Results of lifestyle modification promotion and reproductive/general health check for male partners of couples seeking conception.

Purpose: Male infertility is partially caused by an inappropriate lifestyle and comorbidities. In this study, we analyzed the prevalence of these factors and the effects of lifestyle modifications as part of male preconception care. Methods: Four hundred and two male partners of couples seeking conception with abnormal parameters upon the first semen analysis were enrolled. They were advised to modify their inappropriate lifestyle as male preconception care. Afterward, their general and male reproductive health was examined. Semen quality was compared before and after the promotion. Results: Smoking, chronic alcohol use, and genital heat stress were found in 22.6%, 47.0%, and 75.1% of patients, respectively. Palpable varicoceles, hypogonadism, obesity (body mass index ≧30 kg/m2), hypertension, zinc deficiency, hyperlipidemia, liver dysfunction, and diabetes mellitus were found in 25.9%, 17.0%, 7.0%, 14.9%, 16.2%, 37.0%, 26.9% and 3.4% of the participants, respectively; 98.8% of the patients had at least one factor. After the promotion, semen parameters and sperm DNA fragmentation were improved significantly. Improvement was found in those with palpable varicocele or hypogonadism but not in those with night work shift, abstinence (>3 days), erectile dysfunction, hypertension, obesity, zinc deficiency, or diabetes mellitus. Conclusions: Comorbidities and inappropriate lifestyle choices were common among men with infertility. The promotion of lifestyle modifications as part of male preconception care could improve semen quality without urologic intervention.

A case of discrepancy between three ERA tests in a woman with repeated implantation failure complicated by chronic endometritis.

BACKGROUND: Endometrial receptivity array (ERA) is used to determine the timing of embryo transfer (ET) synchronized with the window of implantation (WOI). The effectiveness and evaluation of ERAs in women with recurrent implantation failure remain controversial. We report the case of a patient with recurrent implantation failure that raises the issue of reproducibility of ERA tests. CASE REPORT: A 36-year-old Japanese woman with secondary infertility who had previously given birth failed to conceive after three frozen-thawed embryo transfer (FET) cycles. An ERA test was conducted to confirm the WOI. The first ERA test was performed 125 h after progesterone exposure. The laboratory reported that the endometrium was in a non-receptive (post-receptive) phase, and recommended retesting 101 h after progesterone exposure. A simultaneous chronic endometritis (CE) test showed a score of 3. After the antibiotics administration to treat CE, the second ERA test was performed after 101 h of progesterone exposure. The laboratory reported that the endometrium had not reached the WOI and estimated the WOI to be 113 ± 3 h after progesterone exposure. The third ERA test was performed 113 h after progesterone exposure. The laboratory reported that the endometrium was in a non-receptive (pre-receptive) phase and estimated the WOI to be 137 ± 3 h after progesterone exposure. A CE test performed at the same time as the second and third ERA tests showed a score of 1 for the collected endometrium. According to the third ERA test results, the vitrified-warmed blastocyst was transferred at 137 h of progesterone exposure. Pregnancy was achieved and the patient had an uncomplicated vaginal delivery at 39 weeks. One year later, another pregnancy was achieved after FET at 137 h of progesterone exposure, and the patient delivered at 33 weeks due to an unexpected membrane rupture. CONCLUSION: Because the results of the ERA test may vary in the presence of CE, CE should be diagnosed simultaneously with or before conducting ERA tests. If CE is diagnosed, ERA testing should be performed after treatment with antimicrobials or other drugs.

PIEZO-ICSI increases fertilization rates compared with standard ICSI: a prospective cohort study.

