfer and frozen-thawed embryo transfer by P4 level were assessed in 1535 cycles. RESULTS: The survival rates of frozen-thawed embryos were 92.5% with slow freezing and 99.1% with vitrification. P4 levels on the day of human chorionic gonadotropin (hCG) injection showed a trend toward an increase with age. The pregnancy rate per mature oocyte with fresh embryo transfer decreased dependently upon P4 level, while that with frozen-thawed embryo transfer was not affected by P4 level. The pregnancy rates with frozen-thawed embryo transfer were higher than those with fresh embryo transfer in patients aged 42 years or younger. CONCLUSIONS: The freeze-all strategy is a valuable treatment option which allows the separation of an embryo transfer cycle from an oocyte retrieval cycle, especially for patients with high P4 levels at oocyte retrieval and patients of advanced maternal age.

Superior clinical pregnancy rates after microsurgical epididymal sperm aspiration.

Purpose: To assess normal fertilization, clinical pregnancy, and live birth rates after the use of microscopic epididymal sperm aspiration (MESA). Methods: One-hundred-and-sixty azoospermic participants who underwent MESA were evaluated. The MESA was performed by using a micropuncture method with a micropipette. In cases in which motile sperm were not obtained after the MESA, conventional or micro-testicular sperm extraction (TESE) was completed. Results: Adequate motile sperm were retrieved in 71 participants by using MESA and in 59 out of 89 participants by using TESE. Of the total number of patients, 123 underwent intracytoplasmic sperm injection. After MESA, the normal fertilization rate was 73.5% and the clinical pregnancy rate per case was 95.7%. Healthy deliveries resulted after MESA in 65 (92.9%) cases and after TESE in 38 (71.7%) cases. Conclusion: The MESA specimen collection does not have any special requirements, such as mincing tissue disposition. The MESA also can reduce the amount of laboratory work that is needed for cryopreservation. In the authors' experience, MESA is a beneficial procedure and should be given priority over TESE.