In-vitro maturation of round spermatids using co-culture on Vero cells.

In an attempt to determine whether co-culture could promote sperm maturation, three patients with non-obstructive azoospermia, two with maturation arrest at the level of primary spermatocytes and one patient with <1% tubules showing complete spermatogenesis, and one patient with total globozoospermia, gave consent to experimentally co-culture round spermatids retrieved from the testicle on Vero cell monolayers. In all azoospermic patients elongating spermatids could be obtained from round spermatids. In one case of maturation arrest, of 37 round spermatids co-cultured for up to 5 days, 30% developed flagella, 46% matured to elongating and 19% to elongated spermatids, with one mature spermatozoon also obtained (3%). In the same patient, primary cultures of three round spermatids with flagella enabled development of one further mature spermatozoon. In the case with total globozoospermia, of six round spermatids co-cultured for up to 5 days, one mature spermatozoon was obtained, with a flagellum and normal head morphology. These preliminary findings suggest that it may be possible to overcome the round spermatid block, and even the triggering of morphological abnormalities arising at the spermiogenic level, by in-vitro maturation under special environmental conditions.     

Fertilization and early embryolgoy: Can Matrigel substitute for Vero cells in promoting the in-vitro development of mouse embryos?

The influences of Vero cells and the basement membrane substratumfor these cells (Matrigel^®) on the rate of hatched blastocystformation from mouse zygotes in vitro were compared. Zygotesobtained from C57BL/6xBALB/c F1 females pretreated with pregnantmare's serum gonadotrophin/human chorionic gonadotrophin matedwith BDF1 males were cultured (120 h) in human tubal fluid mediumsupplemented 0.5% with bovine serum albumin. The rates of earlyhatching and hatched blastocyst formation at 96 and 120 h ofculture were expressed as the percentage of 2-cell embryos visualizedafter the initial 24 h. The rate of total blastocyst formationdid not differ between treatment groups. However, <10% ofembryos cultured for 96 h in medium alone advanced to the hatchingstage compared with 35–40% of blastocysts cultured withVero cells or with Matrigel alone. Similarly, by 120 h of culture,only 20% of embryos cultured in medium alone developed to hatchingor hatched blastocysts compared with >70% for those embryosco-cultured with Vero cells or with Matrigel. In conclusion,Vero cells improved the rate of development of mouse embryosto hatched blastocysts during serum-free culture. Similar improvementswere seen in the presence of Matrigel alone; Matrigel is thebasement membrane substratum used for the Vero cells. Furtherstudies on the means whereby Matrigel promotes early embryonicdevelopment (e.g. appropriate combination of basement membrane-associatedgrowth factors) may lead to a safe, defined medium preparationfor the stimulation of in-vitro development of human embryos.     

Fertilization and early embrology: Vero cell effect on in-vitro human blastocyst development: preliminary results

Asynchrony between embryo and uterine environment is one ofthe major limits in human in-vitro fertilization (TVF). A culturesystem which could prolong culture time and increase embryoniccleavage rate and viability would improve success rates. UsingVero cells, an in-vitro co-culture system was developed to investigateand promote human embryo development. Vero cells provide goodsupport for human early embryos up to the blastocyst stage.When fertilized embryos were co-cultured, 68% of them reachedthe blastocyst stage. Pregnancy rate was 50% per transfer inpatients with several previous failures of implantation. A significantincrease in clinical pregnancy rate was also demonstrated whenzygotes were maintained on Vero cell monolayer for only 24 h.The beneficial effect of the feeder layer may be through therelease of embryotrophic factors and the detoxification of theculture medium by the cells. Co-culture is a new concept inassisted reproduction.     

Improved development of human embryos in vitro by a human oviductal cell co-culture system.

This study was undertaken to determine the effect of co-culture with human oviductal cells on human embryos. Spare embryos from gamete intra-Fallopian transfer (GIFT), pronuclear stage transfer (PROST) and in-vitro fertilization/embryo transfer (IVF/ET) programmes were either cultured in serum-supplemented Earle's balanced salt solution alone, or co-cultured in the same solution with oviductal cells from the pronuclear stage (day 1 post-insemination) or two- to four-cell stage (day 2 post-insemination). The co-cultured embryos appeared to have a higher developmental potential (higher rate of blastocyst formation and lower fragmentation rate), although there was no statistical difference in their rate of development, degree of fragmentation and stages attained, when compared with conventionally cultured embryos. The percentage of hatching blastocysts was significantly higher (P less than 0.05, Fisher's exact test) for embryos co-cultured from day 1 post-insemination (38%) than for embryos which had not been co-cultured (7%). The blastocyst hatching rate for embryos co-cultured from day 2 post-insemination was 15%. It was therefore concluded that co-culture of human embryos with oviductal cells could improve the development of the embryos in vitro. The degree of improvement was more pronounced when the co-culture started at an earlier stage.     

