Evaluation of the spindle apparatus of in-vitro matured human oocytes following cryopreservation

The present study was conducted to determine if the cryopreservationof immature human oocytes has a deleterious effect on the meioticspindle following maturation in vitro. Oocytes were obtainedin excess from in-vitro fertilization patients and divided intofour groups. Groups 1 (n = 98) and 2 (n = 80) consisted of immatureoocytes cryopreserved before or after maturation in vitro respectively.Groups 3 (n = 37) and 4 (n = 9) served as non-frozen controlsand included oocytes matured in vitro and in vivo respectively.The meiotic spindle was identified after incubation in anti-tubulinmonoclonal antibody (1 h, 37°C) and fluorescein-conjugatedgoat anti-mouse immunoglobulin G (IgG) (1 h, RT). Chromosomeswere counterstained with 4‘, 6’-diamidino-2-phenylindole.Following cryopreservation, group 1 oocytes demonstrated a 63%survival rate and 68% maturation rate in vitro. In all, 58%of the oocytes in group 2 survived the thaw. The number of oocyteswith normal spindles in group 1 (81.0%) was not significantlydifferent from control groups 3 (83.8%) and 4 (88.9%), whilethe number of group 2 oocytes with normal structures (43.5%)was significantly lower than groups 1 (P = 0.0004), 3 (P = 0.0002),and 4 (P = 0.025). These results suggest that cryopreservationof the prophase I human oocyte does not significantly increaseabnormalities in the resulting meiotic spindle.     

Differentiation of spermatogenic cells during in-vitro culture of testicular biopsy samples from patients with obstructive azoospermia: effect of recombinant follicle stimulating hormone

In-vitro differentiation of spermatogenic cells is a potentialapproach to the treatment of male sterility due to spermatogenicarrest. This is a pilot study evaluating meiotic, morphogeneticand cytoplasmic maturation of spermatogenic cells from 18 patientswith obstructive azoospermia, during in-vitro culture of partlydisintegrated testicular biopsy samples in the presence or absenceof recombinant follicle stimulating hormone (rFSH). Meioticprogression was detectable only in the presence of rFSH in culturemedium. FSH-dependent condensation, peripheral migration andprotrusion of spermatid nuclei, together with FSH-independentflagellar growth, were the main events indicating post-meioticsperm cell differentiation. rFSH also promoted the progressionof spermatid cytoplasmic maturation, reflected by accelerationof acrosomal development. These differentiation events appearedto be mediated by humoral activity of Sertoli cells, withoutthe need for a direct Sertoli-sperm cell contact. These findingsprovide a background for similar studies in patients with non-obstructiveazoospermia. If reproducible in the latter group, transmeioticin-vitro differentiation of primary spermatocytes may be usefulin cases of complete maturation arrest, whereas the developmentof culture-specific forms may help select viable spermatidsin cases of complete spermiogenesis failure.     

人牙龈成纤维细胞原代培养及冻存和复苏

背景：牙龈成纤维细胞是牙龈固有结缔组织层中主要的细胞,在许多生理和病理过程中起着重要作用。 目的：对人牙龈成纤维细胞进行原代培养、鉴定、冻存及复苏。 方法：采用组织块培养法培养人牙龈成纤维细胞,并进行形态学和免疫学鉴定。对人牙龈成纤维细胞进行冻存与复苏,倒置显微镜下观察细胞形态变化。 结果与结论：人牙龈成纤维细胞原代培养成功率为86.7%,细胞呈梭形或纺锤形。免疫组织化学鉴定抗波性蛋白抗体染色阳性,抗角蛋白抗体染色阴性,为中胚层来源的成纤维细胞。牙龈成纤维细胞冻存与复苏成功,细胞经2次传代后生物学性状与原代相似。提示采用组织块培养法培养原代人牙龈成纤维细胞及冻存与复苏方法可行。     