RESEARCH QUESTION: Is PIEZO-intracytoplasmic sperm injection (ICSI) coupled with a new novel operational fluid (perfluoro-n-octane) superior to standard ICSI? DESIGN: A cohort of patients (n = 69) undertaking microinjection were recruited between January and November 2019 and were then prospectively case-matched. Patients required six or more mature oocytes for inclusion in the study. PIEZO-ICSI uses high-speed microinjection drilling to penetrate the zona and oolemma and deposit the spermatozoa into the cytoplasm, compared with the traditional 'cutting' action of ICSI. The primary outcome was fertilization, with secondary outcomes including oocyte degeneration, abnormal fertilization, embryo cryopreservation and embryo utilization. RESULTS: PIEZO-ICSI resulted in significantly higher fertilization rates (80.5 ± 2.4% vs 65.8 ± 2.3%, P < 0.0001) and lower oocyte degeneration rates (4.4 ± 1.3% vs 8.6 ± 1.2%, P = 0.019) and abnormal fertilization rates (2.9 ± 1.1% vs 7.4 ± 1.1%; P = 0.003) compared with standard ICSI. This improvement in fertilization was of most benefit in patients aged ≥38 years. This increase in fertilization increased the number of good quality embryos that were available for cryopreservation/transfer (3.8 ± 0.2 vs 3.1 ± 0.2; P = 0.038), such that patients on average had one extra usable embryo per cycle compared with standard ICSI. There were no differences to Day 5 embryo development or clinical pregnancy from fresh embryo transfer (57.1% PIEZO-ICSI vs 60.0% ICSI) between microinjection methods, although pregnancy outcomes were underpowered. CONCLUSIONS: PIEZO-ICSI significantly increased fertilization rates, thereby increasing the number of embryos available for cryopreservation compared with standard ICSI. Further prospective studies assessing cumulative pregnancy rates are warranted.

Piezo-ICSI for Human Oocytes.

Since the first successful pregnancies achieved by intracytoplasmic sperm injection (ICSI) were reported, ICSI has become an essential technique in assisted reproductive technology (ART). ICSI uses micropipettes with a spiking tip to penetrate the zona pellucida and membrane. Then, the cytoplasm is usually aspirated into the micropipette for membrane breakage (conventional-ICSI). The survival and fertilization rates of mouse oocytes after conventional-ICSI were as low as 16% and 8%, respectively. Kimura and Yanagimachi applied a piezo drive unit, mercury, and a micropipette with a flat tip for mouse ICSI. The membrane breakage could be performed semi-automatically by combining these types of equipment without cytoplasmic aspiration into the micropipette (piezo-ICSI). These authors reported significantly higher survival and fertilization rates (80% and 78%) compared to those of conventional-ICSI (16% and 8%). Therefore, the piezo-ICSI may be effective not only for mouse oocytes but also for human oocyte ICSI. However, only five papers are available that assessed the effectiveness of piezo-ICSI compared to conventional-ICSI for human oocytes. All of these five papers reported significantly higher fertilization rates compared to those of conventional-ICSI. The goal of the piezo-ICSI protocol described here is to improve the clinical results of ICSI compared to the conventional-ICSI.

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.

Effects of sperm direction in Piezo-ICSI on oocyte survival, fertilization, embryo development and implantation ability in humans: A preliminary study.

AIMS: The objective of this study was to investigate the effect of the head-first or tail-first injection of sperm into the cytoplasm by Piezo-ICSI (PICSI) on oocyte survival, fertilization, embryo development and implantation ability in humans. METHODS: We retrospectively investigated 632 mature oocytes retrieved from 152 infertile patients who attended our PICSI-ET program at the Niji Clinic between October 2010 and January 2014. Of these, 342 mature oocytes retrieved from 75 patients were injected with sperm head first, and 290 mature oocytes retrieved from 77 patients were injected with sperm tail first into the cytoplasm. The rates of oocyte survival, fertilization, good-quality day-3 embryos, pregnancy, implantation and live birth were evaluated in both groups. RESULTS: There were no differences among the two groups with respect to survival, fertilization, good-quality day-3 embryos, pregnancy, implantation and live birth rates. CONCLUSION: Sperm direction (i.e., head first or tail first) does not influence the outcome of PICSI in human oocytes, including oocyte survival, fertilization, embryo development and implantation ability. These findings contribute to an understanding of factors that influence the success of human intracytoplasmic sperm injection (ICSI) techniques.

Erratum to: Clinical efficiency of Piezo-ICSI using micropipettes with a wall thickness of 0.625 µm.

Erratum to: J Assist Reprod Genet (2015) 32:1827­1833, DOI 10.1007/s10815-015-0597-9. The authors would like to apologize for accidentally making a mistake in the inner and outer diameter calculation of the Piezo-ICSI micropipettes.

Clinical efficiency of Piezo-ICSI using micropipettes with a wall thickness of 0.625 µm.

