Relationship between periodontal disease and male infertility: A case-control study.

OBJECTIVES: To compare the prevalence of chronic periodontitis between men who had semen abnormalities and those who had normozoospermia through a case-control study. MATERIALS AND METHODS: Male patients who visited the assisted reproduction clinic of a large general hospital and were diagnosed with semen abnormalities were included in the case group. The control group was composed of patients of the same clinic with normozoospermia. The semen analysis included sperm concentration, count and progressive and total motility, which were measured in the laboratory. A questionnaire and clinical periodontal examination were conducted for all participants. Logistic regression was performed to explore the relationship between chronic periodontitis and male infertility. RESULTS: A total of 192 participants were included: 63 participants (32.8%) had some type of semen abnormality (case group), while 129 participants (67.2%) had normozoospermia (control group). The case group had a significantly higher prevalence of moderate/severe periodontitis than the control group (33.3% vs. 17.8%, p = .012). The logistic regression showed that participants who had moderate/severe periodontitis had a greater chance of having semen abnormalities after adjusting for other confounding factors (OR = 3.377, p = .005). CONCLUSIONS: Periodontitis is associated with semen abnormalities and sperm motility in men.

Enhancement of the efficiency of oocyte vitrification through regulation of histone deacetylase 6 expression.

OBJECTIVE: Successful oocyte vitrification (OV) is critical for cryopreservation of the oocytes from female patients with infertility, polycystic ovaries, and gynecologic cancers. Recent evidence suggests that relatively low levels of histone acetylation are critical for maintenance of the maturation capacity of cryopreserved oocytes. However, previous studies have only demonstrated a key role of histone deacetylases (HDAC) 1 and 2 in the cryopreservation of oocytes. METHODS: In this study, we investigated the role of HDAC6 in these settings. We found that mouse oocytes with low HDAC6 levels decreased survival rate, cleavage rate, and blastocyst rate after OV. Bioinformatics analyses were used to predict HDAC6-targeting microRNAs (miRNAs), while the functional binding of miRNAs to HDAC6 mRNA was evaluated by a dual luciferase reporter assay. RESULTS: Among all HDAC6-targeting miRNAs, we detected expression of miR-558, miR-527, and miR-762 in mouse oocytes. Specifically, we found that only miR-762 significantly inhibited protein translation of HDAC6 via binding to the 3'-UTR of the HDAC6 mRNA. Transfection of oocytes with HDAC6 or antisense of miR-762 significantly increased the survival rate, the cleavage rate, and blastocyst rate after OV. CONCLUSION: As a result, our data suggest that induction of HDAC6 levels by miR-762 suppression may improve the current protocol for OV.

Andrographolide disrupts meiotic maturation by blocking cytoskeletal reorganisation and decreases the fertilisation potential of mouse oocytes.

Andrographolide (AG) is a diterpenoid lactone isolated from the stem and leaves of Andrographis paniculata Nees that is used for the effective treatment of infectious diseases in Asian countries. Previous studies have reported adverse effects of AG on female fertility in rodents; however, the underlying mechanisms are unknown. The aim of the present study was to investigate the effects of AG on the IVM of mouse oocytes and their fertilisation potential. Immature oocytes incubated for 6, 14 or 24h in medium containing 5, 10 or 20µM AG showed time- and dose-dependent decreases in maturation rates compared with the control group. Immunostaining revealed that AG exposure disrupted spindle organisation and migration, as well as actin cap formation and cytokinesis. Furthermore, most oocytes exposed to 20µM AG underwent apoptosis, and the few oocytes exposed to 5 or 10µM AG that reached MII exhibited lower fertilisation rates after intracytoplasmic sperm injection. The findings of the present study suggest that AG may disrupt mouse oocyte meiotic maturation by blocking cytoskeletal reorganisation, and may thus have an adverse effect on female fertility.