Lectin binding sites on human sperm and spermatogenic cells

Testes of sexually mature men were studied histochemically with 20 fluorescein isothiocyanate-labeled lectins. Based on their pattern of reactivity with intratesticular spermatogenic cells, lectins were divided into five groups: 1) lectins reacting with all spermatogenic cells (Suc. ConA, WGA, LCA, PHA-E, PHA-L, STA, MPA, and RCA-II); 2) lectin reacting with spermatocytes, spermatids, and spermatozoa, but not with spermatogonia (RCA-I); 3) lectins reacting with spermatids and spermatozoa only (BPA, PNA, SBA, and VVA); 4) lectins reacting only with spermatozoa (HPA, GSA-I, UEA-II, and GSA-II); and 5) lectins with no distinct staining of spermatogenic cells (DBA, LBA, and UEA-I). All lectins from groups 1-4 were reactive with ejaculated spermatozoa. On the basis of the staining patterns of the head region of ejaculated spermatozoa, four lectin reactivity groups were defined: 1) lectins reacting with the plasma membrane of the whole head (BPA, WGA, LCA, STA, RCA-II, PHA-E, PHA-L, RCA-I, UEA-II, and GSA-II); 2) lectin reacting with the acrosomal cap and postacrosomal region of the plasma membrane (Suc. ConA); 3) lectin reacting with the acrosomal cap region of the plasma membrane (PNA); and 4) lectins reacting with the midregion of the sperm head in a bandlike manner (HPA, VVA, SBA, GSA-I, and MPA). These data provide a map of lectin binding sites on human testicular spermatogenic cells and ejaculated spermatozoa and show that the distribution of glycoconjugate domains of spermatogenic cell changes during differentiation and maturation.     

Zona pellucida damage to human embryos after cryopreservation and the consequences for their blastomere survival and in-vitro viability

The study objective was to quantify zona pellucida (ZP) damage in cryopreserved human embryos. The influence of two different freezing containers was investigated, and the influence of freezing damage on the survival and viability of the embryos evaluated. ZP damage did not differ according to whether embryos originated from in-vitro fertilization (IVF) cycles or from IVF cycles in association with intracytoplasmic sperm injection (ICSI). The freezing container, however, significantly influenced the occurrence of ZP damage after cryopreservation. More damage was observed when the embryos were frozen-thawed using plastic cryovials than using plastic mini-straws (16.6% versus 2.3%; P < 0.0001). A clear association was found between blastomere survival and ZP intactness. Consequently, the percentage of embryos with 100% blastomere survival was higher when embryos were frozen-thawed using plastic mini-straws. The further cleavage of frozen-thawed embryos suitable for transfer was not different whether there was ZP damage or not; however, it was higher when there was 100% blastomere survival as compared with when some blastomeres were damaged (79.0% versus 43.7%; P < 0.0001). Consequently, more embryos suitable for transfer cleaved further when they were frozen-thawed using plastic mini-straws. In conclusion, the aim of a cryopreservation programme should be to have as many fully intact embryos as possible after thawing. Increased ZP damage might indicate a suboptimal cryopreservation procedure.     

Outcome of in-vitro culture of fresh and frozen-thawed human testicular spermatozoa

The effect of in-vitro culture on the motility and morphology of fresh and frozen-thawed human testicular spermatozoa obtained from obstructive azoospermic patients and on the motility of testicular spermatozoa obtained from non-obstructive azoospermic patients was evaluated. The outcome of intracytoplasmic sperm injection (ICSI) with fresh and frozen-thawed human testicular spermatozoa was studied. The results showed that significant improvement of sperm morphology and motility was observed in culture of fresh (n = 17) and frozen-thawed (n = 15) testicular sperm samples obtained from patients with obstructive azoospermia. The motility of cultured testicular spermatozoa reached a peak at 72 h without the need for special media. In six of 20 samples obtained from patients with non-obstructive azoospermia, improvement of sperm motility was observed. When only non-motile testicular spermatozoa were cultured, they all remained non-motile (n = 9). In patients with obstructive azoospermia, fertilization rates of 80 and 81% were obtained using ICSI with fresh and frozen-thawed testicular spermatozoa respectively. Clinical pregnancies were observed in four out of nine patients with fresh testicular spermatozoa and two out of five patients after using frozen-thawed spermatozoa. When fresh testicular spermatozoa obtained from patients with non-obstructive azoospermia were used for ICSI, the fertilization rate was 68% and two out of seven patients achieved clinical pregnancies. In conclusion, the morphology and motility of fresh and frozen-thawed testicular spermatozoa in patients with obstructive azoospermia can be significantly improved after in-vitro culture. The outcome of in-vitro culture of testicular spermatozoa in patients with non-obstructive azoospermia is unpredictable. In-vitro culture of non-motile testicular spermatozoa is not successful so far. The outcome of ICSI with fresh and with frozen-thawed testicular spermatozoa was similar.      