PURPOSE: The purposes of the present study are to assess the clinical efficiency of Piezo-intracytoplasmic sperm injection (ICSI) and to improve the Piezo-ICSI method for human oocytes. METHODS: We examined three ICSI methods to determine their clinical efficiency by comparing the survival, fertilization, good-quality day-3 embryo, pregnancy, and live birth rates. The three ICSI methods tested were conventional ICSI (CI) (using beveled spiked micropipettes with a wall thickness of 1 µm), conventional Piezo-ICSI (CPI) (using flat-tipped micropipettes with a wall thickness of 0.925 µm), and improved Piezo-ICSI (IPI) (using flat-tipped micropipettes with a wall thickness of 0.625 µm). We collectively investigated 2020 mature oocytes retrieved from 437 patients between October 2010 and January 2014. RESULTS: The survival rates after CI, CPI, and IPI were 90, 95, and 99 %, respectively. The fertilization rates after CI, CPI, and IPI were 68, 75, and 89 %, respectively. The good-quality day-3 embryo rates after CI, CPI, and IPI were 37, 43, and 55 %, respectively. The pregnancy rates after the transfer of good-quality day-3 embryo of CI, CPI, and IPI were 19, 21, and 31 %, respectively. The live birth rates of CI, CPI, and IPI were 15, 16, and 25 %, respectively. Significantly higher survival, fertilization, good-quality day-3 embryo, pregnancy, and live birth rates were obtained using IPI. CONCLUSIONS: When comparing the IPI to the CI and CPI, the results revealed that the Piezo-ICSI using flat-tipped micropipettes with a wall thickness of 0.625 µm significantly improves survival, fertilization, good-quality day-3 embryo, pregnancy, and live birth rates.

Impact of the size of zona pellucida thinning area on vitrified-warmed cleavage-stage embryo transfers: a prospective, randomized study.

PURPOSE: The aim of this study was to determine if the size of zona pellucida thinning area by laser assisted hatching could affect the rates of pregnancy and implantation for vitrified-warmed embryo transfers at the cleavage-stage. METHODS: A total of 120 vitrified-warmed cleavage-stage embryo transfers were randomly assigned to either quarter or half of zona pellucida thinning group. RESULTS: The rates of clinical pregnancy (46.7 versus 25.0%) and implantation (32.0 versus 16.2%) were significantly greater in the half thinning group than in the quarter thinning group (P = 0.0218 and P = 0.0090, respectively). CONCLUSIONS: The results of this investigation show that, in vitrified-warmed embryo transfers at the cleavage-stage, the size of zona pellucida thinning area by laser assisted hatching impacts the rate of clinical pregnancy and implantation and that half of zona pellucida thinning significantly increases both of these results compared with quarter of zona pellucida thinning.

Case report: successful delivery following the transfer of a human re-vitrified day-7 spontaneously hatched blastocyst developed from vitrified cleaved embryos.

PURPOSE: To report a successful delivery after the transfer of a re-cryopreserved day-7 hatched blastocyst. METHODS AND RESULTS: A 30-year-old woman underwent a long-treatment protocol for ovarian stimulation. Fourteen mature oocytes were obtained, and twelve were fertilized. On day 3, two cleaved embryos were transferred, but no implantation occurred. The remaining embryos were vitrified. Subsequently, two vitrified day-3 embryos were transferred. The woman became pregnant and delivered a healthy baby. Subsequently, two vitrified day-3 embryos were transferred, but no pregnancy occurred. Subsequently, all the remaining vitrified day-3 embryos were cultured. On day 5, no blastocyst was obtained. The remaining embryos were continued to be cultured. On day 7, a hatched blastocyst was obtained and re-vitrified. Subsequently, the re-vitrified day-7 hatched blastocyst was transferred. The woman became pregnant and delivered a healthy female. CONCLUSIONS: The day-7 hatched blastocyst developed from vitrified embryos can be re-vitrified and have pregnancy potential after re-warming.

Perinatal outcomes following transfer of human blastocysts vitrified at day 5, 6 and 7.

Little information is available on perinatal outcome of cryopreserved day-7 blastocyst transfer. In the present report, perinatal outcomes for transfers of cryopreserved blastocysts by a vitrification method were examined with respect to the day of blastocyst expansion among on day 5, 6 or 7 before cryopreservation. We investigated 263 cycles of vitrified-warmed blastocyst stage embryo transfer performed between April 2005 and April 2009, which were reviewed retrospectively. There were 144 cycles with day-5 blastocyst, 100 cycles with day-6 blastocyst, and 19 cycles with day-7 blastocyst transfers. There were no differences among the vitrified day-5, day-6 and day-7 blastocyst transfer groups regarding mean number of embryos transferred, pregnancy rate, implantation rate and miscarriage rate. At this time, 71 deliveries have occurred with no reported abnormalities. There were 47 infants from 41 deliveries with day-5 blastocyst, 26 infants from 23 deliveries with day-6 blastocyst, and 8 infants from 7 deliveries with day-7 blastocyst. There were no differences among the three groups in the mean gestational age, preterm delivery rate and mean birth weight. These results indicated that blastocysts have similar inherent viability regardless of whether they develop by day 5, 6 or 7.