Incubation of sperm heads impairs fertilization and early embryo development following intracytoplasmic sperm injection (ICSI) by decreasing oocyte activation in mice.

When intracytoplasmic sperm injection (ICSI) is performed in mice, isolation of sperm heads is usually performed prior to injections in order to increase the efficiency of the procedure. Consequently, the isolated sperm heads undergo an inevitable incubation in vitro. However, little is known about the effects of this incubation step on fertilization and embryo development following ICSI. When we incubated sperm heads at 37 °C, there was a significant time-dependent decrease in fertilization and blastocyst formation. Moreover, the DNA integrity of the sperm heads was maintained over 12 h incubation. Using assisted oocyte activation, these defects in fertilization and embryo development were rescued. Taken together, incubation of sperm heads following isolation can affect the oocyte-activating capacity of sperm thereby compromising fertilization and embryo development associated with ICSI.

[Effects of Yupei Qisun Sequential Method of Chinese medicine on correlated indices of repeated implantation failure patients in the fresh cycle].

OBJECTIVE: To study the clinical effects of Yupei Qisun Sequential Method (YQSM, by Shen supplementing and Pi invigorating) of Chinese medicine on correlated indices of repeated implantation failure patients in the fresh cycle. METHODS: Sixty patients with more than three failure cycles of in virto fertilization and embryo transfer (IVF-ET) or intracytoplasmic sperm injection (ICSI) were recruited. They were assigned to the treatment group (treated by IVF/ICSI and Chinese medicine) and the control group (treated by IVF/ICSI alone), 30 in each group. The total dose of gonadotropin (Gn), the days of controlled ovary hyperstimulation (COH), the thickness of endometrium on the day of embryo transplantation, the number of retrieved oocytes, the fertilization number, the embryo number, the high quality embryo number, the pregnancy rate, and the implantation rate were compared. RESULTS: In the treatment group the numbers of embryo and high quality embryo were 7.5 +/- 4.9 and 5.1 +/- 3.2 respectively, which were both higher than those of the control group with significant difference (5.1 +/- 3.2, 3.2 +/- 1.8; P < 0.05). No significant difference existed in aspects of the total numbers of Gn, the days of COH, the thickness of endometrium on the day of embryo transplantation, the numbers of retrieved oocytes, the fertilization number, the pregnancy rate, and the implantation rate between the two groups (P > 0.05). CONCLUSION: YQSM combined with COH could improve the quantity and the quality of embryos, which was promising to increase the accumulative pregnancy rate of RIF patients.

Impact of Maternal Age on Singleton Birthweight in Frozen Embryo Transfer Cycles.

Background: Previous studies have investigated the effect of maternal age on assisted reproductive technology success rates. However, little is known about the relationship between maternal age and neonatal birthweight in frozen embryo transfer (FET) cycles. Whether maternal age influences singleton birthweight in FET cycles remains to be elucidated. Methods: This study was conducted at a tertiary care center, involving singleton live births born to women undergoing frozen-thawed embryo transfer during the period from January 2010 to December 2017. A total of 12,565 women who fulfilled the inclusion criteria were enrolled and grouped into four groups according to the maternal age: <30, 30-34, 35-39, and ≥40 years old. A multivariable linear regression analysis was conducted to reveal the relationship between maternal age and neonatal birthweight with controlling for a number of potential confounders. Results: The highest proportions of low birthweight (LBW, 4.1%), high birthweight (1.2%), preterm birth (PTB, 5.9%), and very PTB (0.9%) were found in the group over 40 years old, but no significant difference was observed among the four groups. Additionally, the 35-39-year-old group had the highest rate of very LBW (0.6%), whereas the 30-34-year-old group had the lowest rate of small for gestational age (SGA, 2.7%). However, multivariate analyses revealed that neonatal outcomes including PTB, LBW, and SGA were similar between the different maternal age groups. Conclusion: Grouping with different maternal age was not associated with mean birthweight and Z-scores of singletons resulting from FET.

