Allelic estrogen receptor 1 (ESR1) gene variants predict the outcome of ovarian stimulation in in vitro fertilization

The outcome of in vitro fertilization (IVF) depends substantially on the effectiveness of controlled ovarian hyperstimulation (COH) induced by administration of follicle-stimulating hormone (FSH). In COH, endogenously produced estrogens extend the action of FSH in stimulating folliculogenesis. We determined the associations between genetic variations in estrogen receptor ESR1 and ESR2 genes and etiology of female infertility, and analysed the influence of these variations on COH outcome-the quantity and quality of oocytes retrieved. ESR1 PvuII T/C (rs2234693) and XbaI A/G (rs9340799) single-nucleotide polymorphisms (SNPs) and (TA)n microsatellite polymorphism, as well as ESR2 RsaI G/A (rs1256049) SNP and (CA)n microsatellite polymorphism were genotyped in 159 IVF patients. The ovarian response to FSH was diminished in patients with endometriosis when compared to tubal factor infertility. ESR1 PvuII and XbaI as well as ESR2 RsaI SNPs were associated with the microsatellite length of the respective genes. Shorter ESR1 (TA)n was linked with a higher risk for unexplained infertility, whereas longer ESR1 (TA)n associated with PvuII*C allele were predictive of a better COH, but not clinical pregnancy outcome in an age-independent manner. These data suggest the variations in ESR1 gene, in addition to the age of a woman, may predict the COH outcome in IVF.     

来曲唑微刺激促排卵在卵巢储备下降患者IVF-ET中的应用

目的研究来曲唑微刺激促排卵应用于卵巢储备降低患者行IVF-ET的效果。方法 346名拟行IVF-ET的卵巢储备降低的不孕患者随机分成来曲唑微刺激促排卵组(A组n=226)和GnRH-a短方案组(B组n=120),比较两组促排卵天数,Gn总量,获卵数,优胚率、临床妊娠率,多胎率和流产率。结果来曲唑促排组促排天数、Gn总量、诱导日E2、获卵数、受精率、可利用胚胎数、种植率、临床妊娠率显著低于短方案组(P<0.05),诱导日LH、P水平高于Gn-RHa短方案组(P<0.05),而两组的诱导日内膜厚度、卵裂率、优质胚胎率、移植胚胎数、多胎率、流产率差异均无显著统计学意义(P>0.05)。结论对于卵巢储备降低的患者行IVF-ET助孕时来曲唑微刺激促排卵可以减少Gn用量,但是在妊娠率上并不优于GnRH-a短方案。     

Association of oestrogen receptor gene polymorphisms with outcome of ovarian stimulation in patients undergoing IVF.

Oestrogen plays an important role in follicular formation and oocyte maturation via its receptor (ER). Many studies have shown association of the ER gene polymorphisms with a variety of pathological conditions. In this study we have examined the relationship of a common PvuII and a rare BstUI polymorphism in the ER gene to the mean numbers of follicles and oocytes, their mean ratios, mean number of embryos, mean oestrogen concentrations, mean size of the follicles and pregnancy rates. Analyses were carried out in 200 local Chinese patients undergoing in-vitro fertilization (IVF) and embryo transfer in three consecutive cycles. The mean follicular number, oocyte number, embryo number, follicular size and pregnancy rate were significantly smaller in patients homozygous for PvuII polymorphism (P < 0.001). These results indicate that PvuII polymorphism may be associated with ovarian follicular development and subsequently with the pregnancy rate. This study supports the view that genetic variability in the ER gene may have a role in the quality of the ovarian follicles in stimulation, which may affect implantation. However BstUI polymorphism was not found in either the IVF or control groups, suggesting that it has no role in the local Chinese population.     