Vitrified human day-7 blastocyst transfer: 11 cases.

This case report describes successful pregnancies after vitrification of human day-7 blastocysts. A total of 16 day-7 blastocysts were vitrified and warmed. All 16 blastocysts survived after warming and were transferred to 11 patients. Six of the women (55%) became clinically pregnant and the implantation rate was 44% (7/16). Among these women, one woman delivered a healthy baby, two pregnancies ended in miscarriage, and three pregnancies are ongoing at 10, 29 and 34 weeks of gestation. This is the first report of successful pregnancies after vitrification of human day-7 blastocysts.

Case report: two successful pregnancies following the transfer of re-vitrified human day 7 blastocysts developed from vitrified cleaved embryos.

PURPOSE: To report successful pregnancies after the transfer of re-vitrified human day 7 blastocysts developed from vitrified cleaved embryos. METHODS AND RESULTS: A total of five day 7 blastocysts developed from vitrified cleaved embryos were re-vitrified and re-warmed. All of five re-vitrified day 7 blastocysts (100%) survived after warming and were transferred to three patients. Two of the women became clinically pregnant. Of these women, one woman delivered a healthy baby and the other pregnancy is ongoing at 26 weeks of gestation. CONCLUSIONS: This is the first report of successful pregnancies after the transfer of re-vitrified human day 7 blastocysts developed from vitrified cleaved embryos.

Effect of the size of zona pellucida opening by laser assisted hatching on clinical outcome of frozen cleaved embryos that were cultured to blastocyst after thawing in women with multiple implantation failures of embryo transfer: a retrospective study.

PURPOSE: To evaluate the effect of the size of zona pellucida opening by laser assisted hatching for frozen cleaved embryo that were thawed after both fresh and frozen cleaved embryo transfer cycles failed and were cultured to blastocyst after thawing in patients with multiple implantation failures. MATERIALS AND METHODS: Of 101 consecutive procedures (October 2003 to June 2006), 30 patients declined to perform assisted hatching and were selected as control group, 40 patients had 40 microm opening of the zona (October 2003 to January 2005), 31 patients had 50% of the zona opening (February 2005 to June 2006). RESULTS: The pregnancy, implantation and delivery rates were significantly higher in 50% opening group (74%, 52% and 65%) compared to control group (17%, 10% and 13%; P < 0.01) and 40 microm opening group (43%, 27% and 38%; P < 0.04). CONCLUSIONS: The size of the zona pellucida opening may affect the outcome of frozen cleaved embryo transfer.

Zona pellucida removal and vitrified blastocyst transfer outcome: a preliminary study.

The objective of this study was to investigate whether a change in assisted hatching technique from partial opening to total removal of the zona pellucida improved the outcome of vitrified blastocyst transfer. This was a preliminary observational study conducted from November 2003 to April 2006. Partial opening using acid Tyrode's solution was performed in 45 cycles, while total removal using a laser and mechanical pipetting was performed in 57 cycles. The clinical pregnancy, implantation, and delivery rates were higher in the total removal group than in the partial opening group (67% versus 42%, P < 0.02; 55% versus 30%, P < 0.01; 56% versus 36%, P < 0.04, respectively). These results suggest that total removal of the zona pellucida is associated with higher pregnancy, implantation and delivery rates compared with partial opening for vitrified blastocyst transfer.

Vitrification of human hatched blastocysts: a report of 4 cases.

BACKGROUND: Vitrification is a fairly well-established technique for cryopreservation of human zona-intact blastocysts. However, little is known about the efficacy of the vitrification technique for zona-free hatched blastocysts. CASE: A total of 4 hatched blastocysts from 4 healthy, infertile women undergoing in vitro fertilization were vitrified and warmed. All 4 hatched blastocysts expanded after warming and were transferred to 4 patients. Three of the women became clinically pregnant. Two healthy infants were born in 2 deliveries, and 1 pregnancy had progressed to 35 weeks at this writing. CONCLUSION: Our results suggest that vitrification is a useful cryopreservation technique not only for zona-intact blastocysts but also for zona-free hatched blastocysts.

