Healthy births and ongoing pregnancies obtained by preimplantation genetic diagnosis in patients with advanced maternal age and recurrent implantation failure

Preimplantation genetic diagnosis (PGD) and subsequent embryo development was evaluated in 72 couples presenting at our centre for intracytoplasmic sperm injection (ICSI) due to severe male factor. The embryo biopsies were performed in Ca(2+)/Mg(2+)-free medium. These patients were further divided into those with advanced maternal age (AMA, n = 49) and those with recurrent implantation failure (RIF, n = 23). Fluorescence in-situ hybridization (FISH) was carried out on 329 blastomeres (91.3%) with probes for the X, Y, 13, 18 and 21 chromosomes. The chromosomal abnormality rate was 41.3% with no significant difference between the AMA and RIF groups. Aneuploidy accounted for the majority (72.8%) of chromosomal abnormalities. Out of 329 embryos, 84.2% had cleaved after 24 h and 15.1% had arrested. Embryos were transferred in 70 patients and 22 pregnancies were achieved (31.4% with an ongoing pregnancy rate of 28.5%). There were no significant differences between the pregnancy rates of the AMA and RIF groups (32.5 and 30% respectively). Therefore PGD should be offered to patients with AMA and RIF. Furthermore, the use of Ca(2+)/Mg(2+)-free medium during the blastomere biopsy facilitates the procedure, while further embryo cleavage, ongoing pregnancies and healthy births are possible.     

A father of four consecutive trisomic pregnancies with elevated frequencies of associated aneuploid sperm

A couple with normal somatic karyotypes had four consecutive trisomic pregnancies, each involving a different chromosome, of which two children were liveborn with confirmed paternal-origin trisomies. The apparently healthy father produced abnormally high frequencies of disomic sperm for each of the four chromosomes involved in the trisomic pregnancies (P< 0.003, by sperm fluorescence in situ hybridization (FISH)). His elevated sperm aneuploidies persisted over a 2-year period and affected all chromosomes evaluated, suggesting that he had a genome-wide defect in meiotic disjunction. He also had the highest frequencies of aneuploid sperm reported for any healthy man to date. His frequencies of aneuploid sperm were comparable to the peak frequencies of the transient responses reported in some cancer patients after receiving aneugenic chemotherapies. These findings indicate that apparently healthy men can produce abnormally high frequencies of sperm aneuploidies that suggest that this condition may contribute to recurrent trisomic pregnancies.     

Testicular sperm retrieval and cryopreservation prior to initiating ovarian stimulation as the first line approach in patients with non-obstructive azoospermia.

The potency for fertilization and successful implantation was compared between fresh and cryopreserved testicular spermatozoa obtained from the same patient with non-obstructive azoospermia. Spermatozoa cryopreserved at the outset were also evaluated. Non-obstructive azoospermic men (n = 55) underwent testicular sperm extraction (TESE); mature spermatozoa were found in 33 (60%) of them. Of 57 intracytoplasmic sperm injection (ICSI) cycles in 25 patients, 15 used fresh spermatozoa (14 patients, group 1), 24 used the excess spermatozoa cryopreserved after 'fresh' ICSI (11 couples who did not conceive in the 'fresh' cycle, group 2) and 18 cycles used cryopreserved spermatozoa at the outset (11 other patients, group 3). Fertilization, cleavage, embryo quality, implantation and take home baby rates were not significantly different in groups 1 and 2, and 6/14 couples ultimately had healthy babies (42.8% cumulative take home baby rate per TESE). In group 3, neither the fertilization rate, embryo development, pregnancy nor implantation rates per embryo transfer were significantly different from groups 1 and 2. The cumulative delivery and ongoing pregnancy rate in this group was 36. 4%. Cryopreservation did not impair the availability of motile spermatozoa for ICSI. When immotile spermatozoa were injected, however, fertilization rate decreased dramatically. Since criteria for predicting the presence of spermatozoa in the testicular tissue of patients with non-obstructive azoospermia are inadequate, it is suggested that TESE be performed prior to initiating ovarian stimulation.     