Effect of oxygen concentration on human in-vitro fertilization and embryo culture

In this prospective randomized study on 1380 consecutive in-vitro fertilization (IVF) treatments, the results were compared of culture of human oocytes and embryos for the first 2 or 3 days of development in microdroplets of medium under oil using a gas phase containing either atmospheric (approximately 20%) or reduced (5%) O2 concentrations. No significant differences were found between the two groups cultured under either 5% or 20% O2 in rates of fertilization (60 versus 61%, respectively), embryo development at day 2 or 3, pregnancy (26.6 versus 25.4%, respectively), and implantation (13.4 versus 14.0%, respectively). Culture of surplus embryos under 5% O2 resulted in a significantly higher mean incidence of blastocyst formation per cycle as compared to the 20% O2 group (25.8 +/- 2.0 versus 20.4 +/- 1.9, respectively). The mean number of cells of embryos classified as blastocysts by microscopic observation of a blastocoel was significantly higher in the 5% O2 group as compared to the 20% O2 group, both in blastocysts fixed on day 5 (39.8 +/- 1.7 versus 31.9 +/- 1.9, respectively), as well as those fixed on day 6 (45.6 +/- 2.6 versus 33.7 +/- 3.4, respectively). This difference was due to the fact that significantly more blastocysts of the 20% O2 group had an abnormal low cell number of < 25 as compared to the 5% O2 group, both in blastocysts fixed on day 5 (39 versus 22%, respectively), as well as those fixed on day 6 (43 versus 22%, respectively). To conclude, although culture under 5% O2 leads to slightly improved preimplantation embryonic viability, this effect is either too marginal to result in higher pregnancy rates, or low O2 concentrations exert an effect during the later stages of preimplantation development only.     

Developmental potential of human spermatogenic cells co-cultured with Sertoli cells.

BACKGROUND: Development of an in-vitro culture system capable of supporting human early germ cell differentiation would be important for treatment of azoospermic patients. METHODS: Sertoli cells, spermatogonia and spermatocytes were isolated from testicular biopsies of 61 non-obstructive azoospermic patients, and co-cultured using Vero cell conditioned medium only or supplemented with recombinant (r)FSH or rFSH plus testosterone. Germ cell purity was checked by fluorescent in-situ hybridization (FISH) analysis. RESULTS: Best results were achieved with both hormones, which elicited 6.9% of meiosis index and 22.7% of differentiation into normal late spermatids after 2-3 weeks of culture. In-vitro matured spermatids were microinjected into oocytes to study their developmental potential. Round spermatids elicited 37.5% of fertilization and 28.6% blastocyst rates. Abnormal elongating and elongated spermatids enabled 8.3 and 27.3% fertilization rates respectively, but none achieved the blastocyst stage. Normal elongating and elongated spermatids elicited 30.5% fertilization and 42.9% of blastocyst rates. FISH analysis showed sex chromosome anomalies in all embryos, except in the case of morulae from normal late spermatids. CONCLUSIONS: Results suggest that meiosis and spermiogenesis can be resumed in vitro, with normal differentiated spermatids showing a low fertilization potential but regular rates of blastocyst formation. However, most of the embryos did not reach the morula stage and showed major sex chromosome abnormalities.     

Morphology and biochemistry of in-vitro produced bovine embryos: implications for their cryopreservation

Examination of some ultrastructural and physiological characteristicsof in-vitro produced bovine embryos may help to explain whysuch embryos are more sensitive to freezing than their in-vivoderived counterparts. Improvement of embryo survival after freezingcan be achieved by changing the conditions of their culture,selection of embryos based on the kinetics of their development,and changing ‘standard’ freezing procedures. Cryopreservationof embryos by vitrification, in particular, seems to yield highersurvival than conventional slow freezing. Further developmentof protocols requires additional embryo transfer studies toensure that the ability of thawed embryos to develop normallyin vivo correlates strongly with in-vitro survival assays.     

Anxiety during pregnancy and fetal attachment after in-vitro fertilization conception

The aim of this study was to compare 70 couples who had conceived by in- vitro fertilization (IVF) with 63 matched controls for the prevalence of anxiety and quality of attachment to the baby during pregnancy. Results for mothers showed no group differences using a global measure of anxiety, the Spielberger State-Trait Anxiety Inventory. However, pregnancy-specific measures revealed significantly higher levels of anxiety in IVF mothers about the survival and normality of their unborn babies, about damage to their babies during childbirth and about separating from their babies after birth. When IVF mothers were differentiated according to the number of treatment cycles, more differences in anxiety level were revealed, with most increases occurring in mothers who had experienced two or more treatment cycles. IVF fathers did not differ from controls on the global anxiety measure. No data on pregnancy-specific anxiety were available for fathers. Neither IVF mothers nor IVF fathers differed from controls on measures of attachment to the baby during pregnancy. Results are discussed in the context of the need for researchers to employ differentiated and issue-specific measures to identify concerns that may be unique to IVF couples. Clinical implications regarding the need for psychological support during pregnancy are also discussed.      

