The utility of human two plus one small pronucleated zygotes (2.1PN) based on clinical outcomes and the focused ploidy analysis.

PURPOSE: Are human embryos arising from two plus one small pronucleated zygotes, called 2.1 pronuclei (PN), clinically useful? METHODS: In a retrospective embryo cohort study and prospective experimental study, a total of 287 cycles in which at least one 2.1PN was identified in the fertilization check were included. Embryonic development and clinical outcome were compared for the 1395 2PN zygotes and 304 2.1PN zygotes that were siblings. All embryos were individually cultured in time-lapse systems. Twenty-five 2.1PN-derived blastocysts, donated for research, were used in focused single-nucleotide variant ploidy analysis to identify the distribution pattern of heterozygosity. RESULTS: The average diameter of PN was 24.9 ± 2.4 µm for large PN and 10.2 ± 2.4 µm for small PN; 79.9% of small PN was derived from female pronuclei. Blastocyst formation rate and good-quality blastocyst rate were significantly lower with 2.1PN embryos than with 2PN embryos (40.0% vs. 57.7%, 21.4% vs. 33.5%, respectively). A total of 13 embryos derived from 2.1PN were transferred, and three healthy babies were born. In ploidy constitutions of trophectoderm (TE), 2.1PN-derived blastocyst TE was shown to be mostly diploid (95.8%, 23/24), and only one blastocyst showed triploid. CONCLUSIONS: It was suggested that 2.1PN embryos have lower embryonic developmental potential than 2PN embryos, but most of the 2.1PN were diploid, indicating that they are likely to be clinically usable. It is recommended to perform embryo transfer following a combination of PGT-A and ploidy analysis.

Micromapping testicular sperm extraction: A new technique for microscopic testicular sperm extraction in nonobstructive azoospermia.

Purpose: In microscopic testicular sperm extraction (mTESE) for nonobstructive azoospermia (NOA), sperm can be recovered relatively easily in some cases, and mTESE may be retrospectively considered excessive. However, mTESE is routinely performed in the majority of NOA patients because of the difficulty in predicting tissue status. A minimally invasive and comprehensive sperm retrieval method that allows on-the-spot tissue assessment is needed. We have developed and evaluated a novel sperm retrieval technique for NOA called micromapping testicular sperm extraction (MMTSE). Methods: MMTSE involves dividing the testis into four sections and making multiple small needle holes in the tunica albuginea to extract seminiferous tubules and retrieve sperm. The sperm-positive group by MMTSE (Group I) underwent additional tissue collection (ATC) via a small incision, whereas the sperm-negative group by MMTSE (Group 0) underwent mTESE. Results: In total, 40 NOA participants underwent MMTSE. Group I included 15 patients and Group 0 included 25 patients. In Group 1, sperm were recovered from all patients by ATC. In Group 0, sperm were recovered in 4 of 25 cases using mTESE. Conclusions: MMTSE shows promise as a simple method that comprehensively searches testicular tissue and retrieves sperm using an appropriate method while minimizing patient burden.

Chlormadinone acetate in progestin-primed ovarian stimulation does not negatively affect clinical results.

Purpose: To investigate whether progestin-primed ovarian stimulation (PPOS) with chlormadinone acetate (CMA) adversely affects clinical results and neonatal outcomes, or causes congenital deformities. Methods: This retrospective study was conducted at private IVF clinic from November 2018 to November 2021. Women underwent oocyte retrieval using gonadotropin-releasing hormone (GnRH) antagonist protocol (n = 835) or PPOS protocol (n = 57) were included. Eligible patients were normal ovarian responders (aged <40, AMH ≧1.0 ng/mL) with freeze-all cycle. Embryo developments, clinical results, or neonatal outcomes of singletons derived from transfer of frozen single blastocysts were compared within each group. Results: Patient characteristics were similar in both groups. The median LH level (mIU/mL) at trigger in the GnRH antagonist group [2.0 (1.2-3.7)] was significantly higher than in the PPOS group [0.9 (0.3-1.7)]. There was no cycle with premature LH surge in the PPOS group. Fertilization and blastocyst formation rates did not differ significantly between groups. Furthermore, clinical outcomes were also similar in the two groups. Congenital abnormality rates did not differ significantly [0.9% (3/329), 0.0% (0/17)]. Conclusions: CMA using ovarian stimulation did not negatively affect clinical results. Our data suggest that PPOS with CMA is an appropriate ovarian stimulation method for normal ovarian responders.