The effects of cryopreservation on sperm morphology, motility and mitochondrial function

BACKGROUND: The effects of cryoinjury were determined simultaneously on the mitochondrial function, motility, morphology and viability of ejaculated human sperm. METHOD: Rhodamine 123 (R123) uptake (% of sperm) and stain intensity were used to determine sperm mitochondrial activity before and after cryopreservation from the semen of 50 men attending for infertility investigation. Morphology was assessed using Tygerberg's strict criteria and viability was assessed by eosin Y. Sperm motility was measured using computer-assisted semen analysis (CASA). RESULTS: Freeze-thawing caused a 37% (P = 0.001) reduction in normal morphological forms of sperm. All CASA sperm motility parameters except amplitude of lateral head displacement were similarly reduced. R123 uptake and intensity within sperm mitochondria decreased by 36 and 47% respectively (both P = 0.001). In addition, there was a similar significant decrease (31%, P = 0.001) in the viability of the sperm. CONCLUSIONS: Sperm morphology, motility, mitochondrial activities and viability are equally susceptible to cryopreservation-induced damage. R123 intensity is a novel and robust indicator of mitochondrial function before and after such trauma.     

The value of sperm pooling and cryopreservation in patients with transient azoospermia or severe oligoasthenoteratozoospermia.

BACKGROUND: A transient state of azoospermia may occur due to toxic, environmental, infectious or iatrogenic conditions. Finding sperm in the ejaculate of such patients is often unpredictable and may be critical in IVF treatment. In the present study, the approach of pooling and cryopreservation of sperm is evaluated. Cryopreservation was performed in a unique group of patients in whom no sperm had been found in at least one previous sperm examination and in patients diagnosed as suffering from non-obstructive azoospermia in whom, occasionally, sperm were found. METHODS: A total of 157 semen pooling and cryopreservation procedures in 53 patients was performed between January 1998 and December 2000 in our centre. Forty five of these patients underwent an IVF-ICSI treatment during the study period. In 32 patients, fresh sperm were used to perform ICSI. In 13 patients no sperm were available, and the previously frozen sperm were used. RESULTS: Using our pooling system, 13 IVF-ICSI cycles were rescued. In seven patients with a previous testicular biopsy due to azoospermia, sperm cryopreservation was possible. Overall, 13 pregnancies (10 deliveries, two ongoing pregnancies and one missed abortion) were achieved. CONCLUSION: The introduction of semen banking for patients with transient azoospermia may increase the chance of pregnancy using their own sperm.     

Terminology associated with vitrification and other cryopreservation procedures for oocytes and embryos

Over the past 30 years many cryopreservation procedures have been applied to oocytes, embryos, sperm, ovarian and testicular tissue. Over this time many, often specialized, terms have been developed for all aspects of these procedures. This can make it difficult for readers who are not familiar with the terminology or protocols to compare and evaluate different procedures. This paper describes the main cryopreservation procedures, the terminology associated with them, and briefly explains the underlying physical, chemical and biological processes. The aim is to help readers understand and interpret other papers on slow cooling, rapid cooling, ultrarapid cooling and vitrification.     

Andrology: Successful fertilization and establishment of pregnancies after intracytoplasmic sperm injection in patients with globozoospermia

Globozoospermia or round-headed spermatozoa is a rare type ofteratozoospermia where the acrosome is absent resulting in maleinfertility with no known therapy. A few studies have shownthat round-headed spermatozoa cannot bind to or penetrate thezona pellucida, and no normal fertilization has been observedin in-vitro fertilization (IVF) after insemination of humanoocytes with round-headed spermatozoa. In this study, the fertilizationcapacity of round-headed spermatozoa after intracytoplasmicsperm injection (ICSI) into human oocytes has been examined.In pre-clinical experiments, 45 oocytes were injected; 41 oocyteswere intact after injection, 15 oocytes were fertilized normally,and 13 of these 15 oocytes developed further in vitro. ICSIwas carried out in 11 treatment cycles of seven infertile coupleswith globozoospermia. Normal fertilization and embryo transferoccurred in four cycles (three patients). Positive serum humanchorionic gonadotrophin was observed in three cycles (two patients);one patient had a pre-clinical abortion and the other patientbecame pregnant twice: the first pregnancy was ectopic and thesecond pregnancy is a twin pregnancy which is currently at 16weeks of gestation.     

