Complementary Critical Functions of Zfy1 and Zfy2 in Mouse Spermatogenesis and Reproduction.

The mammalian Y chromosome plays a critical role in spermatogenesis. However, the exact functions of each gene in the Y chromosome have not been completely elucidated, partly owing to difficulties in gene targeting analysis of the Y chromosome. Zfy was first proposed to be a sex determination factor, but its function in spermatogenesis has been recently elucidated. Nevertheless, Zfy gene targeting analysis has not been performed thus far. Here, we adopted the highly efficient CRISPR/Cas9 system to generate individual Zfy1 or Zfy2 knockout (KO) mice and Zfy1 and Zfy2 double knockout (Zfy1/2-DKO) mice. While individual Zfy1 or Zfy2-KO mice did not show any significant phenotypic alterations in fertility, Zfy1/2-DKO mice were infertile and displayed abnormal sperm morphology, fertilization failure, and early embryonic development failure. Mass spectrometric screening, followed by confirmation with western blot analysis, showed that PLCZ1, PLCD4, PRSS21, and HTT protein expression were significantly deceased in spermatozoa of Zfy1/2-DKO mice compared with those of wild-type mice. These results are consistent with the phenotypic changes seen in the double-mutant mice. Collectively, our strategy and findings revealed that Zfy1 and Zfy2 have redundant functions in spermatogenesis, facilitating a better understanding of fertilization failure and early embryonic development failure.

Random-start ovarian stimulation with aromatase inhibitor for fertility preservation in women with Japanese breast cancer.

PURPOSE: Fertility preservation is an important issue for young cancer patients. Random-start controlled ovarian stimulation and double ovarian stimulation have been proposed for efficient oocyte retrieval within the limited time before cancer therapy. We aimed to clarify the efficacy of these new protocols within the Japanese population. METHODS: We performed a retrospective observational study at a multicenter from February 2012 to August 2017. The study entailed 50 cycles with 34 patients who underwent fertility preservation due to breast cancer. Follicular phase or luteal phase ovarian stimulation with aromatase inhibitor was performed. A second ovarian stimulation was started with or without waiting until the next menstruation. We measured the number of retrieved oocytes and cryopreserved oocytes/embryos, the ratio of mature oocytes, and the fertilization rate. RESULTS: The numbers of retrieved oocytes and frozen oocytes/embryos were not significantly different between follicular phase and luteal phase ovarian stimulation. The number of retrieved oocytes was not reduced at the second ovum pick up compared to the first ovum pick up in the double ovarian stimulation. CONCLUSIONS: Random-start controlled ovarian stimulation and double ovarian stimulation with aromatase inhibitor for breast cancer patients were effective protocols for retrieving a greater number of oocytes within the limited time.

Validity for assisted hatching on pregnancy rate in assisted reproductive technology: analysis based on results of Japan Assisted Reproductive Technology Registry System 2010.

AIM: The aim of this study was to assess the efficacy of assisted hatching (AH) in assisted reproductive technology (ART) treatment. MATERIAL AND METHODS: In this retrospective observational study, the data of patients who were registered in the National ART Registry System of Japan between January and December 2010 were analyzed. The descriptive statistics and validity of AH in fresh embryo transfer (ET) and frozen-thawed ET were assessed by using multiple logistic regression analyses. RESULTS: From a total of 105,450 single ET, 46,029 (43.7%) cycles underwent AH. A total of 9737 (21.3%) and 36,292 (60.9%) cycles underwent AH from 45,818 fresh single ET and 59,632 frozen-thawed single ET, respectively. In the fresh ET patients that underwent AH, the clinical pregnancy and live birth rate were significantly decreased in patients of all ages compared with that of the non-AH group. In the frozen-thawed ET patients, there was no significant difference in pregnancy and live birth rate between the AH group and the non-AH group. CONCLUSION: AH treatment was more frequently performed in frozen-thawed ET patients than in fresh ET patients, and in the blastocyst stage than in the early cleavage stage. A significantly decreased pregnancy and live birth rate was observed in the fresh ET patients who underwent AH. In the frozen-thawed ET patients who underwent AH, improvement in the clinical pregnancy and live birth rate was not observed. Further studies on the indication and application of AH in ART treatment are required.

The incidence of monozygotic twinning in assisted reproductive technology: analysis based on results from the 2010 Japanese ART national registry.

PURPOSE: To assess the incidence of monozygotic twinning (MZT) among cases undergoing assisted reproductive technology (ART) treatment. METHODS: We performed a retrospective observational study and analyzed the data of patients who were registered in the national ART registry system of Japan from January to December 2010; only the data of patients with single embryo transfer (ET) were included. RESULTS: Of 30,405 pregnancies, 425 resulted in MZT following fresh and frozenthawed ET. The MZT incidence among women undergoing ART was 1.4 %. Multiple logistic regression analysis indicated that cases undergoing fresh and frozen-thawed ET, blastocyst transfer had a significantly increased MZT rate (P < 0.01). Assisted hatching (AH) and frozen-thawed ET and maternal age did not significantly affect the MZT incidence. Of 8510 fresh ET pregnancies, 104 resulted in MZT. Multiple logistic regression analysis indicated that blastocyst transfer significantly increased the MZT rate in cases undergoing fresh ET. Ovarian stimulation, intracytoplasmic sperm injection, AH, and maternal age did not significantly affect the MZT incidence. CONCLUSIONS: Blastocyst transfer was associated with an increased MZT incidence. We have to be aware of the potential risk of MZT caused by blastocyst transfer. However, further studies are required to assess the correlation among specific AH types, embryo culture conditions, and MZT incidence.

