Spindle and chromosome configurations of human oocytes matured in vitro in two different culture media.

In-vitro maturation can have deleterious effects on spindle formation and proper chromosome alignment in human oocytes and can be profoundly affected by culture conditions. This study compared the spindle presence and location with the maturation rate of germinal vesicle (GV) oocytes cultured in two different media: G1.2 and G1.2 supplemented with follicle-stimulating hormone, human chorionic gonadotrophin and 17beta-oestradiol. A total of 304 oocytes were retrieved from 101 women undergoing IVF treatment with intracytoplasmic sperm injection. Spindle presence was recorded using the Polscope. Spindle morphology was evaluated with immunocytological staining for alpha-tubulin and chromatin. Twenty-one in-vitro matured oocytes with the presence of spindle and ten of their corresponding polar bodies (PB) were also assessed for aneuploidy. A significantly increased maturation rate (69.7%) was observed after 24h in the supplemented culture media compared with the G1.2 media (56.6%; P<0.05). The proportions of metaphase II (MII) oocytes with spindle presence and abnormal spindle morphology were similar in the two culture media. Also, 76.9% of MII and 70% of PB had chromosomal abnormalities. In conclusion, supplementing culture media may increase the oocyte maturation rate in vitro, but does not necessarily indicate the presence of a birefringent spindle, or normal spindle and chromosomal alignment.

Long-term follow-up after cervical cancer treatment and subsequent successful surrogate pregnancy.

Preservation of fertility is a major concern for premenopausal women after diagnosis of cervical cancer. Successful surrogate pregnancy after treatment for cervical cancer has very rarely been reported. In the present report, a case of successful surrogate pregnancy after radical hysterectomy, lymphadenectomy and ovarian transposition for cervical cancer, followed by radiation therapy, is presented. After stimulation of the transposed ovaries using the short gonadotrophin-releasing hormone (GnRH) analogue protocol, four oocytes were retrieved transabdominally from the genetic mother. IVF followed and two embryos were transferred to the surrogate mother, leading to an uneventful singleton pregnancy, and ultimately normal vaginal delivery of a healthy female infant at term. The unique aspect in this case is the long-lasting favourable outcome for both genetic mother and child, observed during 8.5 years of follow-up, the longest follow-up period reported to date in such cases.

Pregnancies following blastocyst stage transfer in PGD cycles at risk for beta-thalassaemic haemoglobinopathies.

BACKGROUND: Preimplantation genetic diagnosis (PGD) usually involves blastomere biopsy 3 days post-insemination (p.i.), followed by genetic analysis and transfer of unaffected embryos later on day 3 or 4. We evaluate a strategy involving embryo biopsy on day 3 p.i., genetic analysis on day 4 and, following culture in blastocyst sequential media, transfer of unaffected embryos on day 5 p.i. METHODS: PGD cycles were initiated in 15 couples at risk of transmitting beta-thalassaemia major. Oocyte retrieval and ICSI were performed according to standard protocols. Embryo culture used blastocyst sequential media. Embryos were biopsied on day 3 p.i. using acid Tyrode's for zona drilling, and the single blastomeres were genotyped by a protocol involving nested polymerase chain reaction and denaturing gradient gel electrophoresis analysis. RESULTS: Forty of 109 (37%) embryos biopsied on day 3 p.i. developed to blastocysts by day 5 p.i., with at least one blastocyst available for transfer in 12 cycles (80%). Genotype analysis characterized 51/109 (47%) embryos unaffected for beta-thalassaemia major, of which 28 were blastocysts. Transfer of 37 day 5 p.i. embryos (blastocysts and non blastocysts) initiated eight clinical pregnancies. Implantation rate per embryo transferred was 12/37 (32%). CONCLUSIONS: Embryo biopsy on day 3, followed by delayed transfer until day 5 p.i. offers a novel and effective strategy to overcome the time limit encountered when performing PGD, without compromising embryo implantation.

A widely applicable strategy for single cell genotyping of beta-thalassaemia mutations using DGGE analysis: application to preimplantation genetic diagnosis.