Fertilization and early embryology: Ongoing pregnancies and birth after intracytoplasmic sperm injection with frozen--thawed epididymal spermatozoa

In seven patients who did not become pregnant following microsurgicalepididymal sperm aspiration (MESA) and intracytoplasmic sperminjection (ICSI), a subsequent ICSI was performed using previouslycryopreserved supernumerary epididymal spermatozoa without re-operatingon the husband. During the original MESA procedure a mean spermconcentration of 12.3x10⁶/ml was achieved. The supernumeraryspermatozoa were cryopreserved for later use. After thawingfrozen epididymal spermatozoa a mean concentration of 1.9x10⁶spermatozoa/ml was obtained in straws containing a total volumeof sperm suspension of 250 µl. From 68 intact oocytesinjected with frozen—thawed epididymal spermatozoa, atwo pronuclear fertilization rate of 45% and a cleavage rateof 82% were obtained. A total of 17 embryos were replaced inthe seven patients, resulting in two ongoing singleton pregnanciesand one twin delivery. Six embryos were cryopreserved. In conclusion,it would appear mandatory to cryopreserve supernumerary spermatozoaduring a MESA in order to avoid subsequent further scrotal surgery.     

First report of management and outcome of pregnancies associated with hereditary orotic aciduria

Two pregnancies in a 25-year-old woman with hereditary orotic aciduria who was managed prenatally on uridine therapy are described. The first pregnancy resulted in an infant with multiple congenital anomalies and a 47, xx, inv(4)(p12q25), + der(22)t(11;22)(p23;q11) karyotype. The proposita was found to be a carrier of a de novo 11;22 translocation and a pericentric inversion of chromosome 4. Subsequently, several carriers of orotic aciduria in this family were identified with the inverted chromosome 4. The second pregnancy resulted in a normal male with an inverted chromosome 4.     

Outcome of first and repeated testicular sperm extraction and ICSI in patients with non-obstructive azoospermia

BACKGROUND: It is unclear whether or not testicular sperm extraction (TESE) should be repeated for patients in whom no sperm were found during their first TESE attempt. METHODS AND RESULTS: The outcome of repeated TESE was evaluated in patients with non-obstructive azoospermia (NOA) after failing to obtain sperm in their first extraction attempt, or having used all available cryopreserved testicular tissue. Out of 83 patients with NOA, patients repeated TESE two (n = 22), three (n = 8), four (n = 6) and five (n = 3) times. Distribution of main testicular histology included germ cell aplasia (55%), maturation arrest (29%) and germ cell hypoplasia (16%). The first TESE yielded mature sperm for ICSI in 39% of patients (sp+), and failed in the remaining 61% (sp-). A second TESE yielded mature sperm in 1/4 from the sp- group and in 16/18 from the sp+ group. At the third, fourth and fifth trials, 8/8, 5/6 and 3/3 of the original sp+ patients were sp+ again respectively. Compared with the outcome of the first trial, all further trials did not differ statistically in the rate of fertilization (54 versus 49%), implantation (9.5 versus 5.4%), or clinical pregnancy/cycle (19 versus 15%). No pregnancies were achieved among the three patients after their fifth TESE. Pregnancies occurred in all histological groups, except maturation arrest. CONCLUSIONS: The outcome of repeated TESE cycles, up to the fourth trial, justifies the procedure.     

The first case of familial Mediterranean fever associated with renal amyloidosis in Korea

Familial Mediterranean fever (FMF) is an auto-inflammatory disease characterized by periodic episodes of fever and recurrent polyserositis. It is caused by a dysfunction of pyrin (or marenostrin) as a result of a mutation within the MEFV gene. It occurs mostly in individuals of Mediterranean origin; however, it has also been reported in non-Mediterranean populations. In this report, we describe the first case of FMF in a Korean child. As eight-year-old boy presented recurrent febrile attacks from an unknown cause, an acute scrotum and renal amyloidosis. He also showed splenomegaly, lymphadenopathy, pleural effusion, ascites and elevated acute phase reactants. After MEFV gene analysis, he was diagnosed as FMF combined with amyloidosis.     

The length of the fertile window is associated with the chance of spontaneously conceiving an ongoing pregnancy in subfertile couples

BACKGROUND: The period in each menstrual cycle during which sexual intercourse can result in conception is called the "fertile window". Although the fertile window closes on the day of ovulation, little is known about the moment it opens. We defined the first day of normal sperm-mucus interaction as the opening of the fertile window. We hypothesized that length of the fertile window varies between couples and that the number of days the fertile window is "open" is related to the time to spontaneous conception. METHODS Serial post-coital tests and sperm-mucus penetration tests were performed to detect the first normal sperm-mucus interaction day. Ovulation was confirmed by serial ultrasound. Using Cox' regression analysis, we determined whether the fertile window length was associated with time to ongoing pregnancy. This association was expressed in fecundability ratios (FR). RESULTS The fertile window length was determined in 410 subfertile couples. The fertile window length varied among couples from <1 to >5 days. The FR increased with increasing fertile window length and varied between 0.11 (95% CI: 0.03-0.45) for a fertile window of 1 day, to 2.4 (95% CI: 1.1-5.2) for a fertile window of 5 days or more. CONCLUSIONS The longer the fertile window in subfertile couples, the higher is the probability of spontaneously conceiving an ongoing pregnancy.     