[A 10-year survey on birth defects after in vitro fertilization-embryo transfer in Shanghai].

OBJECTIVE: To survey birth defects of neonates conceived by using various types of in vitro fertilization and embryo transfer (IVF-ET) between 1998 and 2007 in Shanghai. METHODS: From 1998 to 2007, 8507 neonates from 6551 pregnancies conceived through assistant reproductive technology (ART) from 7 reproductive medicine center in Shanghai were enrolled in this retrospective study, including Shanghai Ji-Ai Genetics and IVF Institute, Shanghai Jiaotong University School of Medicine affiliated Renji Hospital, Ruijin Hospital, China Welfare Institute International Maternal and Infant Health Hospital, Shanghai First Maternity and Infant Health Hospital, Shanghai the Ninth People's Hospital and the Second Military Medical University affiliated Changhai Hospital. The clinical data about the type and incidence of birth defect were analyzed. Meanwhile, the factors associated with birth defect were investigated, such as various ART, gender, mother age, numbers of gestational sac, the source and quality of sperm. RESULTS: The birth defect rate was 1.08% (92/8507) and the most remarkable malformation occurred in circulation system [34% (31/92)]. The rates of major congenital malformations were 1.21% (34/2799) in fresh conventional IVF-ET, 1.07% (20/1871) in IVF-frozen-thawed embryo transfer (IVF-FET), 1.04% (23/2212) in fresh intracyto plasmic sperm injection (ICSI)-ET and 0.92% (15/1625) in ICSI-FET, which did not show statistical difference (P > 0.05). There was also no significantly different incidence of malformations between 1.12% (49/4371) in male neonates and 1.02% (42/4136) in female neonates (P > 0.05). However, the occurrence of congenital malformation is positively related with maternal age, the rates were 0.84%(41/4884) in mother's age less than 30 years and 1.77% (16/902) in mother' age more than 35 years, which reached statistical difference (P < 0.05). It also showed remarkable different incidence among 0.53% (25/4679) in singletons, 1.59% (57/3576) in twins and 3.97% (10/252) in triplets (P < 0.05). No remarkable difference of malformation rate among sperm sources used in fertilization were observed, including 1.09% (81/7419) in ejaculated sperm, 1.21% (7/579) in percutaneous epididymal aspiration (PESA) and 0.79% (4/509) in donor sperm (P > 0.05). CONCLUSIONS: The overall incidence of birth defect in neonates from ART is similar to those conceived naturally and is not associated with different types of IVF-ET, embryo cryopreservation, sperm sources. However, maternal age and multiple pregnancies confer the higher possibility of birth defect.

Conhecimento, atitude e prática de emergência pré-hospitalar e seus fatores de influência entre cuidadores de idosos: pesquisa transversal / Conocimientos, actitudes y prácticas de emergencias prehospitalarias y sus factores de influencia en cuidadores de personas mayores: estudio transversal / Knowledge, attitude, and practice of pre-hospital emergency and their influencing factors among caregivers for older adults: a cross-sectional survey

Resumo Objetivo Investigar a situação atual e analisar os fatores influenciadores do conhecimento, atitude e prática de emergência pré-hospitalar entre cuidadores de idosos. Métodos Estudo transversal com amostragem por conveniência, conduzido entre dezembro de 2021 e junho de 2022, e seleção de 133 cuidadores de idosos em instituições de longa permanência na província de Guangdong, China, como participantes. Todos receberam um questionário de informações gerais e um questionário de conhecimento, atitude e prática de emergência pré-hospitalar. Na análise dos dados foi utilizada estatística descritiva e inferencial. Resultados As pontuações nas dimensões conhecimento, atitude e prática foram 24,65 ± 4,49, 24,52 ± 4,34 e 24,05 ± 4,67, respectivamente. A análise de regressão mostrou que a idade, o nível de habilidade profissional e a experiência em educação em saúde dos cuidadores foram os principais fatores que influenciaram seu conhecimento de emergência pré-hospitalar. A presença/ausência dos cuidadores na participação direta na emergência pré-hospitalar foi o principal fator de influência na atitude, enquanto o nível educacional e a situação profissional foram os fatores que influenciaram principalmente a prática na emergência pré-hospitalar. Conclusão O atual nível de conhecimento, atitude e prática em emergência pré-hospitalar dos cuidadores de idosos é de baixo a médio. Para os cuidadores chineses, os principais fatores que afetam a implementação da emergência pré-hospitalar são a idade avançada, os baixos níveis de escolaridade, o emprego temporário e as deficiências do sistema de segurança ocupacional.

