A novel solution for freezing individual spermatozoa using a right angular cryopiece embedded in a grooved petri dish.

Herein, we introduced a novel individual sperm freezing device named SpermCD, which consists of a right angular cryopiece (RA-Cryopiece, or "C") and a grooved petri dish ("D"). SpermCD allows embryologists to transfer sperm and perform ICSI on the same focal plane. Thirty-five patients underwent single sperm cryopreservation using SpermCD, including four patients with non-obstructive azoospermia (NOA), 14 patients with virtual azoospermia and 17 patients with cryptozoospermia. One hundred and twenty-five cryopreserved spermatozoa from nine patients were thawed on the day of the oocyte retrieval and 121 spermatozoa were found, with a sperm recovery rate of 97.1 ± 4.6%. Sixty-five MII oocytes from their spouse were injected with thawed sperm. Normal fertilization and high-quality embryo rates were 68.0% ± 33.2% and 24.4% ± 22.2%. Nineteen transplantable embryos were formed after fertilization with frozen sperm, eight of which were transplanted in five couples, resulting in four successful deliveries. SpermCD is a simple and practical individual sperm freezing device.

Slow freezing should not be totally substituted by vitrification when applied to day 3 embryo cryopreservation: an analysis of 5613 frozen cycles.

PURPOSE: This study aimed to compare slow freezing (SF) and vitrification (VT) techniques for day 3 embryo cryopreservation in infertile couples. METHODS: This retrospective cohort study enrolled 5613 infertile patients, with 7862 frozen-thawed day 3 embryos and 3845 vitrified-warmed day 3 embryos, from 2010 to 2014, at a single center. The rates of embryo survival, pregnancy, implantation, miscarriage, live birth, and live birth weight were compared between the two groups. RESULTS: A total of 5613 cycles with 5520 transfers were analyzed. Using SF, the rates of overall embryo survival and fully intact blastomeres were lower than those in VT (91.5 vs. 97.4 % and 68.7 vs. 92.3 %, respectively). The rate of good quality embryos after thawing/warming was lower in SF than in VT. In single frozen embryo transfer cycles (FETs), the pregnancy and implantation rates were similar between the two groups (35.0 vs. 40.8 % and 34.6 vs. 35.9 %, respectively). In double FETs, the pregnancy rate per cycle was also similar between the groups (58.8 vs. 58.4 %). The implantation rate per embryo transfer was significantly higher with SF than with VT (38.8 vs. 34.6 %). With adjustment for maternal age and the number of good quality embryos, differences in implantation rate remained significant (adjusted P value, SF vs. VT P < 0.05). No independent effect was found for the method of cryopreservation on the pregnancy rate. No significant differences in the rates of miscarriage, live birth, and live birth weight were observed between the two techniques. CONCLUSIONS: Despite the significantly low embryo survival rate, fully intact blastomere rate, and good quality embryo rate in SF, the pregnancy and implantation rates were not adversely affected in single and double FETs. SF yielded an equivalent miscarriage rate, live birth rate, and live birth weight compared with VT. The SF protocol to cryopreserve day 3 embryos still should be considered.

Are testicular sperms superior to ejaculated sperms in couples with previous ART failure due to high rate of fragmented embryos? A retrospective cohort study.

