A multi-centre, retrospective case series of oocyte cryopreservation in unmarried women diagnosed with haematological malignancies.

STUDY QUESTION: Is oocyte cryopreservation an applicable option for fertility preservation in unmarried patients with haematological malignancies? SUMMARY ANSWER: Oocyte cryopreservation via the vitrification method is accessible and may be considered an option for fertility preservation in unmarried patients with haematological malignancies. WHAT IS KNOWN ALREADY: Haematological malignancies are most commonly observed amongst adolescent and young adult women. Although the survival rate and life expectancy of those with haematological malignancies have improved, chemotherapy and radiotherapy may impair their reproductive potential. Oocyte cryopreservation is thus an ideal option to preserve their fertility. STUDY DESIGN SIZE DURATION: This study retrospectively evaluated 193 unmarried patients (age: 26.2 ± 0.4 years) with haematological malignancies, who consulted for oocyte cryopreservation across 20 different fertility centres in Japan between February 2007 and January 2015. The primary outcome measures were the oocyte retrievals and oocyte cryopreservation outcomes. The secondary outcome measures were the outcomes following oocyte warming for IVF. PARTICIPANTS/MATERIALS SETTING METHODS: The patients had commenced ovarian stimulation cycles via antagonist, agonist, natural and minimal methods for oocyte retrievals, defined according to the treatment strategy of each respective fertility centre. A vitrification method using the Cryotop safety kit was used for oocyte cryopreservation. ICSIs were used for insemination of warmed oocytes. The endometrial preparation method for embryo transfer was hormonal replacement therapy, except in the case of a patient who underwent a spontaneous ovulatory cycle. MAIN RESULTS AND THE ROLE OF CHANCE: Among 193 patients, acute myeloid leukaemia (n = 45, 23.3%) was most common, followed by acute lymphoid leukaemia (n = 38, 19.7%) and Hodgkin's lymphoma (n = 30, 15.5%). In total, 162 patients (83.9%) underwent oocyte retrieval, and oocytes were successfully cryopreserved for 155 patients (80.3%). The mean number of oocyte retrieval cycles and cryopreserved oocytes were 1.7 ± 0.2 and 6.3 ± 0.4, respectively. As of December 2019, 14 patients (9.2%) had requested oocyte warming for IVF. The survival rate of oocytes after vitrification-warming was 85.2% (75/88). The rates of fertilisation and embryo development were 80.0% (60/75) and 46.7% (28/60), respectively. Ten patients (71.4%) had successful embryo transfers, and seven live births (50.0%) were achieved. LIMITATIONS REASONS FOR CAUTION: This study was limited by its retrospective nature. Additionally, there remains an insufficient number of cases regarding the warming of vitrified oocytes to reliably conclude whether oocyte cryopreservation is effective for patients with haematological malignancies. Further long-term follow-up study is required. WIDER IMPLICATIONS OF THE FINDINGS: Oocyte retrieval and oocyte cryopreservation were accessible for patients with haematological malignancies; however, the number of oocyte retrievals may have been limited due to the initiation of cancer treatments. Acceptable embryonic and pregnancy outcomes could be achieved following oocyte warming; therefore, our results suggest that oocyte cryopreservation can be considered an option for fertility preservation in patients with haematological malignancies. STUDY FUNDING/COMPETING INTERESTS: This research received no specific grant from any funding agency in the public, commercial or not-for-profit sectors. The authors declare no conflict of interest. TRIAL REGISTRATION NUMBER: N/A.

Comparison of Residual Dimethyl Sulfoxide (DMSO) and Ethylene Glycol (EG) Concentration in Bovine Ovarian Tissue During Warming Steps Between Slow Freezing and Vitrification.