External validation of prognostic models for ongoing pregnancy after in- vitro fertilization

This study aimed to validate prognostic models for predicting ongoing pregnancy after the first and second in-vitro fertilization cycles. Models were developed using data from the University Hospital, Nijmegen, 1991-1994 and tested using more recent data from the same centre and data from two other centres. Although the variables included in the models seemed plausible, the predictions of the models were unsatisfactory. The models did not discriminate between women who had achieved pregnancy and women who did not achieve pregnancy; neither could they indicate which women had a (very) low probability of ongoing pregnancy. Taking into account the success rate of a specific clinic or the success rate during a specific period did not show any advantage. The predictions were even inaccurate in the same hospital during another period. It is obvious that these prognostic models should not be used. This study shows the importance of validating prognostic models before their implementation in clinical practice.      

Parental human leukocyte antigens and implantation failure after in- vitro fertilization

At present, it is well accepted that maternal recognition of paternally derived fetal antigens occurs during normal pregnancy and may be beneficial for implantation and maintenance of gestation. Thus, we have investigated the compatibility of human leukocyte antigens (HLA) in couples with successive failed in-vitro fertilization (IVF) cycles. Study group 1 included 50 couples with prior primary infertility who had not achieved a pregnancy after > or = 3 (range 3-7, mean 3.7) IVF cycles where at least two embryos (mean 3.3, range 2-4) were transferred in each attempt. An infertile control group (group 2) included 50 infertile couples undergoing IVF with the same indications as couples in group 1, who achieved a viable pregnancy with their first IVF attempt. The results were compared with those found in a population sample including 100 men and 100 women from the local population (group 3). We found a statistically significant (P < 0.05) excess of HLA sharing (> or = 2 antigens) between partners in group 1 as compared to groups 2. There was a trend toward increased HLA sharing in group 1 when groups 1 and 3 were compared. We conclude that some cases of implantation failure after IVF and embryo transfer might be caused by underlying close histocompatibility between partners.      

Recurrent abnormal follicular maturation and ovarian stimulation for in-vitro fertilization

A total of 1078 cycles were stimulated for in-vitro fertilization between 1 January 1986 and 31 July 1987. One-hundred-and-ninety-six cycles had to be cancelled because of an abnormal ovarian response (18.2%). The majority of these cancellations were due to the phenomenon of follicular atresia (45%) or the absence of follicular growth (30%). Bad responder patients who had undergone cancellation during at least two successive cycles, when compared with normal responder patients, were somewhat older and had more frequent periovarian adhesions and ovulatory defects. The use of a GnRH agonist allowed correction of a certain number of ovarian response inadequacies, but follicular atresia and absence of folliculogenesis remained frequent phenomena.     

Premature luteinization of follicles during ovarian stimulation for in-vitro fertilization

The structure and growth of developing follicles was monitored using vaginal ultrasound scanning in an outpatient programme of in-vitro fertilization (IVF) and embryo transfer (ET). Patients received either human menopausal gonadotrophin (HMG) alone or clomiphene citrate (CC) + HMG for controlled ovarian stimulation. Ultrasound data were compared with pre-ovulatory oestradiol (E2), luteinizing hormone (LH) and progesterone (P) levels. Hormonal parameters and results were classified according to the main indications of IVF-ET treatment. Twenty-one of the 271 patients in the study showed ultrasonic evidence of premature luteinization (PL) of follicles, thickening of the follicular wall and the appearance of irregular echogenic structures in the follicle. PL was preceded in eight cases by an indisputable LH surge and subsequent P elevation. In the remaining 13 cases PL occurred either due to an abortive LH surge not exceeding by 3-fold the baseline values or as a result of HMG administration. Special attention was paid to the P pattern prior to and after human chorionic gonadotrophin (HCG) administration. PL cycles demonstrated significantly (P less than 0.05) higher P levels before HCG administration and at the time of oocyte retrieval as well. Because implantation was not achieved in these cases, the cancellation of PL cycles is recommended. Vaginal ultrasound scanning seems to be helpful in the evaluation of minor changes in the follicular structure, correlating frequently with hormonal findings.     