Objective: The aim was to clarify whether using testicular sperm reduces embryo fragmentation and improves cycle outcomes. Methods: Fragmented embryo was defined as an embryo in which fragments account for more than one third of the embryonic surface area. High rate of fragmented embryos was defined by a proportion of fragmented embryos higher than 50%. We recruited infertile couples who had undergone at least one ovarian stimulation cycle using ejaculated sperm but failed to conceive due to high rate of fragmented embryos in each previous cycle. After fully informed consent, the couples agreed to obtain testicular sperm by testicular puncture and use testicular sperm for intracytoplasmic sperm injection (ICSI). The normal fertilization rate, transferable embryo rate, fragmented embryo rate and cycle outcomes were compared between ejaculated sperm group (EJA-sperm group) and testicular sperm group (TESTI-sperm group). Results: Twenty-two couples who agreed to participate in our study underwent 32 ICSI cycles with ejaculated spermatozoa and 23 ICSI cycles with testicular spermatozoa. Embryo transfers were cancelled in 8 ejaculated cycles and 4 testicular cycles because of no transferable embryos. There were no significant differences in age, normal fertilization rate and high-quality embryo rate between ejaculated and testicular groups. The transferable embryo rate and implantation rate in TESTI-sperm group were significantly higher than those in EJA-sperm group (36.9% vs. 22.0%, p < 0.01; 34.2% vs. 0%, p < 0.001). The fragmented embryo rate in TESTI-sperm group was significantly lower than that in EJA-sperm group (61.2% vs. 75.7%, p < 0.05). Conclusion: Our small retrospective cohort study suggests that using testicular sperm may be a recommended option for couples with previous ART failure because of high rate of fragmented embryos. Large samples, multicenter studies or randomized controlled trial (RCT) are needed to further confirm the superiority of testicular sperm.

[Analysis of low-quality embryo transfer strategy in in vitro fertilization and embryo transfer cycle].

OBJECTIVE: To investigate the transfer strategy of low-quality embryo in in vitro fertilization and embryo transfer (IVF-ET) cycle. METHODS: A retrospective analysis was performed for the clinical data of 621 IVF-ET cycles under controlled ovarian hyperstimulation, including 574 fresh embryo transfer (ET) cycles (Group T1) and 47 frozen-thawed embryo transfer (FET) as the first ET cycles (Group C1). Logistic regression was used to model the probability of clinical pregnancy rate based on the cycle-specific factors. RESULTS: The clinical pregnancy rate was significantly higher in Group C1 than Group T1 [38.3% (18/47) vs 22.1% (127/574), P < 0.05]. Multivariate logistic regression analysis revealed that patient age and ET method were significantly associated with the clinical pregnancy rate. After adjusting for patient age, the clinical pregnancy rate remained significantly higher in Group C1 than Group T1 (OR: 2.107, 95%CI: 1.128 - 3.939, P < 0.05). CONCLUSION: The use of FET instead of fresh ET may improve the clinical pregnancy rate in low-quality embryo cycles.

[Risk factors associated with twin pregnancy in double embryo transfer].

OBJECTIVE: To explore the risk factors associated with twin pregnancy in double embryo transfer. METHODS: A retrospective analysis was performed for 2970 double embryo transfer cycles, including 1984 cycles of fresh embryo transfer and 986 cycles of frozen-thawed embryo transfer (FET). Multiple factor Logistic regression was used. The twin pregnancy rate was studied in group of age < 35 or age ≥ 35 years old, in group of zero, single or double high-quality embryos transferred and group of fresh embryo transfer or FET cycles. RESULTS: (1) There was a significantly negative correlation between women's age and twin pregnancy according to the analysis of multiple factor Logistic regression (P < 0.01). And a significantly positive correlation existed between the number of high-quality embryos transferred, FET and twin pregnancy (P < 0.01); (2) the twin pregnancy rate of both fresh embryo transfer and FET cycles was higher in group of age < 35 years than that in group of age ≥ 35 years (16.0% vs 8.0%, P < 0.01; 26.9% vs 14.2%, P < 0.01); (3) the twin pregnancy rate of fresh embryo transfer cycles with double high-quality embryos transferred was higher than that with zero or single high-quality embryos transferred (19.1% vs 5.4%; 19.1% vs 11.0%, P < 0.01); the twin pregnancy rate of FET cycles with double high-quality embryos transferred was also higher than that with zero or single high-quality embryos transferred (32.7% vs 10.8%; 32.7% vs 20.7%, P < 0.01); (4) the twin pregnancy rate of FET cycles was significantly higher than that of fresh embryo transfer cycles (24.7% vs 14.9%, P < 0.01). CONCLUSION: Women's age, the number of high quality embryos transferred and FET are risk factors associated with twin pregnancy. At reproductive centers with a mature technique of FET, we recommend performing single high-quality embryo transfer in FET cycles to reduce the occurrence of twin pregnancy.