BACKGROUND: DMSO and EG have been used as cryoprotectants for human ovarian tissue cryopreservation, but residual cryoprotectants concentration and safety have rarely been reported. OBJECTIVE: We aimed to compare residual cryoprotectants (DMSO, EG) concentration in bovine ovarian tissue during warming steps between one kind of common slow freezing method and two kinds of vitrification methods, which are usually used for cryopreservation of human ovarian tissue in Japan. MATERIALS AND METHODS: In this study, we used five bovine ovaries with an average age of 24.2 months divided into three kinds of cryopreservation methods. All ovarian cortices cut to 1 mm thickness were cryopreserved in slow freezing and two kinds of vitrification methods. Residual cryoprotectants before, during and after warming of cryopreserved ovarian cortices were measured using GC-MS and compared. RESULTS: Concentrations of residual cryoprotectants in the ovarian tissue just before transplantation into the body after warming were high after both vitrification methods but almost zero with the slow freezing method. CONCLUSION: We are concerned about the residual cryoprotectants in ovarian tissue, and continue to study the safety of cryopreservation methods to the woman after reimplantation and her baby.

A transportation network for human ovarian tissue is indispensable to success for fertility preservation.

PURPOSE: The purpose of this study was to examine the efficacy of an ovarian tissue transportation network for fertility preservation (FP) for cancer patients in Japan. METHODS: PubMed was searched for papers on transportation of human ovarian tissue for FP. We analyzed population, area, number of cancer patients for ovarian tissue cryopreservation (OTC), quality control/assessment and safety, cost of a cryopreservation center for the building for 30 years, and medical fees of cancer patients (operation, cryopreservation, and storage of ovarian tissue). RESULTS: More than twenty babies have been born in Denmark and Germany through a transportation system. Up to 400 new patients a year need OTC. The fees for removal, cryopreservation, and storage for 5 years, and transplantation of ovarian tissue are around €5,000, €4,000, and €5,000, respectively. It costs more than €5 million to establish and maintain one cryopreservation center for 30 years. If we have a few cryopreservation centers in Japan, we can cryopreserve 400 patients' ovarian tissue per year by safer slow freezing and maintain quality control/assessment. We need to lighten the patients' burden for easy to use FP by a government subsidy and medical insurance coverage. CONCLUSIONS: This model has been termed the Danish model ("the woman stays - the tissue moves"). This is truly patient-centered medicine. We can have maximum effects with the minimum burden. A transportation network like those of Denmark and Germany is the best strategy for FP in Japan. It may be the best system for cancer patients, medical staff, and the Ministry of Health, Labor, and Welfare.

Pregnancy achieved following ICSI from a man with Klinefelter's syndrome and spinal cord injury.

Klinefelter's syndrome and spinal cord injury are major causes of male infertility. Intracytoplasmic sperm injection (ICSI) is a relatively new method of assisted reproduction. A testicular biopsy was obtained from a patient with the double complications of non-mosaic 47,XXY Klinefelter's syndrome and spinal cord damage, and motile spermatozoa were collected. ICSI was then performed. Of the four sperm-injected oocytes, three became fertilized and cleaved. Two embryos were implanted, resulting in a single pregnancy with visible evidence of a heartbeat appearing at 6 weeks gestation. The pregnancy is now entering its 20th week. To the best of our knowledge, this is the first case of a pregnancy resulting from the sperm of a patient with double complications.

Efficiency of using frozen-thawed testicular sperm for multiple intracytoplasmic sperm injections.

PURPOSE: To compare fertilization and pregnancy rates of fresh and frozen-thawed testicular sperm injections (TESE-ICSI). METHODS: Sperm collected from the testes of 28 azoospermic patients by an open testicular biopsy technique was used for initial ICSI or cryopreserved. RESULTS: Fresh-sperm ICSI treatment (28 cycles) resulted in a 58.1% fertilization rate and a 32.1% clinical pregnancy rate per embryo transfer, while frozen-thawed sperm (24 subsequent cycles) had rates of 54.5 and 29.2%, respectively. The PR was lower using frozen-thawed sperm from nonobstructive azoospermia patients (9.1%) than from obstructive azoospermia patients (46.2%). PR declined to 0% upon the fourth ICSI attempt. CONCLUSIONS: Fertilization, embryo cleavage, and pregnancy rates were unaffected by fresh or frozen-thawed sperm use. A 57.1% cumulative clinical PR was achieved using the latter. The PR was significantly lower using frozen-thawed sperm from nonobstructive azoospermia patients than from obstructive azoospermia patients.