Resumen Objetivo Investigar la situación actual y analizar factores influyentes de los conocimientos, actitudes y prácticas de emergencias prehospitalarias en cuidadores de personas mayores. Métodos Estudio transversal con muestreo por conveniencia, llevado a cabo entre diciembre de 2021 y junio de 2022. Se seleccionaron 133 participantes cuidadores de personas mayores de instituciones de larga estadía en la provincia de Guangdong, China. Todos recibieron un cuestionario de información general y un cuestionario de conocimientos, actitudes y prácticas de emergencias prehospitalarias. En el análisis de los datos se utilizó estadística descriptiva e inferencial. Resultados El puntaje en la dimensión conocimientos fue 24,65 ± 4,49, en actitudes fue 24,52 ± 4,34 y en prácticas 24,05 ± 4,67. El análisis de regresión demostró que los principales factores que influyeron en los conocimientos de los cuidadores sobre emergencias prehospitalarias fueron la edad, el nivel de habilidad profesional y la experiencia en educación para la salud. La presencia/ausencia de los cuidadores en la participación directa en emergencias prehospitalarias fue el factor principal de influencia en la actitud, mientras que el nivel educativo y la situación profesional fueron los que más influyeron en la práctica de emergencias prehospitalarias. Conclusión El nivel actual de conocimientos, actitudes y prácticas en emergencias prehospitalarias de los cuidadores de personas mayores es de bajo a mediano. En los cuidadores chinos, los principales factores que afectan la implementación de emergencias prehospitalarias son la edad avanzada, los bajos niveles de escolaridad, el empleo temporario y las deficiencias del sistema de seguridad laboral.

Abstract Objective To investigate the status quo and analyze the influencing factors of the knowledge, attitude, and practice of pre-hospital emergency among caregivers for older adults. Methods In this cross-sectional study, 133 caregivers for older adults in Guangdong province, China, nursing homes were selected as survey participants from December 2021 to June 2022 via convenience sampling. All participants were administered a general information questionnaire and a Pre-Hospital Emergency Knowledge, Attitude, and Practice Questionnaire. For data analysis, we used descriptive and inferential statistics. Results The scores on the knowledge, attitude, and practice dimensions were 24.65 ± 4.49, 24.52 ± 4.34, and 24.05 ± 4.67, respectively. Regression analysis showed that the age, professional skill level, and healthcare education experience of the caregivers were the main influencing factors of their pre-hospital emergency knowledge. Additionally, the presence/absence of direct participation in the pre-hospital emergency of the caregivers was the primary influencing factor of attitude, while education level and employment status were the factors mainly influencing pre-hospital emergency practice. Conclusion Caregivers for older adults currently have a low-to-medium level of knowledge, attitude, and practice of pre-hospital emergency. The main factors affecting the implementation of pre-hospital emergency for caregivers in China are their older age, low education levels, temporary employment and imperfect occupational security system.

[Application of fallopian tube embolization before in vitro fertilization and embryo transfer dealing with the hydrosalpinx].