CFTR is essential for sperm fertilizing capacity and is correlated with sperm quality in humans.

BACKGROUND: Our previous studies have demonstrated the cystic fibrosis transmembrane conductance regulator (CFTR) is important for capacitation and male fertility in mouse and guinea pig spermatozoa. However, the exact function of CFTR on human sperm fertilizing capacity, and correlation with sperm quality has not been established. The present study may shed light on some unexplained male infertility, and on a possible new method for diagnosis of male infertility and strategy for male contraception. METHODS: To assess the effect of CFTR on human sperm fertilizing capacity, we examined sperm capacitation and the acrosome reaction using chlortetracycline staining, analyzed sperm hyperactivation by computer-assisted semen analysis (CASA), measured intracellular cAMP levels using ElA and evaluated sperm penetration of zona-free hamster eggs assay in fertile men. The percentage of spermatozoa expressing CFTR from fertile, healthy and infertile men (mainly teratospermic, asthenoteratospermic, asthenospermic and oligospermic) was conducted by indirect immunofluorescence staining. RESULTS: Progesterone significantly facilitated human sperm capacitation and ZP3 triggered the acrosome reaction, both were significantly inhibited by CFTR inhibitor-172 (CFTRinh-172; 10 nM-1 microM) in a dose-dependent manner. The presence of 100 nM CFTRinh-172 markedly depressed intracellular cAMP levels, sperm hyperactivation and sperm penetration of zona-free hamster eggs. In addition, the percentage of spermatozoa expressing CFTR in the fertile men was significantly higher than healthy and infertile men categories (P < 0.01). CONCLUSIONS: CFTR is essential for human sperm fertilizing capacity and the impairment of CFTR expression in spermatozoa is correlated with a reduction of sperm quality. These results suggest that defective expression of CFTR in human sperm may lead to the reduction of sperm fertilizing capacity.

[Fertilizing ability, cleavage potential and inheritance risk of globozoospermia].

OBJECTIVE: To explore the fertilizing ability, cleavage potential and inheritance risks of globozoospermia. METHODS: A globozoospermic patient was diagnosed by sperm morphological staining and transmission electron microscope. From his wife the investigators obtained 26 oocytes in which 6 oocytes were donated and the other 19 (in 20) MII oocytes injected into 19 round-headed sperms. Six donated oocytes accepted in vitro fertilization (IVF). This patient's chromosome and microdeletion in AZFa, AZFb and AZFc in Y chromosome were checked through 6 sequence tag sites of sY84, sY86, sY127, sY134, sY254 and sY255. RESULTS: Only 4 (in 19) were normally fertilized and cleaved. All 6 donated oocytes were normally fertilized and cleaved. The fertilizing rate was significantly higher than that of this patient (100% vs 21.1%, P < 0.01). But the cleavage rate has no statistical difference (100% vs 100%, P > 0.05). This patient had normal chromosome (46, XY) and there was no deletion in Y chromosome. His wife became pregnant after accepting two thawed embryos and then gave birth to a boy whose chromosome was normal (46, XY). The acceptor also gave birth to a healthy boy after accepting two thawed embryos. CONCLUSIONS: Despite a lower fertilizing rate, intracytoplasmic sperm injection is still an effective therapy for globozoospermic infertility.

[Comparison between the results of ICSI with fresh and with frozen-thawed sperm obtained by PESA to treat azoospermia].