Development of human embryos to the hatched blastocyst stage in the presence or absence of a monolayer of Vero cells

In a prospective randomized study, excess embryos from 100 womenundergoing in-vitro fertilization were cultured from the 2-cellto the hatched-blastocyst stage in the presence or absence ofa confluent monolayer of Vero cells. The frequencies of fragmentation,developmental arrest, multi-nucleation and blastocyst formationwere observed for 254 embryos over 7 days in culture. The numberof nucleated cells, and fine structure of trophectoderm andinner cell mass were analysed at the expanded blastocyst stageon day 5.5 post-insemination. The frequency of hatching fromthe zona pellucida was determined between days 6 and 7 post-insemination.With respect to these developmental parameters, the findingsindicate that no overt or statistically significant improvementin early human embryogenesis occurs in the co-culture system.     

Andrology: Co-culture with Vero cell monolayer maintains the motility of asthenozoospermic semen samples

The clinical effectiveness of co-culture with Vero (Green monkeykidney) cell monolayer in maintaining the motility and viabilityof fresh asthenozoospermic semen (18 samples) and frozen–thawedsemen with poor motility (motility fraction <50%) (15 samples)in a 24-h period was evaluated. Co-culture with Vero cell monolayerin human tubal fluid (HTF) medium for 24 h resulted in a statisticallybetter maintenance of motility percentage (P < 0.005), meanamplitude of lateral head displacement (ALH) (P < 0.005),and mean track speed (VCL) (P < 0.05) than culture in HTFmedium alone. However, these motility parameters (motility percentage,ALH, VCL) declined soon after removal of spermatozoa from themonolayer. Co-culture with Vero cell monolayer also maintainedthe viability percentage of these sperm samples (52% of theoriginal value) after the 24-h period compared with culturein HTF medium alone (22% of the original) (52% versus 22%, P< 0.05).It is concluded that Vero cell monolayer is effectivein the maintenance of motility and viability of asthenozoospermicsemen or frozen–thawed semen with poor motility.This co-culturesystem may be beneficial in enhancing the in-vitro performanceof asthenozoospermic semen samples in the practice of assistedreproductive technology.However, its safety needs further evaluation.     

Improved hatching in mouse embryos brought about by combined partial zona dissection and co-culture

In this study 874 mouse embryos were allocated to six groupsincluding a control, co-culture, and four groups that underwentpartial zona dissection (PZD): at the 2-cell (PZD-2) and morulastages (PZD-M) both with and without co-culture. Rates of completeblastocyst hatching on day 5 increased in the following order:control, co-culture alone, PZD-2 alone, PZD-M alone, PZD-2 withco-culture and PZD-M with co-culture (P < 0.00001). PZD-Mled to significantly higher rates of complete blastocyst hatchingcompared to PZD-2 (P < 0.03). This study showed also thatco-culture apparently compensates for any minor damage incurredduring the PZD technique at the 2-cell and morula stages, (P< 0.01 and P < 0.01) respectively. Therefore PZD and co-cultureseem mutually beneficial techniques that promote early blastocysthatching in the mouse.     

Comparison of human blastulation rates and total cell number in sequential culture media with and without co-culture