A prospective randomized trial of blastocyst culture and transfer in in- vitro fertilization

The effectiveness of blastocyst culture and transfer in human in-vitro fertilization (IVF) was evaluated in a prospective randomized trial in patients having a moderate to good response to gonadotrophin stimulation. Embryos were transferred either on day 3 after culture to around the 8-cell stage in Ham's F-10 medium supplemented with fetal cord serum, or on day 5 after culture to the blastocyst stage in the sequential serum-free media G 1.2 and G 2.2. The pregnancy rates after transfer on day 3 or day 5 were equivalent, 66 and 71% respectively; however, significantly more embryos were transferred on day 3 (3.7) than on day 5 (2.2). The number of blastocysts transferred did not affect the implantation rate, and pregnancy rates when either two or three blastocysts were transferred were 68 and 87% respectively. The implantation rate of the blastocysts (50.5% fetal heart beat) was significantly higher compared to the cleavage stage embryos transferred on day 3 (30.1%). The percentage of blastocyst development was not affected by the number of 2-pronuclear embryos, or by maternal age. Irrespective of the number of blastocysts formed, pregnancy rates were similar. Furthermore, the pregnancy rate following blastocyst transfer in patients with 10 or more follicles at the time of human chorionic gonadotrophin administration was not affected by patient age. More than 60% of patients having blastocyst culture and transfer had supernumerary embryos for cryopreservation. The establishment of a pregnancy following thaw and transfer confirmed the viability of cryopreserved blastocysts cultured in the absence of serum or co- culture. The ability to transfer just two blastocysts while maintaining high pregnancy rates will therefore help to eliminate high order multiple gestations and improve the overall efficiency of human IVF.      

Cumulative pregnancy rates and selective drop-out of patients in in- vitro fertilization treatment

The validity of the cumulative pregnancy rate (CPR) calculated by life- table approach, obtained in a transport in-vitro fertilization (IVF) programme, was tested by the determination of possible influence of selective drop-out of patients with a poor treatment prognosis. A cohort of 1211 patients who had a first IVF cycle was followed, and the CPR after three IVF cycles was assessed. First cycles of patients who discontinued treatment after failed IVF, and of those who did not achieve a pregnancy but proceeded to a subsequent cycle, were compared for fertilization rate and for occurrence of prognosticators of poor treatment outcome: oocyte yield < or =2, and replacement of <2 embryos. The CPR after three cycles was 54.9%. No differences were found in the first and second cycles of patients who continued treatment and those who dropped out. Selective drop-out of patients with a poor treatment prognosis was not found. Therefore, although calculations of CPR using life-table analysis generally overestimate the real probability of pregnancy after successive IVF cycles, the calculated CPR after three IVF cycles gives a reliable indication of the chance of occurrence of a pregnancy for the population studied.      

Does ovarian stimulation for in-vitro fertilization induce a hypercoagulable state?

Effects on blood coagulation and fibrinolytic activity during ovarian stimulation for in-vitro fertilization (IVF) were examined in 12 women. Blood samples were taken prior to hormonal stimulation (days 2-3 of the menstrual cycle, mean serum oestradiol concentration 0.16 nmol/l) and the day after ovulation induction with human chorionic gonadotrophin (HCG) (days 10-12, mean serum oestradiol concentration 5.35 nmol/l). We measured whole blood clotting time, whole blood clot lysis time, plasma fibrinogen, factor VII and antithrombin III. The whole blood clotting time was slightly, but not significantly shortened after ovarian stimulation. A significant rise in plasma fibrinogen (P less than 0.001) and reduction in antithrombin III (P less than 0.001) were observed, whereas no change in factor VII was found. The blood fibrinolytic activity was significantly reduced as evaluated by an increase in the clot lysis time (P less than 0.02). These results indicate that ovarian stimulation for IVF may create a state of hypercoagulability.     