Unusual features of the nuclear envelope in human spermatogenic cells

Different types of human germ cells show unusual features of the nuclear envelope. Spermatogonial nuclei demonstrate two kinds of modifications. The first one is a series of intranuclear flattened cisterns, parallel to each other and to the inner aspect of the nuclear envelope. The second one is a nuclear envelope protrusion into the cytoplasm occupied by a double membrane-limited vesicle. Pores are found on the membrane of the vesicle facing the interior of the nucleus. In spermatocytes the nuclear pores are concentrated over certain areas and completely absent from others. In the regions where they are absent a single cytoplasmic cistern of rough endoplasmic reticulum is closely apposed to the outer membrane of the nuclear envelope. Early modifications of the nuclear surface appear in spermatids before the attachment of the acrosomic vesicle and may indicate an active role of the nuclear envelope in the morphogenesis of the acrosome. In round spermatids nuclear pores are absent from the area which is first related to the Golgi and later covered by the acrosomal cap. Single or multiple layers of cytoplasmic annulate lamellae are closely associated with the nuclear envelope over the pore rich areas. Frequently there are intranuclear accumulations of dense material adjacent to the annulate lamellae-nuclear pore complex. The chromatoid body is usually present on the cytoplasmic side of this complex. In the elongating spermatids most annulate lamellae are free in the cytoplasm, often in relation with Golgi and chromatoid body remnants near the axial filament. Few stacks of annulate la-mellae are noted adjacent to the pore rich nuclear regions. It is suggested that the described modifications are related to an active nuclear-cytoplasmic interaction.     

Cleavage anomalies in early human embryos and survival after prolonged culture in-vitro

This study examines the relationship between common morphological anomalies of cleaving embryos and their ability to form apparently normal blastocysts in vitro. The impact of cleavage rate, fragmentation, and multinucleation on compaction, cavitation, along with inner cell mass and trophectoderm formation has been assessed. The study population consisted of 102 patients who elected or were selected to have a day 5 embryo transfer. Clinical pregnancy and implantation rates were 66.7 and 49% respectively. Slow and fast cleavage had a significant negative association with normal blastocyst formation. Only 13.8% (67/484) of embryos with <7 cells and 27.5% (25/91) of those with >9 cells on day 3 formed blastocysts with apparently normal morphology, compared to 41.9% (252/602) with 7-9 cells on day 3 (P < 0.001). Fragmentation had a negative impact on normal blastocyst formation. Embryos with >15% fragmentation formed normal blastocysts at a significantly lower rate (46/279; 16.5%) than embryos with 0-15% fragmentation (311/935; 33.3%) (P < 0. 001). Furthermore, the pattern of fragmentation was associated with blastocyst formation. Type IV fragmentation led to a significant reduction in blastocyst formation (25/170 or 14.7%), compared to types I, II and III which performed much better (38.6, 32.9 and 32.4% respectively). Only 15.9% (22/138) of embryos with one or more multinucleate cells on day 2 and/or 3 formed normal blastocysts compared with 31.9% (335/1051) (P < 0.001) of those without multinucleation. Collectively, the data suggest that cleavage anomalies, some of which do not preclude development after short-term culture, may reduce the developmental competence of embryos after prolonged culture.     

Follicular development and early luteal function of conception and non-conceptional cycles after human in-vitro fertilization: endocrine correlates

Two-hundred patients, half of whom had on-going pregnancies,were examined in terms of follicular growth, urinary oestrogenand LH output, oocytes recovered and embryos replaced. The twogroups were identical in all parameters measured except thaturinary LH output was significantly higher (P>0.01) in non-pregnantpatients on the two days prior to HCG administration. Duringthe early to mid-luteal phase, plasma progesterone concentrationswere related to the number of follicles aspirated at oocyterecovery, but the overall pattern of secretion was similar inboth groups. It is concluded that monitoring urinary LH output,a non-invasive technique, may be of great value for assessingoocyte quality and predicting the outcome of in-vitro fertilizationand embryo replacement.     