Recent interest in delayed embryo transfers necessitated the evaluation of two improved in-vitro systems that could generate viable blastocysts. A total of 178 two-pronucleated embryos (entire cohorts) from 19 patients was cultured in IVF50 medium (100 microl) under oil for 24 h until day 2. Each patient's day 2 embryos were then equally allotted to two in-vitro systems. Embryos in system A were grown until the morning of day 3 on Vero cells covered with IVF50 medium (100 microl) under oil. The medium was then replaced on day 3 with a 1:1 mixture (100 microl) of IVF50:S2 medium and on day 4 with S2 medium only. The same culture protocol was used for system B without Vero cells. Throughout the 5 days all dishes were housed in sealed humidified modular chambers containing a triple gas atmosphere. Separately, 175 spare embryos from 80 patients were grown in system A and B up to days 6 and 7 for total cell number (TCN) analysis. Blastulation rates were not significantly different between system A and B (67.4 versus 68.5%; P > 0.01) although co-cultured embryos cleaved slightly faster by day 4. The overall pregnancy and implantation rates were 52.0% and 32.1% for the 19 patients each of whom received a mixed cohort of three day 5 embryos from both systems. TCN values for the day 6 and 7 blastocysts from both systems were high and increased steadily from days 6-7 and from expanded to hatching stages. There were no significant differences in TCN for day 6 expanded blastocysts between the two systems although day 6 hatching and hatched co-cultured blastocysts had greater values than non-co-cultured blastocysts (246.0 +/- 18.5 and 236.7 +/- 17.8 versus 173.0 +/- 13.5 and 166.5 +/- 16.0; P < 0.01). The results demonstrated that the culture protocol using the sequential IVF50-S2 media combination was a good substitute for Vero cell co-culture for the transfer of viable day 3-6 embryos.     

Insulin-like growth factor-binding proteins produced by Vero cells, human oviductal cells and human endometrial cells, and the role of insulin-like growth factor-binding protein-3 in mouse embryo co-culture systems

Co-culturing embryos on helper cells can mimic the in-vivo environment,thereby enhancing embryo development in vitro. Insulin-likegrowth factors (IGF) and their binding proteins (IGFBP) alsoenhance embryo development To investigate the kinds of IGFBPproduced by various cell monolayers and the effects of IGFBP-3on mouse embryo co-culture systems, 2-cell ICR mouse embryoswere cultured in either human tubal fluid medium alone or inthe presence of Vero cells, human oviductal cells or endometrialcells. The helper cells were analysed immunohisto-chemicallyto investigate the types of IGFBP produced by various cell monolayers.The concentrations of IGF-I and IGFBP-3 in media obtained fromthe culture of embryos alone, cells alone or cells plus embryoswere determined by radioimmunoassays. On day 7, more blastocystshatched in the co-culture groups (73% in the Vero cell group,76% in the endometrial cell group and 74% in the oviductal cellgroup) than in the control group (43%) (P < 0.0001). Theresults of immunohistochemistry revealed that (i) all threecell groups produced a lot of IGFBP-1, -2 and -3, but only alittle of IGFBP-4 and -5; and (ii) IGFBP-1, -2 and -3 were presentin blastocysts in either the presence or absence of helper cells.The IGF-I secreted by cell monolayers or embryos was undetectable(detection limit 0.83 ug/1). The IGFBP-3 concentrations in mediaobtained from co-cultured embryos and cells were significantlyhigher than in media without embryos (median values in oviductalcell culture medium, 165 versus 127 µg/1, P = 0.04; medianvalues in endometrial cell culture medium, 277.5 versus 183.5µg/1, P = 0.0002; median values in Vero cell culture medium,219 versus 120 µg/1, P = 0.011). Although IGFBP-3 concentrationin the medium that contained embryos alone was undetectableby radioimmuno-assay (detection limit 1.1 µg/1), immunohistochemistrydemonstrated the presence of IGFBP-3 in the embryos. Co-culturein systems in which there was an increased production of IGFBP-3led to an improved development of mouse embryos. IGFBP can improvethe binding of IGF to cell surface receptors of target tissue,and thus enhance the effect of limited IGF concentrations inpromoting embryo development in a co-culture system. We concludethat Vero cells, human endometrial cells and oviductal cellsproduce IGFBP-1, -2, -3, -4 and -5. IGFBP-3 may play a rolein embryotrophic potential by either regulating the action ofIGF or directly enhancing embryo development     

In-vitro co-culture of early stage caprine embryos with oviduct and uterine epithelial cells.