Assisted reproductive technologies are slightly associated with maternal lack of affection toward the newborn: The Japan Environment and Children's Study.

AIM: To evaluate the association between use of assisted reproductive technology (ART) and mother-to-infant bonding. METHODS: Using nationwide birth cohort study with periodical follow-ups for mothers and children during pregnancy and at 1 year after delivery, mothers were classified following three groups; infertility group with ART: 2792 mothers; infertility group with non-ART treatment (ovulation induction and intrauterine insemination): 3835 mothers and unaided pregnancy group: 78 726 mothers. Data on maternal and child health as well as basic characteristics were collected via medical records and self-administered questionnaires. The Japanese version of Mother-to-Infant Bonding Scale was used to evaluate maternal bonding style. To evaluate the association between ART and maternal bonding toward babies, multivariate analysis was used with adjustment for potential confounders such as babies' sex, socio-economic status and history of maternal mental disorders. RESULTS: Multivariate logistic regression analysis indicated that ART was slightly but significantly associated with an increased risk of maternal lack of affection toward their newborn (adjusted odds ratio 1.10, 95% confidence interval 1.02-1.20), while non-ART was significantly associated with both maternal lack of affection and anger/rejection toward the newborn. No substantial association was observed between ART and overall Mother-to-Infant Bonding Scale score (adjusted odds ratio 1.03, 95% confidence interval 0.94-1.12). Very slight negative correlations were observed between poor mother-to-infant bonding and good infant mental and physical development at both 6 months and 1 year. CONCLUSION: ART may exert a negative influence on maternal emotion after delivery, but this does not have strong correlation with child development evaluated by mothers.

The risk of secondary sex ratio imbalance and increased monozygotic twinning after blastocyst transfer: data from the Japan Environment and Children's Study.

BACKGROUND: Some studies have suggested that blastocyst transfer is associated with i) imbalance in the secondary sex ratio (SSR) (which favors male offspring), ii) increased incidence of monozygotic twins (MZT). In contrast, others have not found these changes. In this study, we evaluated the association between blastocyst transfer and SSR and MZT, considering potential parental confounders. METHODS: The Japan Environment and Children's Study is a large, nationwide longitudinal birth cohort study funded by the Ministry of the Environment of Japan. We used this large dataset, including 103,099 pregnancies, to further investigate the association between blastocyst transfer, SSR and MZT, using spontaneously conceived pregnancies, non-assisted reproductive technology (non-ART) treatment (intrauterine insemination and ovulation induction with timed intercourse) and cleavage stage embryo transfer for comparison. We evaluated the association with each group, the SSR, and the frequency of MZT, calculating the adjusted odds ratio (AOR) using multivariable logistic regression analyses, adjusting for potential parental confounders such as basic health and socioeconomic status. RESULTS: For each group (spontaneous conception vs. non-ART treatment vs. cleavage stage embryo transfer vs. blastocyst transfer), the percentages of males were 51.3% vs 50.7% vs 48.9% vs 53.4% and the monozygotic twinning rates per pregnancy were 0.27% vs 0.11% vs 0.27% vs 0.99% respectively. Multivariate logistic regression analyses indicated that blastocyst transfer was significantly associated with a higher SSR and higher incidence of MZT than the other three groups (SSR: AOR 1.095, 95% CI1.001-1.198; MZT: AOR 4.229, 95% CI 2.614-6.684). CONCLUSIONS: There are significant relationships between blastocyst transfer and SSR imbalance and a higher occurrence of MZT.

Association of four imprinting disorders and ART.