OBJECTIVE: To explore the effectiveness of the fallopian tubes embolization for the hydrosalpinx before in vitro fertilization and embryo transfer (IVF-ET). METHODS: The fallopian tubes embolization was performed on 46 hydrosalpinx patients. First, the hysterosalpingography was performed to show the positions and shape of the hydrosalpinx. Then the hydrops was drawn into the celiac cavity in order to be absorbed after performing fallopian tube recanalization. Finally, transvaginal therapy was performed, putting the embolization microcoils into the fallopian tube through a micro-catheter One month after the fallopian tubes embolization, hysterosalpingography was conducted to check for the effectiveness of the embolization. After 3 months, all the 46 patients received the treatment of IVF-ET. RESULTS: The interventional treatment of 82 fallopian tubes obtained one time success among 46 cases of fallopian tubes embolization. Among them, obvious results were achieved in 72 fallopian tubes, taking up 88% of the total; effective results were seen in 10 fallopian tubes, accounting for 12% of the total. No one was invalid. In the same period, compared with the 91 cases of non-hydrosalpinx as the control group, the embolization group of patients achieved a higher fertilization rate (69%), and clinical pregnancy rate (41%), compared with the control group (63% and 39% respectively), but without a significant difference ( P>0.05). However, the ectopic pregnancy rate (0) and the abortion rate (8%) were significantly lower than the control group (8%, 16% respectively; P<0.05). CONCLUSIONS: Fallopian tubes embolization used in hydrosalpinx treatment before IVF-ET is an innovative approach, simple, safe, economical, with no negative impact on ovarian function. It can significantly increase the clinical pregnancy rate and prevent the occurrence of tubal pregnancy. It is a feasible and effective method.

Association between induced abortion history and later in vitro fertilization outcomes.

OBJECTIVE: To establish an effective and safe clinical fertility strategy by investigating the relationship between abortion history and pregnancy outcomes of in vitro fertilization (IVF) treatment. METHODS: In the present retrospective cohort study, data from IVF treatment cycles performed at a reproductive center in China between October 1, 2014, and October 31, 2015, were assessed. Outcomes were compared between women with a history of induced abortion and those without. RESULTS: There were 1532 IVF treatment cycles included; 454 patients had a history of induced abortion and 1078 did not. The spontaneous abortion rate was significantly higher (30/170 [17.6%] vs 41/420 [9.8%]; P=0.002) and the endometrium was significantly thinner (8.8 ± 1.8 vs 9.7 ± 1.8 cm; P=0.001) among patients with a history of induced abortion compared with those without. In a subgroup analysis of patients with a history of induced abortion, women who had undergone surgical abortions had a lower live delivery rate compared with medical abortions (29/76 [38%] vs 101/378 [27%]; P=0.039). Further, women who had a history of more than two surgical abortions had lower live delivery and clinical pregnancy rates (both P<0.05). CONCLUSION: A history of induced abortion was associated with worse IVF outcomes, especially a history of more than two surgical abortions.

Transcriptome profiling of human oocytes experiencing recurrent total fertilization failure.

There exist some patients who face recurrent total fertilization failure during assisted reproduction treatment, but the pathological mechanism underlying is elusive. Here, by using sc-RNA-seq method, the transcriptome profiles of ten abnormally fertilized zygotes were assessed, including five zygotes from one patient with recurrent Poly-PN zygotes, and five zygotes from a patient with pronuclear fusion failure. Four zygotes with three pronuclear (Tri-PN) were collected from four different patients as controls. After that, we identified 951 and 1697 significantly differentially expressed genes (SDEGs) in Poly-PN and PN arrest zygotes, respectively as compared with the control group. KEGG analyses indicated down regulated genes in the Poly-PN group included oocyte meiosis related genes, such as PPP2R1B, YWHAZ, MAD2L1, SPDYC, SKP1 and CDC27, together with genes associated with RNA processing, such as SF3B1, LOC645691, MAGOHB, PHF5A, PRPF18, DDX5, THOC1 and BAT1. In contrast, down regulated genes in the PN arrest group, included cell cycle genes, such as E2F4, DBF4, YWHAB, SKP2, CDC23, SMC3, CDC25A, CCND3, BUB1B, MDM2, CCNA2 and CDC7, together with homologous recombination related genes, such as NBN, XRCC3, SHFM1, RAD54B and RAD51. Thus, our work provides a better understanding of transcriptome profiles underlying RTFF, although it based on a limited number of patients.