Early stage caprine embryos were incubated with goat oviduct and uterine cells to evaluate whether these cells could be used as a somatic cell culture system to enhance development through the developmental block at the 8- to 16-cell stage during in-vitro culture. Following gonadotrophin treatment and natural mating, 2- to 4-cell embryos were surgically recovered from donor females for in-vitro culture studies. In Experiment 1, embryos were equally and randomly allotted to culture treatments of either culture medium plus caprine oviduct cells or culture medium alone. In both treatment groups, embryos were incubated in Medium-199 with 10% fetal bovine serum, 0.25% lactalbumin and 1% antibiotic-antimycotic at 37 degrees C in a humidified atmosphere of 5% CO2 in air. In Experiment 2, similar embryos were cultured in the same medium with either caprine oviduct cells, caprine uterine cells or sequentially incubated with oviduct cells and then uterine cells during a corresponding incubation interval. The culture conditions in Experiment 2 were the same as in Experiment 1. Following 72 h in culture, (Experiment 1), significantly more embryos developed through the in-vitro developmental block into blastocysts and hatched blastocysts when cultured with oviduct cells compared with no embryos developing through the in-vitro block when incubated with medium alone. In Experiment 2, caprine embryos co-cultured with oviduct cells alone resulted in more embryos developing into blastocysts and hatched blastocysts compared with those co-cultured with uterine cells alone.(ABSTRACT TRUNCATED AT 250 WORDS)     

Co-culture of 1-cell mouse embryos on different cell supports

The development of 1-cell mouse embryos in explanted oviducts, on mouse and bovine oviduct epithelial cells and on two established cell line supports is compared. The best rates of blastocyst formation were obtained using explanted oviducts; mouse and to a lesser extent, bovine oviduct epithelial cells allow good embryonic development, associated with high viability after transfer of the blastocysts obtained in co-culture. MDBK (from bovine kidney) and Vero (from Green monkey kidney) have been tested. MDBK allows high rates of blastocyst formation (67%) and the blastocysts obtained are viable. Vero does not allow the 2-cell block to be overcome. Maintenance of cell polarity for all the feeder layers did not improve embryo development. A preliminary study on the metabolic modifications induced by the feeder layers showed no modifications at all related to a decrease in glucose, an increase in lactate and early embryonic development. On the other hand, for the free amino acids, cellular supports with high embryotrophic activity seem to mimic tubal secretions, especially with a high level of glycine. Neither a genital tract origin, nor a hormonal contribution are strictly necessary for embryo co-culture, as already demonstrated by co-culture with trophoblastic tissue. Established cell lines, which are easy to handle and control, could be useful tools in embryo biotechnology.     

Fertilization and early embryology: Effects of embryo density and co-culture of unfertilized oocytes on embryonic development of in-vitro fertilized mouse embryos

We have evaluated the effects of embryo density and the co-cultureof unfertilized (degenerating) oocytes on the development ofin-vitro fertilized (IVF) mouse embryos. In experiment 1, groupsof one, five, 10 or 20 zygotes were cultured in 20 µldrops of modified human tubal fluid (HTF) medium for 168 h at38.7°C in 5% CO₂ and 95% air. As the embryo density increased,significantly (P < 0.05) higher rates of embryos reachedhatched blastocyst stage. In addition, the time required forhatching after IVF was significantly (P < 0.05) shortenedby the increase in embryo density. In experiment 2, 10 IVF zygoteswere cultured with or without 10 unfertilized (degenerating)oocytes in 20 µl drops of HTF medium. The rates of IVFembryos that developed to morula, blastocyst, expanded blastocystand hatched blastocyst stages were decreased significantly (P< 0.01) by culturing embryos with unfertilized oocytes comparedwith culturing embryos alone. In experiment 3, groups of oneor 10 IVF zygotes or 10 IVF zygotes plus 10 unfertilized oocyteswere cultured in 20 µl drops of HTF medium and the numberof cells per blastocyst was examined at 120 h after IVF. Increasingembryo density resulted in a significant (P < 0.05) increasein the number of cells per blastocyst. In contrast, the cellnumber of IVF embryos that developed to blastocyst decreasedsignificantly (P < 0.05) when they were cultured with unfertilizedoocytes. The results suggest that in-vitro development of IVFmouse embryos is enhanced by increasing embryo density and isimpaired by co-culture with unfertilized (degenerating) oocytes.     