OBJECTIVE: Retrospective study of the results of ICSI (intracytoplasmic sperm insemination) with frozen sperm obtained by PESA (percutaneous epididymal sperm aspiration) was performed in 27 patients. METHODS: With conventional freezing method, sperm from diagnosing PESA and the remaining motile sperm after treating cycle were frozen. After frozen-thawed and ICSI process, fertilization rate, implantation rate, clinical pregnancy rate were compared and other outcomes including pregnant combinations and parameters of newborns of experimental group (which used frozen-thawed sperm) and control group (which used fresh PESA sperm) were analyzed respectively. RESULTS: One hundred and sixty three and 1 157 oocytes of stage M II were injected respectively in the experimental group (15 cycles) and control group (100 cycles), and fertilization rate of experimental group was prominently higher than that of control group (84.05% vs 73.29%, P < 0.05), while implantation rate and clinical pregnancy rate were of no difference from the control, respectively (23.07% vs 15.73%; 53.33% vs 37.00%, P > 0.05). The differences in newborn's weights between two groups were of no statistical significance (P > 0.05). In the experimental group, eight clinical pregnancies were achieved including 5 live deliveries and 3 ongoing pregnancies, 37 clinical pregnancies including 30 deliveries with only 1 fetal death, 3 ongoing pregnancies and 4 abortions in the control group. Neither vital pregnant combinations nor neonate malformations were found in both groups. CONCLUSION: ICSI using frozen-thawed sperm obtained by PESA is an economic effective and safe method to treat azoospermia. Recovering rates of frozen sperm form PESA should be further increased.

Characteristics of nonylphenol and bisphenol A accumulation by fish and implications for ecological and human health.

Fish populations constitute an important part of aquatic ecosystems. Thus, their accumulation of nonylphenol (NP) and bisphenol A (BPA) may pose risks to ecosystems and human health. This study analyzed the concentrations of NP and BPA in four types of fishes (i.e., wild/farmed freshwater fishes and wild/farmed marine fishes). Wild freshwater fishes contained higher concentrations of NP and BPA than the other three types of fishes. The concentrations of NP in the wild freshwater fishes ranged from 1.01 to 277 µg/kg ww, with bioconcentration factors (BCFs) and biota-sediment accumulation factors (BSAFs) ranging from 74.0 to 2.60 × 10(4)L/kg and from 0.003 to 18.3, respectively. The wild freshwater fishes contained relatively low amounts of BPA, varying from ND to 25.2 µg/kg ww, with the BCFs and BSAFs ranging from 1.00 to 274L/kg and from 0.003 to 3.40, respectively. Five fish species particularly showed high BCFs and BSAFs, indicating that they could be an important source of NP for higher trophic levels, most likely resulting in ecological risks. The demersal fishes showed a greater ability to accumulate NP than the pelagic ones. The fact that the 95th percentile values of the risk quotient (RQ) for NP and BPA were higher than the acceptable threshold indicated that these two compounds would have adverse effects on aquatic organisms in Taiwanese rivers. The consumption of wild marine fishes had the highest 95th percentile values of hazard quotient (HQ) for NP and BPA among the four types of fishes, particularly for the population aged 0-3 years. However, the 95th percentile values of HQ for NP and BPA were all less than 1, suggesting that exposure to NP and BPA through fish consumption posed no remarkable risk to human health in Taiwan.

Intracytoplasmic Sperm Injection Fertilization Rate Does Not Depend on the Proportion of Round-headed Sperm, Small-acrosomal Sperm, or Morphologically Normal Sperm in Patients with Partial Globozoospermia.

BACKGROUND: Generally, intracytoplasmic sperm injection (ICSI) may be the preferable method to treat partial globozoospermia, but whether there exist some correlations between ICSI fertilization rate and the proportion of round-headed sperm or morphologically normal sperm remains open. This study was to explore the correlation between ICSI fertilization rate and the sperm morphology in patients with partial globozoospermia. METHODS: Thirty-four patients diagnosed with partial globozoospermia accepted the following assisted fertilization treatments - 2 cases accepted in-vitro fertilization (IVF) alone, 26 cases accepted ICSI alone, and 6 accepted split IVF/ICSI. Detailed morphological characteristics were described using Diff-Quik rapid staining. Sixty cases accepting IVF or ICSI treatment in our reproductive center were considered as the control group after being matched by relevant criteria. Fertilization rate, embryo quality, embryo implantation rate and clinical pregnancy rate were calculated. RESULTS: Besides very high proportion of round-headed sperm, partial globozoospermia also showed very high proportion of small-acrosomal sperm and very low proportion of morphologically normal sperm. Fertilization rate of IVF (IVF alone plus split IVF) was very low in partial globozoospermia (25.4% ± 17.4%), but ICSI (ICSI alone plus split ICSI) achieved satisfying fertilization rate compared with the control group (66.2% ± 22.5% vs. 68.8% ± 29.4%, P > 0.05). In patients with partial globozoospermia, there were no correlations between ICSI fertilization rate and the proportion of round-headed sperm, small-acrosomal sperm, or morphologically normal sperm. CONCLUSIONS: There was high proportion of small-acrosomal sperm in partial globozoospermia. For patients with partial globozoospermia, ICSI is more preferable than IVF. ICSI fertilization rate does not depend on the proportion of round-headed sperm, small-acrosomal sperm, or morphologically normal sperm.