Observation of the influences of diosgenin on aging ovarian reserve and function in a mouse model.

BACKGROUND: The aim of this study was to investigate the impact of diosgenin, an important monomer of sapogenins in yams, on ovarian reserve in a natural aging mice model. STUDY DESIGN: This randomized controlled trial included 60 9-month-old C57 naturally aging female mice. Twenty-one mice were assigned to the dio group and were fed a single dose of diosgenin (200 mg/kg/day) suspended in 0.3% CMC. Twenty mice were assigned to the DHEA group and were fed a single dose of DHEA (1.25 mg/kg/day) suspended in 0.3% CMC. The remaining 20 mice were assigned to the old control group and were fed a single dose of 0.3% CMC. Three months later, the reproductive performance of these female mice was determined by evaluating ovarian follicles and oocyte number and quality in IVF and comparing age-matched and young controls. The impact of NOBOX, GDF9 and BMP15 mRNA expression was also evaluated. RESULTS: Diosgenin improves ovarian reserve in naturally aging mice in terms of increasing the number of primary follicles (P < 0.05) and serum levels of AMH (P < 0.05). CONCLUSIONS: Diosgenin could counteract age-associated ovarian dysfunction by improving the ovarian reserve in a natural aging mice model.

Elevated progesterone on the trigger day does not impair the outcome of Human Menotrophins Gonadotrophin and Medroxyprogesterone acetate treatment cycles.

To demonstrate the incidence and effects of elevated progesterone (P) on the trigger day on the outcome of in-vitro fertilization (IVF)/intracytoplasmic sperm injection (ICSI) cycles using Medroxyprogesterone acetate (MPA) co-treated with Human Menotrophins Gonadotrophin (hMG + MPA), we performed a retrospective analysis including 4106 IVF/ICSI cycles. The cycles were grouped according to the P level on the trigger day: <1 ng/mL, between 1-1.5 ng/ml (including 1), between 1.5-2 ng/mL (including 1.5), and ≥2 ng/mL. The primary outcome measure was live birth rate. The prevalence of P level categories was 12.93% (531/4106), 2.92% (120/4106), and 1.92% (79/4106) in women with P between 1-1.5 ng/mL, between 1.5-2 ng/mL, and ≥2 ng/mL, respectively. The mean stimulation duration, total hMG dose, serum follicle stimulating hormone (FSH), estrogen(E2) on the trigger day and the number of oocytes in patients with elevated P were significantly higher than patients with P < 1 ng/mL (P < 0.05). However, there were no significant differences in the oocyte retrieval rates, fertilization rates, implantation rates, clinical pregnancy rates and live birth rates between the groups based on frozen embryo transfer (FET). We concluded that elevated P on the trigger day had no negative effect on the final outcome of the hMG + MPA treatment cycles based on FET.

Clinical observations of sequential therapy with Chinese medicine and hysteroscopic mechanical stimulation of the endometrium in infertile patients with repeated implantation failure undergoing frozen-thawed embryo transfer.