Successful dizygotic twin pregnancy after recryopreservation by vitrification of human expanded blastocysts developed from frozen cleaved embryos on day 6: a case report.

BACKGROUND: Vitrification has evolved into an established technique for cryopreservation of human blastocysts. However, it is still unclear whether the blastocysts developed from frozen embryos can be cryopreserved a second time by vitrification for further embryo transfer. CASE: A 31-year-old woman underwent a long-treatment protocol for ovarian stimulation. Twenty-seven mature oocytes were obtained, and 21 were fertilized with intracytoplasmic sperm injection. On day 3, 2 cleaved embryos were transferred, but no implantation occurred. The remaining 19 embryos were cryopreserved with the slow freezing method. Three months after oocyte retrieval, 5 frozen day 3 embryos were thawed, the surviving 2 were transferred, but no implantation occurred. Six months after oocyte retrieval, the remaining 14 frozen day 3 embryos were thawed and the surviving 12 cultured. On day 5, 2 embryos reached the expanded blastocyst stage and were transferred, but no implantation occurred. On day 6, 5 of the nontransferred embryos became expanded blastocysts and were cryopreserved again by vitrification. Eight months after oocyte retrieval, 2 recryopreserved day 6 blastocysts were warmed and transferred. Implantation resulted in a dizygotic twin pregnancy. The pregnancy resulted in delivery of normal, healthy male and female infants weighing 2,155 and 2,590 g at birth, at 36 weeks of gestation. CONCLUSION: The blastocysts developed from frozen embryos on day 6 can be recryopreserved by vitrification and have pregnancy potential after warming.

Blastocoele collapse by micropipetting prior to vitrification gives excellent survival and pregnancy outcomes for human day 5 and 6 expanded blastocysts.

BACKGROUND: Manual puncture of the trophectoderm of human blastocysts with a needle before vitrification increases their survival rate, but the embryos take a long time to re-expand. This study examined whether causing human blastocysts to collapse by manual pipetting before vitrification would allow more rapid re-expansion and improve pregnancy rates. METHODS: After embryo transfer in IVF cycles, surplus embryos that developed to the expanded blastocyst stage were placed in cryoprotectant and then artificially shrunk by mechanical pipetting with a fine hand-drawn glass pipette slightly smaller in diameter than the blastocyst. The shrunken embryos were placed in a small volume of vitrification solution and plunged into liquid nitrogen on a cryotop. The blastocysts were thawed by warming and then dilution in 1 mol/l sucrose. RESULTS: Of 49 expanded vitrified blastocysts, 48 (98%) re-expanded within 3 h after warming. Following transfer (48 blastocysts in 28 cycles), 14 women (50%) became clinically pregnant, and the implantation rate was 33% (16/48). Eight healthy babies have been born in six deliveries, and the other eight pregnancies are ongoing. To date, there have been no spontaneous abortions. CONCLUSIONS: The results suggest that artificial shrinkage with pipetting is a simple and effective technique to assist successful cryopreservation of expanded blastocysts by vitrification.

Case report: successful pregnancy after vitrification of a human blastocyst that had completely escaped from the zona pellucida on day 6.

This case report describes a successful pregnancy after vitrification of a human hatched blastocyst. A 31-year-old woman, after failed stimulated and thaw cycles, underwent short-treatment protocol stimulation, and oocytes were recovered transvaginally with ultrasound guidance. Eight mature oocytes were obtained and six were fertilized with conventional IVF. Consecutive embryo transfer was performed, in which two cleaved embryos were transferred on day 3 and a single blastocyst was transferred on day 5, but no implantation occurred. On day 6, one of the non-transferred embryos developed into a blastocyst that had completely escaped from the zona pellucida. The zona-free hatched blastocyst was vitrified using a cryotop procedure after artificial shrinkage, which in our clinical experience has proved to be effective for zona-intact blastocysts. Six months after the previous retrieval cycle, the cryopreserved hatched blastocyst survived the warming process and was transferred to the patient's uterus. Implantation resulted in a healthy pregnancy; the pregnancy is ongoing at 33 weeks. This is the first report of a pregnancy after vitrification of a human blastocyst that had completely escaped from the zona pellucida.