Preimplantation genetic diagnosis (PGD) allows the selection of unaffected IVF embryos for transfer in couples that are at risk for transmitting genetic diseases. For monogenic diseases, polymerase chain reaction (PCR)-based diagnosis is usually performed on single blastomeres. In Greece, up to 10 per cent of the population are carriers for beta-thalassaemia and related haemoglobinopathies, and more than 20 pathological mutations in the beta-globin gene have been described. In this study we report a strategy which includes a first round of PCR, allowing subsequent nested PCR and DGGE analysis for at least 95 per cent of beta-thalassaemia major genotypes in the Greek population. The use of DGGE for beta-globin genotype analysis is advantageous: it facilitates simultaneous analysis of more than one mutation in a single PCR fragment, it detects the presence of normal alleles and monitors the occurrence of allelic drop-out (ADO) through the expectation that heterozygous samples have more than one electrophoretic band on DGGE analysis. The optimization, accuracy and reliability of the method was evaluated by genotyping 325 single blastomeres, 110 amniocytes and 55 lymphocytes. Results confirmed that PCR efficiency and occurrence of ADO are improved by higher denaturation temperatures in the first cycles of first-round PCR, influenced by the size of the fragment amplified in the first round of PCR and additionally by the quality and type of cells being genotyped. The proposed strategy was accurate and reliable, and thus for application to PGD should ensure the transfer of unaffected embryos. Furthermore it is widely applicable in most of the populations worldwide where beta-thalassaemia is common.

Preimplantation genetic diagnosis in 10 couples at risk for transmitting beta-thalassaemia major: clinical experience including the initiation of six singleton pregnancies.

Preimplantation genetic diagnosis (PGD) offers couples at risk for transmitting an inherited disorder the possibility to avoid the need to terminate affected pregnancies, since it allows the selection of unaffected IVF embryos for transfer. PGD for monogenic diseases is most commonly accomplished by blastomere biopsy from cleavage stage embryos, followed by polymerase chain reaction (PCR)-based DNA analysis. However, PCR-based DNA analysis of single cells is subject to several problems including sample contamination, total PCR failure, or failure of one allele to amplify--a phenomenon known as allelic drop-out (ADO). Furthermore, the molecular heterogeneity of many monogenic diseases requires a diagnostic strategy capable of detecting a spectrum of mutations and compound genotypes. With the above considerations we developed an accurate and reliable strategy for analysis of beta-globin gene mutations, applicable for PGD for the wide spectrum of beta-thalassaemia major genotypes in the Greek population. The strategy involves nested PCR followed by denaturing gradient gel electrophoresis (DGGE) analysis. DGGE is an advantageous method for mutation detection since it facilitates simultaneous analysis of more than one mutation in a single PCR fragment, it identifies the presence of normal alleles and in addition can monitor the occurrence of ADO. This report describes the application of the DGGE-based diagnostic strategy in 11 clinical IVF/PGD cycles, in 10 couples at risk for transmitting beta-thalassaemia major. The transfer of at least one embryo diagnosed as unaffected for beta-thalassaemia major in nine couples has resulted in the initiation of six pregnancies. Four pregnancies have so far been confirmed as unaffected for beta-thalassaemia major by first or second-trimester prenatal diagnosis, two of which have resulted in the birth of two healthy babies. Three singleton pregnancies are still on-going and one ectopic pregnancy was terminated.

Fertility Outcome after Outpatient Hysteroscopic Removal of Endometrial Polyps and Submucous Fibroids

We assessed the value of hysteroscopic removal of polyps and submucous fibroids from women requesting fertility treatment. Between December 1991 and June 1995 we recruited 146 women (age 28-44 yrs) with such lesions who were undergoing investigations for in vitro fertilization (IVF). In most of them, intrauterine pathology was not the sole cause of subfertility. In all women the lesions were diagnosed by hysterosalpingography or vaginal ultrasound examination with fluid instillation and subsequent hysteroscopy. In 122 patients the lesion was removed hysteroscopically with the resectoscope under light sedation in an outpatient setting. No complications occurred during or after surgery. The remaining 24 patients with polyps smaller than 2 cm underwent IVF treatment directly. In the 122 women who proceeded to treatment, further diagnostic office hysteroscopy was also performed. Group A consisted of 82 women with up to three polyps (65 <2 cm, 17 >2 cm); group B, 40 women with up to five fibroids smaller than 4 cm; and group C, 24 women with ultrasound diagnosis of polyps smaller than 2 cm for whom no treatment was carried out. In groups A and B the diagnosis was confirmed histologically. All these women subsequently underwent IVF. In group A (<2 cm polyps) the pregnancy rate was 28% per embryo transfer, and in the rest of group A (>2 cm) it was 40%. In group B the pregnancy rate was 46% and in group C 35%. We think that polyps less than 2 cm diameter do not require removal before IVF and do not affect the outcome of the subsequent pregnancy.