Clinical results of fetal obstructive uropathy treated by vesicoamniotic shunting.

OBJECTIVES: To report the clinical results of 5 fetuses after a vesicoamniotic shunting procedure (VASP). METHODS: Between 1995 and 1998, 5 patients (two with prune belly syndrome, one with a cloacal anomaly, one with urethral stenosis, and one with a sacrococcygeal teratoma) underwent VASP using a double-basket catheter. We used the following selection criteria: a fetus without chromosomal defects, with oligohydramnios, and with a predicted good renal function from sequential or single fetal urinalysis (sodium concentration, chloride concentration, and osmolarity at less than 100 mEq/L, less than 90 mEq/L, and less than 210 mOsm, respectively). RESULTS: The mean gestational age was 20.8 +/- 6.9 weeks at diagnosis, 24.2 +/- 6.0 weeks at VASP, and 30.6 +/- 6.2 weeks at delivery. In our study, 2 of 5 patients survived. One of the patients with prune belly syndrome was 18 months old at last follow-up, with hydrocephalus and a creatinine level of 0.2 mg/dL. The patient with the cloacal anomaly was 4 years old at last follow-up and had signs of clonic convulsion. However, psychomotor development was delayed in both. Of the 3 patients who died, 2 died after birth, and the autopsy determined death was due to pulmonary insufficiency. The patient with urethral stenosis died in utero. CONCLUSIONS: Although the principal purpose of VASP is to prevent pulmonary hypoplasia and dysfunctional kidneys, VASP was not always effective, as the outcomes were poor in most of our patients. A greater standardization of patient selection and a large cohort study in the future should be considered to assess VASP.

Detection of hepatitis C virus helicase activity using the scintillation proximity assay system.

The C-terminal two-thirds of the nonstructural protein 3 (NS3) of hepatitis C virus (HCV) possesses RNA helicase activity. This enzyme is considered to be involved in the viral replication and is expected to be one of the target molecules of anti-HCV drugs. The conventional method for the measurement of RNA helicase activity includes the step of gel electrophoresis which makes the screening of multiple samples inconvenient. In this study, to establish a high-throughput screening system for HCV helicase inhibitors, we applied the scintillation proximity assay (SPA) system to the detection of this enzymatic activity. We could detect the helicase activity using the NS3 protein purified by an immunoaffinity column. The activity was dependent on the concentration of the enzyme and the reaction time. The RNA helicase activity measured by the SPA system was in a good correlation with that obtained by the conventional method. Furthermore, the SPA system showed better reproducibility and less deviation of the data than the conventional method, which makes the former suitable for quantitative analysis. Since any separation step is not required and microtiter plates can be used in this method, it has the advantage of dealing with multiple samples.

Cloning and characterization of Zymomonas mobilis genes encoding extracellular levansucrase and invertase.

The genes encoding the extracellular levansucrase and invertase of Zymomonas mobilis have been cloned and sequenced. The levansucrase gene, sucZE2, spans 1269 bp and encodes an M(r) 46,790 polypeptide, and the invertase gene, sucZE3, is of 1239 bp and encodes an M(r) 46,110 polypeptide. The 5'-terminal sequences of both genes corresponded to the N-terminal amino acid sequences of the secreted levansucrase and invertase, implying that the secretion of both enzymes does not involve proteolytic processing of the N-terminals. Both enzyme molecules appear to carry no typical N-terminal secretion signal. Significant homology between sucZE2 and sucZE3 was observed, but both genes showed no homology to the gene encoding an intracellular invertase coexisting in Z. mobilis. Two genes, sucZE2 and sucZE3, are possibly placed in an operon because the expression of two genes were simultaneously controlled by the regulator gene zliE, previously identified.

A study of the fertilizing capacity of human spermatozoa: the hamster test.