BACKGROUND: Human-assisted reproductive technologies (ART) are a widely accepted treatment for infertile couples. At the same time, many studies have suggested the correlation between ART and increased incidences of normally rare imprinting disorders such as Beckwith-Wiedemann syndrome (BWS), Angelman syndrome (AS), Prader-Willi syndrome (PWS), and Silver-Russell syndrome (SRS). Major methylation dynamics take place during cell development and the preimplantation stages of embryonic development. ART may prevent the proper erasure, establishment, and maintenance of DNA methylation. However, the causes and ART risk factors for these disorders are not well understood. RESULTS: A nationwide epidemiological study in Japan in 2015 in which 2777 pediatrics departments were contacted and a total of 931 patients with imprinting disorders including 117 BWS, 227 AS, 520 PWS, and 67 SRS patients, were recruited. We found 4.46- and 8.91-fold increased frequencies of BWS and SRS associated with ART, respectively. Most of these patients were conceived via in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI), and showed aberrant imprinted DNA methylation. We also found that ART-conceived SRS (ART-SRS) patients had incomplete and more widespread DNA methylation variations than spontaneously conceived SRS patients, especially in sperm-specific methylated regions using reduced representation bisulfite sequencing to compare DNA methylomes. In addition, we found that the ART patients with one of three imprinting disorders, PWS, AS, and SRS, displayed additional minor phenotypes and lack of the phenotypes. The frequency of ART-conceived Prader-Willi syndrome (ART-PWS) was 3.44-fold higher than anticipated. When maternal age was 37 years or less, the rate of DNA methylation errors in ART-PWS patients was significantly increased compared with spontaneously conceived PWS patients. CONCLUSIONS: We reconfirmed the association between ART and imprinting disorders. In addition, we found unique methylation patterns in ART-SRS patients, therefore, concluded that the imprinting disorders related to ART might tend to take place just after fertilization at a time when the epigenome is most vulnerable and might be affected by the techniques of manipulation used for IVF or ICSI and the culture medium of the fertilized egg.

Long-term clinical outcomes of testicular sperm extraction and intracytoplasmic sperm injection for infertile men.

Purpose: To find the best methods to achieve the highest pregnancy and birth rates for couples needing testicular sperm extraction (TESE)-intracytoplasmic sperm injection (ICSI). Methods: Retrospectively studied were 801 patients with male factor infertility who had undergone TESE-ICSI between April, 1996 and July, 2016 and who had been categorized into four groups: obstructive azoospermia (OA); non-obstructive azoospermia (NOA); Klinefelter syndrome (KS); and cryptozoospermia (Crypt). The sperm retrieval rate, hormone levels, fertilization rate (FR), pregnancy rate (PR), and birth rate (BR) after ICSI among three groups were compared: fresh testicular sperm (FS)-fresh oocytes (FO) (Group I); frozen-thawed testicular sperm-FO (Group II); and FS-vitrified-warmed oocytes (Group III). Results: The testicular sperm recovery rate was 57.8% (463/801): 89.6% in the Crypt, 97.1% in the OA, 28.9% in the NOA, and 42.2% in the KS groups. The follicle-stimulating hormone levels were significantly higher in the NOA and KS groups and the testosterone levels were significantly lower in the KS group. The FR, PR, and BR were: 65.2%, 43.2%, and 28.5% in group I; 59.2%, 33.4%, and 18.7% in group II; and 56.4%, 33.8%, and 22.1% in group III. Conclusion: Intracytoplasmic sperm injection with FS-FO achieved the best PR and BR. It should be considered what to do in cases with no testicular sperm by TESE. The authors hope that ICSI with donor sperm will be allowed in Japan in the near future.

Genome-wide microRNA expression profiling in placentae from frozen-thawed blastocyst transfer.

BACKGROUND: Frozen-thawed embryo transfer (FET) is increasingly available for the improvement of the success rate of assisted reproductive technologies other than fresh embryo transfer (ET). There have been numerous findings that FET provides better obstetric and perinatal outcomes. However, the birth weight of infants conceived using FET is heavier than that of those conceived via ET. In addition, some reports have suggested that FET is associated with perinatal diseases such as placenta accreta and pregnancy-induced hypertension (PIH). RESULTS: In this study, we compared the microRNA (miRNA) expression profiles in term placentae derived from FET, ET, and spontaneous pregnancy (SP). We identified four miRNAs, miR-130a-3p, miR-149-5p, miR-423-5p, and miR-487b-3p, that were significantly downregulated in FET placentae compared with those from SP and ET. We found that DNA methylation of MEG3-DMR, not but IG-DMR, was associated with miRNA expression of the DLK1-DIO3 imprinted domain in the human placenta. In functional analyses, GO terms and signaling pathways related to positive regulation of gene expression, growth, development, cell migration, and type II diabetes mellitus (T2DM) were enriched. CONCLUSIONS: This study supports the hypothesis that the process of FET may increase exposure of epigenome to external influences.

Deliveries of babies with normal health derived from oocytes with smooth endoplasmic reticulum clusters.