Villous explant culture using early gestation tissue from ongoing pregnancies with known normal outcomes: the effect of oxygen on trophoblast outgrowth and migration

BACKGROUND: Early placental and embryo development occur in a physiologically low oxygen environment, with a rise in oxygen tension within the placenta towards the end of the first trimester. Oxygen is implicated in the regulation of trophoblast differentiation and invasion. This study examined the effects of oxygen tension on extravillous trophoblast outgrowth and migration from normal pregnancies free of significant pathology. METHODS: Early gestation villous tissue (11-14 weeks gestation), obtained by chorionic villus sampling, was cultured in 3 or 20% oxygen. Maternal and fetal outcomes were ascertained for all samples. The frequency and amount of trophoblast outgrowth and migration from villi were measured for up to 192 h. RESULTS: Significantly fewer explants produced outgrowths in 3% compared with 20% oxygen. The number of sites of trophoblast outgrowth and the extent of migration were also significantly less in 3% compared with 20% oxygen. In vitro hypoxia/reoxygenation further reduced trophoblast growth compared with 3% oxygen alone. HLA-G expression in extravillous trophoblasts was not affected by oxygen tension, with HLA-G positive extravillous trophoblasts being universally Ki67 negative. CONCLUSION: Human placental villi and extravillous trophoblasts in the late first trimester of pregnancy are sensitive to oxygen tension, with low oxygen inhibiting extravillous trophoblast outgrowth and migration.     

急性期精神分裂症缓解后的认知功能及相关因素

目的:考察首发精神分裂症患者急性期临床缓解率,并且探索认知和临床因素对急性期临床缓解的预测作用.方法:选取符合美国精神障碍诊断与统计手册第4版(DSM-Ⅳ)精神分裂症诊断标准的55名门诊和住院首发患者,给予利培酮(2 ～6 mg/d)、奥氮平(10～20mg/d)或阿立哌唑(10 ～ 30mg/d)治疗8周,分别在基线、第4周末和第8周末使用阳性与阴性症状量表(PANSS)评估患者的疾病严重程度和疗效,基线期使用霍普金斯词语学习测验修订版(HVLT-R)、词语流利测验(VFL)、范畴流利测验(VFC)、彩色连线测验(CTT-1和CTT-2)、STROOP色词测验(SCWT)、威斯康星卡片分类测验(WCST-PE和WCST-CC)来评估与记忆和执行功能相关的认知功能指标,使用SCID筛查来确定未治疗精神病期(DUP).8周末满足PANSS量表的8个核心条目评分均小于或等于3的患者被认为“缓解”.结果:共有30名(54.5％)患者在治疗8周末达到临床缓解.单因素分析表明,达到缓解的患者具有较短的未治疗精神病期(DUP)[(8.1±7.7)vs.(17.0±12.1),P＜0.01],较高的HVLT-R分数[(20.4±6.6)vs.(19.1±6.1),P＜0.05]和较少的阴性症状[(20.5±7.4)vs.(25.0±5.5),P＜0.05].多元logistic回归分析表明,HVLT-R得分高(OR=1.15)和DUP较短(OR =0.91)是临床缓解的独立预测因素.结论:在基线期记忆功能较好和未治疗精神病期较短的患者更易达到缓解,未发现临床缓解与本研究所用抗精神病药种类、剂量的相关性.     

Familial deletion within NLGN4 associated with autism and Tourette syndrome

Neuroligin 4 (NLGN4) is a member of a cell adhesion protein family that appears to play a role in the maturation and function of neuronal synapses. Mutations in the X-linked NLGN4 gene are a potential cause of autistic spectrum disorders, and mutations have been reported in several patients with autism, Asperger syndrome, and mental retardation. We describe here a family with a wide variation in neuropsychiatric illness associated with a deletion of exons 4, 5, and 6 of NLGN4. The proband is an autistic boy with a motor tic. His brother has Tourette syndrome and attention deficit hyperactivity disorder. Their mother, a carrier, has a learning disorder, anxiety, and depression. This family demonstrates that NLGN4 mutations can be associated with a wide spectrum of neuropsychiatric conditions and that carriers may be affected with milder symptoms.