Expression and Distribution of Calcium-Binding Protein S100P in Human Placenta during Pregnancy.

BACKGROUND: S100P is a member of the S100 family of calcium-binding proteins, and it participates in pathophysiological events, such as tumor growth and invasion. Based on the striking similarities between trophoblast cells and tumor cells with regard to proliferative and invasive properties, we raised the question of whether and how S100P expresses in trophoblast cells during development. This study aimed to investigate the expression pattern of S100P in the human placenta during pregnancy development. MATERIALS AND METHODS: In this experimental study, we collected 16 first-trimester placental tissues, 10 second-trimester placental tissues, and 12 term placentas. The mRNA expression levels of S100P were detected by reverse-transcription-polymerase chain reaction (RT-PCR) and quantitative real-time PCR, the protein expression levels were detected by western blot, and the localization of S100P was measured by immunohistochemical staining. The values obtained from PCR and western blot analysis were expressed as the mean ± SD. Levene's test was used to test equal variances, and one-way analysis of variance (ANOVA) was used to evaluate differences between groups. RESULTS: Protein and mRNA expression of S100P could be detected in placenta during pregnancy, with minor higher levels in first-trimester (p>0.05). Immunohistochemical staining revealed that S100P protein was strongly expressed in syncytiotrophoblasts, and moderate expression was detected in villous cytotrophoblasts and cytotrophoblast columns. The S100P protein was localized to both cytoplasm and nuclei in syncytiotrophoblasts, while it only existed in the cytoplasm of cytotrophoblasts. CONCLUSION: S100P was strongly detected in human placenta during pregnancy. The specific expression and distribution of S100P in human placenta throughout gestation suggested that S100P function might vary with its location in the placenta.

Fertility-preserving treatment and pregnancy outcomes in the early stage of endometrial carcinoma.

OBJECTIVE: This study aimed to review the available literature on fertility-preserving treatment and pregnancy outcomes in patients with early-stage endometrial carcinoma who desired to preserve their fertility. DATA SOURCES: The PubMed database (1992-2012) was searched for the words "conservative "OR" fertility sparing "OR" fertility preserving" AND "endometrial neoplasms" (MeSH). All relevant articles in English and the relevant references were collected. STUDY SELECTION: Data from published articles about fertility-preserving treatment of endometrial cancer, including the response and recurrence rate of conservative treatment, strategies of infertility treatment, pregnancy, and obstetric outcomes, were selected. Data were mainly extracted from 41 studies, which are listed in the reference section of this review. RESULTS: Hormone therapy was the most common method used for early-stage endometrial carcinoma in patients who wished to preserve fertility. Sixty percent of the patients became pregnant after remission of the carcinoma. The percentage of patients who conceived in the assisted reproductive technology group was higher than that of the natural pregnancy group (80.0% vs. 43.2%, P < 0.01). A higher rate of preterm labor and multiple pregnancies was observed in the assisted reproductive technology group than that in the natural pregnancy group. The majority of pregnancies (71.4%) in the assisted reproductive technology group were achieved by in vitro fertilization-embryo transfer. The clinical pregnancy rate of transfer cycles in patients with endometrial carcinoma was 34.1%. CONCLUSIONS: Assisted reproductive technology is a good option in well-selected patients with early-stage endometrial carcinoma who have completed conservative treatment. In vitro fertilization-embryo transfer offers an opportunity to achieve an immediate pregnancy.