PURPOSE: To examine the impact on development of derived embryos from smooth endoplasmic reticulum clusters (SERC) in human metaphase II (MII) oocytes. METHODS: Retrospective analysis at Kyono ART Clinic. Comparison of embryological development, pregnancy, live birth and fetal malformation between oocytes with SERC (the SERC(+) group) and those without (the SERC(-) group) in 2,158 patients (3,758 cycles) after ICSI. RESULTS: Fertilization and implantation rate were significantly lower in SERC(+) MII oocytes than in SERC(-) MII oocytes. After the transfer of fresh and vitrified embryos derived from SERC(+) oocytes, 14 pregnancies resulted in 14 healthy babies, including 2 from fresh embryo transfer (ET) and 12 from vitrified-warmed ET, with no malformations. CONCLUSION(S): The presence of SERC in MII oocytes was associated with significantly lower fertilization rates and implantation rates than seen in SERC(-) MII oocytes within SERC (+) cycles. However, SERC had no impact on post-implantation development as well as neonatal outcome.

Birth of a healthy infant after intracytoplasmic sperm injection using pentoxifylline-activated sperm from a patient with Kartagener's syndrome.

OBJECTIVE: To describe the delivery of a healthy female infant after intracytoplasmic sperm injection (ICSI) using pentoxifylline-activated sperm from a patient with Kartagener's syndrome. DESIGN: Case report. SETTING: Private assisted reproductive technology clinic in Japan. PATIENT(S): A couple with male factor infertility due to Kartagener's syndrome. INTERVENTION(S): Intracytoplasmic sperm injection using ejaculated sperm activated by pentoxifylline. MAIN OUTCOME MEASURE(S): Semen characteristics, sperm ultrastructure, fertilization, pregnancy, and birth after ICSI. RESULT(S): The fertilization rate was 7 of 12 (58.3%), and the blastocyst formation rate was 4 of 7 (57.1%); all blastocysts were vitrified. After a single blastcyst transfer, a pregnancy ensued and progressed to term; a healthy female infant was delivered. CONCLUSION(S): With ejaculated sperm, which was activated by pentoxifylline, successful fertilization was accomplished by ICSI; thus, fertilization, vitrification, pregnancy, and delivery are attainable with sperm obtained from men with Kartagener's syndrome.

A birth from the transfer of a single vitrified-warmed blastocyst using intracytoplasmic sperm injection with calcium ionophore oocyte activation in a globozoospermic patient.

OBJECTIVE: To present the effectiveness of diagnostic heterologous intracytoplasmic sperm injection (ICSI), mouse oocyte activation test (MOAT), and ICSI combined with assisted oocyte activation (AOA) in a globozoospermic patient. DESIGN: A case report. SETTING: A private IVF center, Japan. PATIENT(S): A patient with globozoospermia. INTERVENTION(S): MOAT in a mouse and ICSI combined with AOA in a human. MAIN OUTCOME MEASURE(S): Ultrastructure, MOAT, fertilization, and pregnancy. RESULT(S): The transmission electron micrographs showed 100% round-headed spermatozoa lacking an acrosome. MOAT showed that the fertilization rate was 68.4% (13/19) when AOA was used but 0% (0/19) when AOA was not used. After the diagnosis of globozoospermia and sperm-related activation deficiency, 17 human mature oocytes were activated with calcium ionophore after ICSI was performed. The fertilization rate was 88.2% (15/17), and 11 blastocysts were cryopreserved using the vitrification method to prevent severe ovarian hyperstimulation syndrome. A single vitrified-warmed blastocyst was transferred. A gestational sac with fetal heart movements was recognized, and a healthy boy weighing 3180 g was born at 40 weeks of gestation by cesarean section without any congenital abnormality. CONCLUSION(S): MOAT allows discrimination between sperm- and oocyte-related fertilization failures and shows the effectiveness of AOA.

Spectroscopic properties and bacteriochlorophyll c isomer composition of extramembranous light-harvesting complexes in the green sulfur photosynthetic bacterium Chlorobium tepidum and its CT0388-deleted mutant under vitamin B12-limited conditions.

The effects of exogenous vitamin B12 on the green sulfur photosynthetic bacterium Chlorobium (Chl.) tepidum were examined. Wild-type cells and mutant cells lacking a gene CT0388 (denoted as VB0388) of Chl.tepidum were grown in liquid cultures containing different concentrations of vitamin B12. The VB0388 cells hardly grew in vitamin B12-limited media, indicating that the product of CT0388 actually played an important role in vitamin B12 biosynthesis in Chl. tepidum. Both wild-type and VB0388 cells in vitamin B12-limited media exhibited absorption bands and CD signals at the Qy region that were shifted to a shorter wavelength than those of cells grown in normal media. BChl c isomers that had S-stereochemistry at the 3(1)-position tended to increase in Chl. tepidum grown in vitamin B12-limited media.