An improved use of buserelin in ovarian stimulation for in-vitro fertilization

In earlier IVF programmes, subcutaneous buserelin (Suprefact, Hoechst) was initially administered three times per day (200 micrograms x 3); then twice daily (300 micrograms x 2). We now suggest that a single administration of 600 micrograms daily may be equally effective. In a preliminary study, 20 patients were selected on the basis of tubal or idiopathic infertility and received 0.6 ml buserelin subcutaneously once a day, beginning on day 1 or 2 of the cycle. A sufficient pituitary desensitization was obtained on day 10 in 75% of patients and on day 16 for 100% and the ongoing pregnancy rate was 35% per treatment cycle. A randomized study comparing the effect of 600 micrograms of buserelin administered in one (n = 50) or two injections (n = 46), has been carried out and indicates that the results in terms of the ovarian suppression and pregnancy rates, were similar. Therefore, this protocol represents a simplification of the treatment with buserelin.     

A prospective, longitudinal study of emotions and relationships in in- vitro fertilization treatment

Emotional and relationship assessments were completed by 144 couples at intake for in-vitro fertilization (IVF) and 6 months after either the identification of pregnancy or the discontinuation of treatment following three unsuccessful cycles. Women also completed emotional assessments at the time of pre-oocyte recovery and post-embryo replacement within each treatment cycle. At intake, women were more anxious than their partners and comparative norms, and were less positive than men about their marital and sexual relationships. Initial emotional assessments were not related to subsequent pregnancy, but at follow-up those who were pregnant were less depressed and more positive about their relationships. Within treatment cycles scores for women were higher after embryo replacement and the failure of pregnancy. First and last treatment cycles were associated with greater anxiety. High levels of confusion and bewilderment found during the initial cycle may indicate the need for better pretreatment information. Services must recognize the presence of high anxiety at intake and provide psychological care for those identified as particularly distressed. Emotional difficulties after failure of IVF treatment can be considered to be iatrogenic effects, and psychological services should be provided to minimize any negative psychological consequences of treatment.      

The application of neural networks in predicting the outcome of in- vitro fertilization

Infertility affects one in six couples at some time in their lives, with 48% of these couples requiring assisted conception techniques in order to achieve a pregnancy. Whilst the overall clinical pregnancy rate per embryo transfer is 23%, this varies widely between clinics. The Human Fertilisation and Embryology Authority has attempted to analyse the results of all units, with weighting of different factors affecting assisted conception, and the published data have invariably led to comparisons between units. However, statistical models need to be developed to eliminate bias for valid comparisons. Neural networks offer a novel approach to pattern recognition. In some instances neural networks can identify a wider range of associations than other statistical techniques due in part to their ability to recognize highly non-linear associations. It was hoped that a neural network approach may be able to predict success for individual couples about to undergo in-vitro fertilization (IVF) treatment. A neural network was constructed using the variables of age, number of eggs recovered, number of embryos transferred and whether there was embryo freezing. Overall the network managed to achieve an accuracy of 59%.      

ESHRE consensus on the definition of 'poor response' to ovarian stimulation for in vitro fertilization: the Bologna criteria

The definition presented here represents the first realistic attempt by the scientific community to standardize the definition of poor ovarian response (POR) in a simple and reproducible manner. POR to ovarian stimulation usually indicates a reduction in follicular response, resulting in a reduced number of retrieved oocytes. It has been recognized that, in order to define the poor response in IVF, at least two of the following three features must be present: (i) advanced maternal age or any other risk factor for POR; (ii) a previous POR; and (iii) an abnormal ovarian reserve test (ORT). Two episodes of POR after maximal stimulation are sufficient to define a patient as poor responder in the absence of advanced maternal age or abnormal ORT. By definition, the term POR refers to the ovarian response, and therefore, one stimulated cycle is considered essential for the diagnosis of POR. However, patients of advanced age with an abnormal ORT may be classified as poor responders since both advanced age and an abnormal ORT may indicate reduced ovarian reserve and act as a surrogate of ovarian stimulation cycle outcome. In this case, the patients should be more properly defined as 'expected poor responder'. If this definition of POR is uniformly adapted as the 'minimal' criteria needed to select patients for future clinical trials, more homogeneous populations will be tested for any new protocols. Finally, by reducing bias caused by spurious POR definitions, it will be possible to compare results and to draw reliable conclusions.     