Embryonic morphology and rate of implantation of human embryos following co-culture on bovine oviductal epithelial cells

A study was undertaken to evaluate embryonic development andestablish pregnancies with human embryos after in-vitro culturein two different systems. Treatment A consisted of culturingzygotes in serum-supplemented human tubal fluid culture medium(HTF). Treatment B consisted of culturing zygotes on a monolayerof bovine oviductal epithelial cells with HTF. At the time ofembryo replacement, embryos in treatment B had 4.11 blastomerespresent, which was greater (P < 0.05) than the 3.81 presentfor embryos in treatment A. In addition, the cellular fragmentationrate for treatment A embryos was 1.10, which was greater (P< 0.05) than the fragmentation rate of 0.38 for embryos withintreatment B. The incidence of ongoing pregnancy was higher afterreplacement of co-cultured embryos (treatment B) (43%) thanreplacement of conventionally cultured embryos (treatment A)(29%). The implantation rate per embryo increased (P < 0.05)from 11.5 to 18.4% after co-culture. In treatment B the proportionof ‘spare’ embryos developing to expanded blastocystswas 58.5%, which was greater (P < 0.05) than the blastocystdevelopment rate of 29.3% observed for embryos within treatmentA.     

In vitro maturation of human oocytes and cumulus cells using a co-culture three-dimensional collagen gel system

BACKGROUND: Deficiencies remain in the ability of in vitro-matured human oocytes to acquire full developmental competence and give rise to a healthy pregnancy. A clear deficiency of current systems utilizing human oocytes has been the absence of cumulus cells. In the present study, a three-dimensional (3D) co-culture system exploiting an extracellular matrix was developed and compared to conventional methods for its ability to support maturation of human oocytes. METHODS AND RESULTS: Cumulus cells were embedded into a 3D collagen gel matrix with individual oocytes added to each gel. Oocytes from the same patient cultured in the gel matrix matured to metaphase II at rates similar to those of cumulus-free oocytes cultured in individual microdrops. Following maturation of oocytes and fixation of intact gels, chromatin and cytoskeletal elements were assessed in oocytes and cumulus cells. The activities of the key cell cycle kinases, maturation-promoting factor (MPF) and mitogen-activated protein kinase (MAPK), were compared in oocytes matured under the two culture conditions. Compared with denuded oocytes, co-cultured oocytes exhibited increased MAPK activity, but no difference in MPF levels. CONCLUSIONS: This work characterizes a novel and efficacious culture system that takes advantage of the unique properties of the extracellular matrix, a 3D microenvironment, and the presence of cumulus cells for maturing human oocytes in vitro.     

Vero cell co-culture counteracts the detrimental effects of hydrosalpinx fluid on the development of mouse embryos in vitro

Recent studies have suggested that the hydrosalpinx has a negative effect on pregnancy outcome, with markedly diminished implantation and increased early pregnancy loss. Fluid from the hydrosalpinx may leak into and accumulate in the uterine cavity. It is not clear, however if this creates a hostile local environment in the uterus for embryo implantation or exerts a direct embryotoxic effect. This study was conducted to investigate the detrimental effects of hydrosalpinx fluid (HSF) on the development of mouse embryos in vitro and to demonstrate whether Vero cells overcome these adverse effects. HSF was collected from three women with bilateral hydrosalpinx at the time of laparoscopic surgery. Collected fluid was centrifuged and the supernatant was frozen at -20 degrees C. For co-culture, Vero cells were commercially obtained in a frozen state and cultured using Ham's F10 medium. Single-cell mouse embryos (B6CBAF1) were cultured for 5 days in 0, 0.4, 0.8, and 1.2% of HSF in media with and without Vero cells and examined daily to record the number of embryos reaching expanded blastocyst and hatching stage. Co-culture of mouse embryos with Vero cells at 0.8% HSF concentration significantly enhanced embryo development, but not at 1.2% hydrosalpinx fluid concentration. These results suggest that HSF is highly embryotoxic and Vero cells are likely to overcome these detrimental effects to some degree.     

狂犬病北京3aG-V生产株的特性研究

目的 研究狂犬病北京固定毒Veto细胞适应株3aG-V生产株的生物学特性。方法 观察毒株形态、培养条件、致病性、免疫原性、毒力试验及其检查在中枢神经系统是否形成病毒包涵体(尼氏小体)。结果 狂犬病北京aG固定毒3aG-V株具有抗原性好、培养产毒量高、保持有aG固定株弱毒性、传代稳定、无变异的特性。结论 狂犬病北京aG固定毒3aG-V株可作为替代地鼠肾细胞狂犬病疫苗aG毒株，用于Veto细胞培养病毒生产出毒液毒力高、灭活后效力高、安全性好的纯化Veto细胞狂犬病疫苗的生产用疫苗株。     