The use of ICSI in all cases of in-vitro conception

Dear Sir, We are pleased that our article (Oehninger and Gosden, 2002)has stimulated a debate about the indications for ICSI and aregrateful for the opportunity to respond to the letter of DrsAbu-Hassan and Al-Hasani who advocate general application     

Predictive value of human chorionic gonadotrophin in the outcome of early pregnancy after in-vitro fertilization and spontaneous conception

This prospective study analyses the value of the -subunit ofhuman chorionic gonadotrophin (-HCG) in 120 pregnancies obtainedafter in-vitro fertilization (IVF)-embryo transfer. Spontaneousconception cycles (n = 16) were also analysed allowing a comparisonbetween these two forms of conception. Of the 120 clinical pregnancies,48 started as single gestations and 50 started with two or moresacs. There were 14 clinical abortions and eight ectopic pregnancies.All subjects had blood samples taken under a fixed protocolon days 11, 14, 17, 20 and 23 after follicular aspiration. Weeklysamples were obtained thereafter until day 60 from ovum retrieval.Transvaginal ultrasounds were performed at weekly intervals,starting on day 23 after follicular aspiration. In spontaneousconception cycles blood samples were obtained daily, startingon the day of follicular rupture. In spontaneous conceptioncycles and in IVF– embryo transfer conceptions, the doublingtime (DT) of ;-HCG was 1.4 ± 0.3 and 1.6 ± 0.4days respectively. This difference was not significant. In multigesta–tions,the DT was 1.5 ± 0.3 days. The absolute values of -HCGin early spontaneous gestations were significantly higher thanin IVF—embryo transfer cycles, suggesting that the blastocystimplants with less cellular mass when initiated in vitro ascompared with the in-vivo condition. The early prediction ofectopic pregnancy and spontaneous clinical abortion was analysedby the -HCG profile as well as the absolute values in comparisonto normal pregnancies. Both parameters showed significant differencesas early as the interval between days 11 and 23 from follicularaspiration. This study provides a comprehensive approach tothe evaluation of the outcome of early gestation in terms ofthe predictability of single and multigestation, ectopic pregnancyand early abortion.     

Live human germ cells in the context of their spermatogenic stages

BACKGROUND: Various types of live, dispersed, human testicular cells in vitro were previously compared with the morphologic characteristics of human spermatogenic germ cells in situ within seminiferous tubules. The current study extends those observations by placing live human germ cells in the context of their developmental steps and stages of the spermatogenic cycle. METHODS: Live human testicular tissue was obtained from an organ-donating, brain-dead person. A cell suspension was obtained by enzymatic digestion, and dispersed cells were observed live with Nomarski optics. Testes from 10 men were obtained at autopsy within ten hours of death, fixed in glutaraldehyde, further fixed in osmium, embedded in Epon, sectioned at 20 microm, and observed unstained by Nomarski optics. RESULTS: In both live and fixed preparations, Sertoli cells have oval to pear-shaped nuclei with indented nuclear envelopes and large nucleoli, which makes their appearance distinctly different from germ cells. For germ cells, size, shape, and chromatic pattern of nuclei, the presence of meiotic metaphase figures, acrosomic vesicles/structures, tails, and/or mitochondria in the middle piece are characteristically seen in live dispersed cells and those in the fixed seminiferous tubules. These lead to identification of live germ cells in man and placement of each in the context of their developmental steps of spermatogenesis at corresponding stages of the spermatogenic cycle. CONCLUSIONS: This comparative approach allows verification of the identity of individual germ cells seen in vitro and provides a checklist of distinguishing characteristics of live human germ cells to be used in clinical procedures or by scientists interested in studying live cells at known steps in spermatogenic development characteristic of germ cells in specific stages of the spermatogenic cycle.     

Normal survival and in-vitro development after cryopreservation of zona-drilled embryos in mice

The Importance of the zona pellucida on survival after freezingand thawing was Invesligated. Zona-drilled and zona Intact mouseembryos were fertilized in vitro, cultured to the 2, 4 and 8-cellstages and frozen using conventional methods. Zona drillingdid not affect the survival or development of frozen embryosto the blastocyst stage in vitro. We conclude that partial damageto the zona pellucida during micromanipulation procedures Lscompatible with rates of survival and development which arenot different to those observed In zona-Intact control embryos.