There are many reports on the hamster test as a test of the fertilizing capacity of human spermatozoa, and its usefulness is widely known. However, the results obtained cannot be simply compared because of the different conditions used for the test. According to our studies, the type of serum albumin present in incubated specimens was considered to affect particularly the rate of sperm penetration. Penetration rates of human spermatozoa were determined under the most appropriate test conditions using good-quality human serum albumin. The penetration rate was 69 +/- 9% (range: 33-98%) in men whose fertility had been confirmed within 2 years before test, while it was 34 +/- 5% (range: 0-100%) in infertile men, showing a significantly low rate. Eight of nine men who achieved fertilization within one year after the examination showed a penetration rate of over 30%. The remaining one showed a rate of 16%, but he underwent surgery for varicocele and treatment with methylcobalamin and Hochuekkito after the test. Men thus seem difficult to conceive women when the penetration rate of their spermatozoa is below 30%.

Relationship between follicular fluid steroid concentrations and in vitro fertilization.

Steroid concentrations were measured in follicular fluid obtained from patients fertilized in vitro. Progesterone and estradiol-17 beta concentrations showed positive correlations with the growth of follicles, whereas the testosterone concentration had negative correlations not only with follicle growth but also with progesterone and estradiol-17 beta concentrations. The testosterone concentration was significantly lower in follicles with cleaved oocytes than in those with uncleaved oocytes or those with cleaved but degenerated oocytes. After the luteinizing hormone (LH) surge, the fluid contained significantly higher progesterone and lower testosterone concentrations than after human chorionic gonadotropin (hCG) injection. These results indicate that steroid concentrations vary with the growth of follicles, and that the oocyte that can cleave well tends to be associated with a low testosterone concentration. Furthermore, the LH surge seems to provide a better hormonal condition for cleavage than does hCG injection.

[The influence of various molecules on the occurrence of the human sperm acrosome reaction].

The acrosome reaction is essential for fertilization, but the mechanism of the acrosome reaction of human spermatozoa is not clear at the present time. We studied the mechanism to analyze the cause of unexplained infertility, the appropriate timing of insemination, and the environment of spermatozoa prior to fertilization. For this study, we examined the effects of Ca++, Mg++, Kallikrein, Phospholipase A2, p-bromophenacyl bromide (Phospholipase A2 specific inhibitor), Lysophosphatidyl choline, Arachidonic acid, and Glyceryl monooleate using in vitro penetration assay employing zona- free hamster eggs. Results obtained were as follows. When human spermatozoa were incubated in mBWW with Ca++ or (and) Mg++ free medium, the acrosome reaction was inhibited. When human spermatozoa were incubated in mBWW with Kallikrein (1.0-4.0 KU ml), the acrosome reaction was promoted. When Phospholipase A2 was used at concentrations of 0.2 and 2.0 unit/ml, penetration rates showed the same tendency as in the control. But when p-bromophenacyl bromide was tested at concentrations of 1 X 10(-5) - 1 X 10(-3)M, penetration rates were inhibited when compared with the control. When human spermatozoa were incubated in medium containing Lysophosphatidyl choline (50 micrograms/ml), Arachidonic acid (5-50 micrograms/ml), and Glyceryl monoleate (300-400 micrograms/ml), the acrosome reaction was accelerated.

[Effects of the addition of serum albumin on penetration of human spermatozoa into zona-free hamster eggs].

Since Yanagimachi et al. (1976) suggested that human spermatozoa were capable of penetrating into zona-free hamster eggs, this in vitro assay has been used to analyse the fertilizing ability of human spermatozoa. Serum albumin is an important constituent of the medium used for the assay. However, a great variation in the rate of sperm penetration was observed in the use of different albumin preparations at different concentrations. Therefore we examined the effects of three different kinds of albumin preparations on the rate of human sperm penetration into zona-free hamster eggs. The results obtained were as follows. The percentages of eggs being penetrated by spermatozoa from three fertile donors A,B, and C, were assessed. When Fraction V, Globulin Free and Fatty Acid Free albumin preparations were tested at a concentration of 3.5% (W/V) by the assay using sperm from donor A, penetration rates were 13.3%, 97.4%, and 8.7% respectively. Dilution of the albumin concentration to 0.3% considerably changed the penetration rates to 64.4%, 78.8% and 12.1% in that order. In cases B and C, penetration rates showed the same tendency as in case A. alpha 1 and alpha 2 globulin fractions contaminated in the Fraction V preparation possibly inhibit the human sperm penetration into zona-free hamster eggs. An appropriate quantity of fatty acid is necessary for human spermatozoa to penetrate into zona-free hamster eggs, because penetration rates were low in the percentage of Fatty Acid Free albumin regardless of the concentration added. It is concluded that use of the same preparation of good quality is mandatory for human sperm penetration tests using zona-free hamster eggs to evaluate the results with reproducibility and accuracy.