Fertilization and early embryology: Granulosa cell co-culture enhances human embryo development and pregnancy rate following in-vitro fertilization

A preliminary study and related clinical trial were performedto evaluate the effects of granulosa-lutein cell co-cultureon human embryo development and pregnancy rates for in-vitrofertilization (IVF). In the study, sibling two-pronuclear zygoteswere randomly allocated to culture with (co-culture) or without(control) autologous granulosalutein cells. After 24 h, embryoswere examined for blastomere number and degree of fragmentation.Co-culture had no effect on the average number of blastomeresper embryo at 24 h; however, fragmentation was significantlydecreased in co-cultured embryos (0.7 ± 0.1) comparedwith controls (1.3 ± 0.2; P < 0.05). In the subsequentclinical trial, all two-pronuclear zygotes were co-culturedfor 48 h prior to embryo transfer. The live birth rate per embryotransfer was 43.4% with an implantation rate per embryo of 17.6%.Of the untransferred embryos, 68% developed to the blastocyststage and were cryopreserved. We conclude that the simple systemof autologous granulosa-lutein cell co-culture improves embryodevelopment, implantation and subsequent pregnancy rates forIVF.     

体外细胞组织重建中共培养技术的应用

细胞共培养体系在维持细胞基本结构和性状的基础上,通过两种或多种细胞组织的相互作用,使体内外环境尽可能相吻合,弥补了单层细胞培养的缺陷,有利于构建更加接近生理状态的重建体外细胞组织,被广泛应用于现代细胞研究,成为角膜、牙周、软骨、心血管以及神经细胞组织等体外构建的重要技术应用.在干细胞研究中,多孔膜两侧直接接触共培养日益受到重视,三维细胞共培养将是未来发展的方向.     

Endometrial stromal cells regulate epithelial cell growth in vitro: a new co-culture model

The regulation of epithelial cell function and morphogenesis by the paracrine effectors from the mesenchyme or stroma has been well established using in-vivo studies. A more complete understanding of these relationships has been delayed due, in part, to a lack of appropriate co-culture models. In this study, we describe a co-culture model which demonstrates that normal paracrine relationships can be reconstituted in vitro and that human endometrial stromal cells regulate both growth and differentiation of primary human endometrial epithelial cells. Interesting differences in the proliferation of stromal and epithelial cells were noted in response to the basement membrane extract, Matrigel((R)). Exposure of stromal cells to Matrigel((R)) enhanced the paracrine capacity of these cells in vitro. When epithelial cells were co-cultured in contact with stromal cells embedded in Matrigel((R)), epithelial cell growth was inhibited by 65-80% compared to controls. Stromal cells in contact with Matrigel((R)) also regulated epithelial cell differentiation, as shown by induction of glycodelin expression. These co-culture studies show great promise as a method to investigate the cellular interactions between endometrial stromal and epithelial cells and their environment and to understand the molecular basis for the regulation of normal growth and differentiation of cells within complex tissues such as the endometrium.     

Assisted hatching with or without bovine oviductal epithelial cell co-culture for poor prognosis in-vitro fertilization patients

Older patients and those who consistently return for embryotransfer but without implnatation were studied to see if a combinationof day 3 assisted hatching and co-culture (AH +CC) might bebeneficial compared to assisted hatching alone (AH-alone). Femalepatients of 38 years and couples who had previously failedto implant embryos three times or more were prospectively andrandomly assigned to either an experimental or a control group.In the experimental group all embryos were co-cultured on partialmonolayers of bovine oviductal epithelial cells for 2 days followedby assisted hatching by zona drilling (AH +CC). All controlembryos were cultured by standard procedures until day 3 whenthey also underwent zona drilling prior to uterine transfer(AH -alone). With 50 cycles in each group there was unfortunatelya marginal bias against the AH + CC group in that these patientshad undergone a higher number of previous transfer cycles. Therewas a marginally lower percentage of fragmentation and a signficantlyhigher degree of zona thickness variablity in the AH + CC embryogroup. Embryonic implantation was significantly increased (P< 0.05) in the AH xCC group (18%) when compared to the AH-alone group (10%). This difference was reflected in a significantlyhigher (P < 0.05) initial pregnancy rate (52 versus 32%)in the AH +CC group, and a higher (not significant) viable pregnancyrate (38 versus 22%).