Successful pregnancy and delivery achieved following intracytoplasmic sperm injection using teratozoospermic sperm exhibiting marked dysplasia of the fibrous sheath: a case report.

A husband and his wife, both 34 years old, consulted our clinic because of primary infertility. Sperm analysis revealed that the sperm concentration, motility, and progressive motility were (42.8±22.8)×106/mL, 23.3%±12.2%, and 12.9%±6.1%, respectively. Based on Krugar strict morphology criteria, 100% of the sperm were teratozoospermic, with 7.9% DNA fragmentation index. Observation of the sperm under a transmission electron microscope revealed that most parts of the fibrous sheath (FS) surrounding the tails of the sperm were missing from midway through the principal piece to the end piece, although the sperm's heads, necks and midpieces were morphologically normal. To collect oocytes, the gonadotropin-releasing hormone antagonist protocol was carried out, and 7 oocytes were retrieved. Intracytoplasmic sperm injection (ICSI) was performed for all the teratozoospermic sperm. Of the 7 oocytes, 3 were fertilized, and one 8-cell embryo and 2 expanded blastocysts were vitrified. Although repeated transfers of expanded blastocysts resulted in no implantation, one 8-cell embryo transfer in a hormone replacement therapy cycle led to pregnancy. The pregnancy using an 8-cell vitrified embryo resulted in the delivery of a healthy female baby at 38 weeks of gestation. No congenital malformations were found until 28 days after birth. Our results demonstrated that healthy birth could be achieved following the transfer of an embryo derived from ICSI using teratozoospermic sperm exhibiting the dysplasia of the fibrous sheath (DFS). Furthermore, while the previous reports on DFS have not investigated male infertility, we evaluated sperms from various aspects such as Kruger sperm function test, chromatin dispersion test, electron microscopy findings, time-lapse images of the obtained embryos, and concluded that ICSI could be desirable as a treatment policy for DFS.

Port site recurrence and unusual diffuse subcutaneous metastases of unexpected early stage ovarian cancer after laparoscopic surgery: a case report.

Port site recurrence is a rare but well-documented adverse event peculiar to laparoscopic surgery. We report an unusual outcome of unexpected early stage ovarian cancer in which port site recurrence occurred after laparoscopic surgery and was followed by diffuse subcutaneous metastases. A 31-year-old Japanese woman with a large tumor in her abdomen visited our hospital. Because no intratumoral solid component was detected on diagnostic imaging, the tumor was diagnosed as a benign ovarian tumor and the patient underwent left ovarian laparoscopic cystectomy. Contrary to our expectations, however, the ovarian tumor was a mucinous carcinoma. We performed additional surgery, but the tumor recurred in the umbilical area, and multiple subcutaneous metastases later appeared. The curative effect of chemotherapy and radiation was limited. This atypical metastatic distribution of an extremely small amount of cancer might have been caused by the laparoscopic procedure. Protection against tumor cell dissemination is necessary during all forms of laparoscopic surgery.

Clinical significance of the serum crosslinked N-telopeptide of type I collagen as a prognostic marker for non-small-cell lung cancer.

INTRODUCTION: Lung cancer is the leading cause of cancer-related death. Many patients with lung cancer are in its advanced stages at the time of diagnosis. The 5-year survival rate for lung cancer is 10% to 20%, and the prognosis for patients with lung cancer is still poor. The crosslinked N-terminal telopeptide of type I collagen (NTx) is a metabolite of type I collagen, the main constituent of bone matrix. PATIENTS AND METHODS: We measured serum NTx levels in patients who underwent staging during hospitalization for the initial treatment of lung cancer in our department. We examined whether serum NTx levels would be relevant to the prognosis of non-small-cell lung cancer (NSCLC). RESULTS: This study included 176 patients with lung cancer (125 men and 51 women), including 109 with adenocarcinoma, 53 with squamous cell carcinoma, 6 with large-cell carcinoma, and 8 with other cancer types. Univariate and multivariate analysis using the Cox proportional hazards model revealed a particularly close association between sex, performance status, disease stage, and serum NTx levels and overall survival (OS). A median OS of 368 days was observed for patients with a serum NTx level < 22 nmol BCE/L, which was significantly longer than the 197 days for patients with a serum NTx level ≥ 22 nmol BCE/L (hazard ratio [HR], 2.02; 95% confidence interval [CI], 1.36-2.99; log-rank P = .00037). CONCLUSIONS: We have revealed that a high serum NTx level (> 22 nmol BCE/L) appears to be a risk factor for a reduction in OS in patients with NSCLC.