Effects of phospholipase A2, lysophosphatidyl choline, and fatty acid on the acrosome reaction of human spermatozoa.

An in vitro penetration assay employing zona-free hamster eggs was used to study the effects of phospholipase A2, lysophosphatidyl choline, and fatty acid on the acrosome reaction of human spermatozoa. Human spermatozoa were preincubated for 4 hr in modified Biggers, Whitten, and Whittingham's medium (mBWW) containing a specific phospholipase A2 inhibitor, p-bromophenacyl bromide (p-BPB: 1 X 10(-5) - 1 X 10(-3) M), lysophosphatidyl choline (LC: 5-500 micrograms/ml), and arachidonic acid (AA: 5-500 micrograms/ml), prior to the addition of zona-free superovulated hamster eggs. Eggs were examined microscopically 2 or 4 hr later for evidence of swelling or decondensing sperm heads in the cytoplasm. Lysophosphatidyl choline increased penetration rates of spermatozoa from 43.2% (control) to 91.4% (LC: 50 micrograms/ml). Arachidonic acid also increased penetration rates from 51.6% (control) to 87.0% (AA: 5 micrograms/ml) and 80.5% (AA: 50 micrograms/ml). p-BPB decreased penetration rates from 90.6% (1% dimethyl sulfoxide) to 16.0% (1% dimethyl sulfoxide+p-BPB 1 X 10(-4) M). These results suggest that endogenous phospholipase A2 may break membrane phosphatidyl choline into lysophosphatidyl choline and fatty acid, when the acrosome reaction occurs.

Induction of ovulation with an estrogen antagonist, tamoxifen.

Tamoxifen (Norvadex), synthesized by the Imperial Chemical Industries Ltd., is a triphenylethylene derivative having a clomiphene-like structure and displays anti-estrogenic activities. In this study we used tamoxifen for the treatment of anovulatory infertility and investigated the clinical results. Induction rate of ovulation recorded 100% in the patients with sporadic anovulatory cycle, 83.3% in those with persistent anovulatory cycle, 70% in those with the first grade amenorrhea type I and 66.7% in those with the first grade amenorrhea type II. However, tamoxifen was absolutely ineffective against the second grade amenorrhea. The patients with sporadic or persistent anovulatory cycle responded to 40 mg-daily tamoxifen-treatment, recording 100% of ovulatory induction rate. The patients with the first grade amenorrhea also responded to 60 mg-daily treatment, indicating 70% of ovulatory induction rate. The rate of pregnancy established in the women desiring pregnancy marked 15.4%, and 2 out of 4 women with successful pregnancy experienced spontaneous abortion. Out of 4 patients who did not respond to the previous clomiphene treatment, 3 women accomplished ovulatory induction with tamoxifen. The endocrinological dynamics in the tamoxifen treatment were similar to those in the clomiphene treatment.

[Prediction of Ovulation and timing of AIH].