Pro-caspase-3 is constitutively expressed in luteinized granulosa cells from women undergoing controlled ovarian stimulation for in vitro fertilization

Apoptosis regulation in luteinized granulosa cells (LGC) during assisted reproduction procedures is still controversial. Caspase-3 is a major apoptosis mediator encoded by CASP3 and formed through cleavage of its precursor pro-caspase-3. The aim of this study was to investigate the expression patterns of pro-caspase-3 (mRNA and protein) and cleaved caspase-3 in human LGC. Thirty-five women undergoing controlled ovarian stimulation for in vitro fertilization were prospectively enrolled in the study. LGC were isolated from follicular fluid during oocyte pickup and evaluated by immunocytochemistry for pro-caspase-3 and cleaved caspase-3, and by real-time PCR for CASP3 mRNA expression. We found a positive staining of pro-caspase-3 in 77 % of the LGC (95 % confidence interval [CI] 60%–84%), whereas cleaved caspase-3 was found in only 4% of the cells (95 % CI 3%–6%). The abundance of cells expressing pro-caspase-3 was independent from CASP3 mRNA levels (r = 0.24, p = 0.255) and did not correlate with the amount of cleaved caspase-3 (r = -0.24, p = 0.186). Multivariable logistic regression showed that pro-caspase-3 positivity was not influenced by clinical characteristics such as age, cause or length of infertility, antral follicle count or hormonal drugs used to induce ovulation. These findings suggest that pro-caspase-3 is constitutively expressed in LGC, allowing quick cleavage into active caspase-3 and apoptosis triggering whenever needed in the course of gonadotropin-induced follicular development.     

The impact of ovarian cystectomy on ovarian response to stimulation during in-vitro fertilization cycles

This study was carried out to investigate whether ovarian cystectomyinterferes with follicular recruitment and the number of oocytesretrieved in an in-vitro fertilization (IVF) cycle. Patientswho had previously undergone unilateral ovarian cystectomy (n= 90) and control patients (n = 90) with no history of ovariansurgery were included in our study. The parameters comparedwere the number of follicles recruited and the number of oocytesobtained from each ovary. In patients who had undergone surgery,the normal ovaries recruited a significantly higher number offollicles (P < 0.001) and yielded a significantly highernumber of oocytes (P < 0.001) compared with the contralat-eralovaries which had undergone cystectomy. In the control patients,no significant differences were identified between the leftand right ovaries. These results demonstrate that ovarian cystectomyreduces follicle and oocyte numbers in ovulation induction cycles.     

人类不同促排卵方案获得的未成熟卵母细胞体外成熟后发育潜能的比较

目的比较体外受精治疗周期中两种不同治疗方案所获得的未成熟卵母细胞经体外成熟培养后的发育潜能。方法根据未成熟卵母细胞来源分为超促排卵组和自然周期组。两组未成熟卵母细胞经未成熟卵母细胞经体外成熟(IVM)后进行常规夫精卵胞浆内单精子注射。比较两组间卵母细胞的成熟率、受精率、卵裂率、优良胚胎率和临床妊娠率的差异。结果超促排卵组受精率、卵裂率和优质胚胎率分别为64.4%、89.9%、17.5%,自然周期组76.7%、100%、33.3%,两组之间的差异无统计学意义(P0.05),但自然周期组的未成熟卵母细胞的成熟率95.6%、可冷冻胚胎率64.2%及临床妊娠率26.3%均显著高于超促排卵组65.5%、26.2%、0,且差异有统计学意义(P0.05)。结论在体外成熟培养中,自然周期获得的未成熟卵母细胞较超促排卵获得的未成熟卵母细胞具有更好的发育潜能。