Comparison of Operative and Fertility Outcome Between Groups of Women with Intrauterine Adhesions after Adhesiolysis

We evaluated intraoperative difficulty and fertility outcome in women with intrauterine synechiae of different severity after hysteroscopic adhesiolysis. The 86 women were recruited before fertility treatment, during hysterosalpingogram (HSG) or diagnostic hysteroscopy, over 3 years. Fifty-eight women had a history of pregnancy terminations or miscarriages, and 28 had undergone myomectomy or correction of congenital uterine anomalies by conventional surgical procedures. Group A (11 women) had over 50% of the fundal cavity obliterated by fibrous tissue, and no tubal ostia could be seen; group B (26 women) had less than 50% of the fundal cavity obliterated by fibrous tissue, and one tubal ostium could be seen; group C (49 women) had a single adhesion thicker than 1 cm. Postoperative hemorrhage requiring treatment occurred in three women from group A and two from group B. All had subsequent HSGs, and three from group A (with previous myomectomies) required a second operation and still failed to achieve an adequate fundal cavity. All patients subsequently received in vitro fertilization. Three women from group A became pregnant. Ten in group B became pregnant, eight of whom delivered or have a continuing pregnancy. In group C, 17 became pregnant, of whom 13 delivered or have a continuing pregnancy. Adhesions after suturing the uterine cavity after open myomectomy or corrective surgery seem to cause maximum damage and yield poor results in both hysteroscopic correction and reproductive performance.

Immunohistochemical expression of placental alkaline phosphatase and vimentin in epithelial ovarian neoplasms.

The immunohistochemical expression of PLAP and vimentin was assessed in 23 benign, 6 borderline malignant and 31 malignant epithelial ovarian neoplasms. PLAP and vimentin were expressed in some benign (3/23 and 5/23 respectively) and borderline malignant (2/6 for both markers) tumours and they were often expressed in malignant tumours (16/31 and 17/31 respectively). There was a significantly increased expression of PLAP and vimentin in serous cystadenomas and serous carcinomas compared to their mucinous counterparts. Although there was no significant correlation between PLAP expression and histologic grade of carcinomas there was a trend towards increased expression in more differentiated carcinomas. No correlation was found between vimentin expression and degree of differentiation.

Value of immunohistochemical demonstration of several epithelial markers in hyperplastic and neoplastic endometrium.

The distribution of prekeratin, vimentin, epithelial membrane antigen (EMA), and secretory component (SC) was demonstrated immunohistochemically in 31 patients with adenomatous hyperplasia (AH), 12 patients with atypical adenomatous hyperplasia (AAH), and 39 patients with endometrial carcinoma. Prekeratin was presented in 94% of AHs, 92% of AAHs, and 87% of adenocarcinomas. Vimentin was detected in 68% of AHs, 50% of AAHs, and 37% of adenocarcinomas, showing decreased expression as the lesion progressed to malignancy (P less than 0.05). EMA was detected in 26% of AHs, 67% of AAHs, and 95% of adenocarcinomas (P less than 0.001). SC demonstrated focal and weak expression in 29% of AHs, but showed increased staining intensity in 67% of adenocarcinomas (P less than 0.01). Well-differentiated tumors expressed SC better than poorly differentiated tumors (P less than 0.01). All markers showed a heterogeneous staining pattern and, for a given histologic hyperplastic or neoplastic state, corresponded to several phenotypes. In conclusion, prekeratin seems to be a good marker for epithelial differentiation in hyperplastic endometrium, and EMA is a good marker in neoplastic endometrium. In hyperplastic lesions, the loss of vimentin expression in the absence of secretory changes gives rise to suspicions regarding their benign process. Also, EMA can help in distinguishing between hyperplastic and neoplastic states, while detection of SC may be of help in more precise grading of endometrial carcinoma.