The relation among ultrasonographic follicular diameter, serum estradiol (E2), luteinizing hormone (LH), cervical mucus changes, and basal body temperature (BBT) was studied. One hundred and eight cycles were examined in forty-two infertile patients. The last low day of the BBT curve was taken as day zero. In 83 of the 85 cycles, 158 of 163 times, follicles could be observed by ultrasonography. Follicular rupture was confirmed in 12 cycles. The mean diameter of the follicles measured the day before estimated ovulation was 24.00 +/- 3.10mm (mean +/- S.D.). Serum E2 levels were more than 400pg /ml from day -2 to day -1 and the peak serum LH levels occurred on day 0. The volume of cervical mucus (C.M.) was more than 100mm3 from day -2 to day +1. The volume was decreased in clomid stimulated cycles and the C.M. displayed a 3+ ferning 23/28 cycles on day 0. Among 5 pregnancies induced by homologous artificial insemination (AIH), three were done on day -2, one on day +1 and one on day +2. The best time to do AIH is when serum E2 is more than 400pg /ml, serum LH is more than 60m IU/ml, cervical mucus is 3+ ferning , and the follicular diameter is more than 20mm.

[Classification of human oocyte maturity].

Maturity of human oocytes was classified into five grades according to the appearance of the oocyte and the cumulus oophorus (Grade I-V). The relation between this "Grade" of oocyte and the state of fertilization was investigated and the following results were obtained. The proportion of fertilized eggs cleaving in vitro was higher when oocytes graded I or II were collected than when oocytes graded III, IV or V were obtained (76% vs. 22%). When oocytes were aspirated from preovulatory follicles 26 hours after the onset of the luteinizing hormone (LH) surge 64% of them were Grade I or II. When oocytes were collected 36 hours after an injection of human chorionic gonadotropin (HCG), 88% of them were Grade I or II. Therefore it seems that the times chosen for oocyte recovery were appropriate. There was no correlation between the "Grade" of oocyte and follicle diameter or volume of follicle fluid.

Isolation and characterization of group B streptococci from genito-urinary tracts in Japan.

The prevalence of group B streptococci in urine specimens submitted from both inpatients and outpatients to the Clinical Microbiology Laboratory were investigated. Out of 3,780 urine specimens, forty-seven strains were identified as group B streptococci, and clinical features revealed that the isolation ratio from outpatients was higher than that from inpatients, and that the majority of isolates were from female urine. Whereas the isolation ratios of group B streptococci from vaginal swabs were 2.9% in 377 pregnant women at 37-40 weeks of gestation, and 4.8% in 335 non-pregnant women. Also, the newborns delivered from mothers tested were investigated. Only two cases were positive in ear, pharyngeal and/or nasal swabs, but no clinical manifestation was observed. An alternative nomenclature system of group B streptococci based on the serological classification of both heat-labile protein and heat-stable polysaccharide antigens was evaluated for the clinical isolates and compared with the original Lancefield's procedure. The results indicated that the method described will provide a simple and reasonable technique for serotyping of group B streptococci.

[Sequential observation of human sperm penetration into zona pellucida-free hamster oocytes by scanning electron microscopy].

Yanagimachi et al. have shown that zona pellucida-free hamster oocytes can be penetrated by capacitated and acrosome-reacted human spermatozoa, and both pronuclei be formed. Recently, it has been said that this technique may be used to test the fertilizing ability of human sperm for clinical use. We have made scanning electron microscopic studies of the interaction of these heterologous gametes, especially the mechanisms of sperm entry into the oocytes. Our results are as follows: The surface of the oocyte is covered with numerous microvilli, which are found to be evenly and densely distributed on the vitellus surface. The surface of the first polar body already released is smooth and microvilli-free. Most spermatozoa lie flat on the vitellus surface, but a few are oriented perpendicularly to the vitellus surface. Most bound sperm had lost their acrosomal caps, because a ridge exists at the leading edge of the equatorial segment. Many microvilli are shown to participate in sperm-egg contact. Initially most microvilli appeared to grasp and immobilize the anterior tip of the sperm head. But as gamete interaction proceeded, microvilli were overlying the postacrosomal region, and observed adjacent to the plasma membrane of the postacrosomal region. The postacrosomal region is first incorporated into the ooplasma, the anterior tip of sperm head being the last portion to be incorporated. The microvilli of the oolenmal surface where sperm penetrated did not show major changes in size or appearance, and the so-called "incorporation cone" was not observed. Our scanning electron micrographs show that the microvillar portion of the oolenma greatly participates in this heterologous gamate interaction.