OBJECTIVES: To investigate the clinical effect of sequential therapeutic intervention Yupei Qisun [compensating for weakness by invigorating Kidney (Shen) and Spleen (Pi) in advance] in Chinese medicine (CM) and hysteroscopic endometrial mechanical stimulation on the treatment of infertile patients with repeated implantation failure (RIF); and to study the differences in patients' endometrial thickness and type on the day of embryo transfer, serum hormone levels on embryo transfer day and clinical pregnancy outcomes. METHODS: In the clinical study, 168 frozen-thawed embryo transfer (FET) cycles for couples with RIF conforming to the research protocol were randomly divided into three groups: a CM group with 56 cycles (CM combined with FET), a hysteroscopy group with 55 cycles (hysteroscopic endometrial mechanical stimulation), and a control group with 57 cycles (conventional FET). Differences in endometrial thickness on the embryo transfer day, levels of serum estradiol (E2) and progesterone (P) on the embryo transfer day, the E2/P ratio on the embryo transfer day, biochemical and clinical pregnancy rates, implantation rate, abnormal pregnancy rate and other indices were compared among the three groups. RESULTS: Endometrial thickness, E2 and P levels, and the E2/P ratio on embryo transfer day and other factors had no significant differences among groups. The biochemical pregnancy, clinical pregnancy, and implantation rates of the CM and hysteroscopy groups were significantly higher than the control group (P<0.05), and there were no significant differences between these two groups. The abnormal pregnancy rate had no significant difference among the three groups. CONCLUSIONS: Sequential therapy of Yupei Qisun could significantly improve the clinical outcomes of rif-fet cycles, being equivalent to hysteroscopic endometrial mechanical stimulation, and provided a reliable method to treat such infertile couples.

Protective effect of quercetin on the development of preimplantation mouse embryos against hydrogen peroxide-induced oxidative injury.

Quercetin, a plant-derived flavonoid in Chinese herbs, fruits and wine, displays antioxidant properties in many pathological processes associated with oxidative stress. However, the effect of quercetin on the development of preimplantation embryos under oxidative stress is unclear. The present study sought to determine the protective effect and underlying mechanism of action of quercetin against hydrogen peroxide (H2O2)-induced oxidative injury in mouse zygotes. H2O2 treatment impaired the development of mouse zygotes in vitro, decreasing the rates of blastocyst formation and hatched, and increasing the fragmentation, apoptosis and retardation in blastocysts. Quercetin strongly protected zygotes from H2O2-induced oxidative injury by decreasing the reactive oxygen species level, maintaining mitochondrial function and modulating total antioxidant capability, the activity of the enzymatic antioxidants, including glutathione peroxidase and catalase activity to keep the cellular redox environment. Additionally, quercetin had no effect on the level of glutathione, the main non-enzymatic antioxidant in embryos.

A novel method for cryopreservation of individual human spermatozoa.

The purpose of this study is to develop a novel method for the cryopreservation and efficient post-thaw recovery of individual or small numbers of human spermatozoa. Spermatozoa equilibrated in cryoprotectant buffer were injected with an intracytoplasmic sperm injection (ICSI) needle into a droplet of cryoprotectant on a homemade cryoleaf. The droplet was of cryoprotectant and seminal plasma at a ratio of 1:1. The sperm-loaded cryoleaf was slowly lowered over and stored in liquid nitrogen. Spermatozoa were thawed in a 37°C oil bath without dilution and centrifugation. To test the fertilizing ability of these spermatozoa, the recovered spermatozoa were injected by ICSI into 1-d-old or in vitro-matured human oocytes. Fresh spermatozoa from the same semen samples served as controls. The trials were performed in two separate experiments. In the first set of experiments, 92 spermatozoa were thawed and carefully investigated. The spermatozoa from percutaneous epididymal sperm aspiration had a motility recovery of 92.9% (13/14); ejaculated spermatozoa had a motility recovery of 61.5% (48/78), and only 1.3% (1/78) was lost. Together in the first and second set of experiments, the fertilization rates for the fresh and frozen-thawed spermatozoa were 67.6% (25/37) and 60.6% (40/66), respectively (P = 0.052). The mean embryo cleavage rates in the fresh and frozen-thawed groups were 88% (22/25) and 85% (34/40), respectively (P = 0.990). This cryopreservation method for individual or small numbers of human spermatozoa was efficient and simple. These findings make this method a promising technique for the clinical application of ejaculated sperm from oligozoospermic patients.