The potential role of water quality parameters on occurrence of nonylphenol and bisphenol A and identification of their discharge sources in the river ecosystems.

Nonylphenol (NP) and bisphenol A (BPA) have attracted great attention due to their estrongenic activities and occurrence in different environments. This study investigated concentrations of NP and BPA in water and sediments of 16 major rivers in Taiwan to determine their association with water quality parameters on their distribution. The sources of NP and BPA discharged into river environments were also identified. The results showed that concentrations of NP and BPA were in the range of 0.02-3.94 and 0.01-44.65µgL(-1) in water and 6.59-47797.69 and 0.37-491.54µgkg(-1) dry weight in sediments. High levels of NP and BPA in water and sediments were measured in sampling sites near highly industrialized and urbanized areas. Construction of the sewage system and wastewater treatment plant has decreased these chemicals discharged into Love River. Dilution effects and erosion of surface sediments in the high-flow season resulted in lower concentrations of NP and BPA detected in sediments of most rivers than those in the low-flow season. Occurrence characteristics of NP and BPA in water and sediments were mainly related to organic carbon contents in sediments and several water quality parameters such as dissolved oxygen, pH, concentrations of ammonia-nitrogen and total organic carbon in water. Effluents from NP/BPA-related factories appeared to be the major discharge sources of NP and BPA. Use and waste of BPA-containing products in household activity was the another discharge source of BPA, but handling NP-containing products became an important source of NP only in the low-flow season.

Outcome of conservative treatment and subsequent assisted reproductive technology for patients with early-stage endometrial adenocarcinoma and poor fertility potential.

Conservative treatment with high doses of progestin is an alternative to standard hysterectomy for young patients with early-stage endometrial adenocarcinoma who desire to preserve their fertility. Here we report a patient with well-differentiated early-stage endometrial adenocarcinoma and poor fertility potential who failed to become pregnant in two in vitro fertilization-embryo transfer cycles and suffered a relapse after conservative treatment. This case illustrates that assessment of fertility potential is critical at the time of initial evaluation and conservative treatment planning for patients with endometrial adenocarcinoma.

Three-dimensional cultures of human endometrial cells on Matrigel mimic in vivo morphology.

BACKGROUND: The regulation of endometrial physiology and morphogenesis by the paracrine effectors has been well established using in vivo studies. A more complete understanding of the endometrial function has been delayed due, in part, to a lack of appropriate culture models. In this study, we aimed to simulate the in vivo three-dimensional (3-D) growth pattern of endometrial cells using a 3-D in vitro culture system. METHODS: Isolated endometrial epithelial cells, stromal cells and RL95-2 cells were seeded into culture chambers coated with the extracellular matrix Matrigel and observed using light microscopy. Fluorescence staining and immunohistochemistry were used to assess the morphology. RESULTS: Depending on the culture conditions, epithelial cells and RL95-2 cells formed multicellular structures on Matrigel; stromal cells remained individually distinguishable or grew together to form 3-D lattice-like structures. CONCLUSIONS: Matrigel provided a good microenvironment for culturing endometrial cells. The cells cultured in the Matrigel-coated chambers closely resembled those seen in vivo.

Maintenance of early pregnancy without early hormone support after frozen-thawed embryo transfer in hormone replacement treatment cycles.

OBJECTIVE: To report two successful pregnancies after frozen-thawed ET in discontinued hormone replacement treatment cycles. DESIGN: Case report. SETTING: University hospital. PATIENT(S): Two infertile patients. INTERVENTION(S): Hormone replacement treatment for endometrial preparation and frozen-thawed ET. MAIN OUTCOME MEASURE(S): Ovarian ultrasonography, serial hormone measurements of serum E2 and P, successful pregnancy, and follow-up visit. RESULT(S): One patient discontinued both E2 and P support 2 weeks after ET. The other discontinued estrogen support on the day preceding ET. No spontaneous follicle development was observed by ultrasonography, and no increase in serum P concentration was observed. Both patients became pregnant and achieved deliveries. CONCLUSION(S): These two cases show that maintenance of pregnancy is possible without early hormone support in hormone replacement treatment cycles.