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Oocyte cryopreservation for age-related fertility loss is gaining interest considering the tendency to postpone motherhood in many societies. Little is currently known about the actual efficiency of this approach. We aimed to explore ovarian response of presumably fertile women undergoing in vitro fertilization for this indication. A total of 105 women underwent 151 stimulation cycles at mean age 37.7 ± 2.4. None had known infertility. Mean daily starting FSH dose was 371 ± 110 (225-600). Mean number of mature oocytes cryopreserved at the first completed cycle was 9.7 ± 7.5 (0-43). However, 21% of started cycles were either cancelled before egg retrieval or resulted in 0-3 mature oocytes retrieved. Therefore, women considering oocyte cryopreservation for prevention of age-related fertility decline should be encouraged to perform this procedure at younger age than, preferably before 35.

Myotonic dystrophy (DM) is the most common form of muscular dystrophy in adults. There are conflicting reports about its effect on female fertility. This study investigated ovarian reserve and IVF-preimplantation genetic diagnosis (PGD) outcome in women with DM1. A total of 21 women undergoing PGD for DM1 were compared with 21 age- and body mass index-matched women undergoing PGD for other diseases. Ovarian reserve markers, response to stimulation, embryo quality and clinical pregnancy and live birth rates were compared. Day-3 FSH concentration was higher, while anti-Mullerian hormone concentration and antral follicle count were lower in the DM1 group (median, range: 6.9 (1.8-11.3) versus 5.7 (1.5-10.7)IU/l; 0.9 (0.17-5.96) versus 2.68 (0.5-9.1)ng/ml; and 13 (0-63) versus 23 (8-40) follicles, respectively, all P < 0.05). Total FSH dose was higher (5200 versus 2250 IU, P = 0.004), while the numbers of oocytes retrieved (10 versus 16, P < 0.04) and metaphase-II oocytes (9 versus 12, P < 0.03) were lower in the DM1 group. The number of cycles with top-quality embryos and the clinical pregnancy rate were lower in the DM1 group. In conclusion, there is evidence of diminished ovarian reserve and less favourable IVF-PGD outcome in women with DM1. Myotonic Dystrophy (DM) is the most common form of muscular dystrophy in adults. There is evidence of subfertility in males affected with the disease but conflicting reports about the effect of the disease on female fertility. The aim of our study was to investigate ovarian reserve and IVF-PGD results in women with DM. Twenty-one women undergoing preimplantation genetic diagnosis (PGD) treatment for DM were compared to 21 age- and BMI matched women undergoing PGD treatment for other diseases. The two groups were compared for antral follicle count (AFC) and serum anti-Mullerian hormone (AMH) levels (the best known markers of ovarian reserve and fertility potential), ovarian response, embryo quality and pregnancy and live birth rates. AFC and the AMH levels were statistically significant lower in the DM group. Total medication dose needed for ovarian stimulation was higher, the number of oocytes and mature oocytes retrieved, and the number of cycles with top quality embryos were lower in the DM group compared to the controls. In conclusion, there is evidence of diminished ovarian reserve, and less favorable IVF-PGD outcome in women with DM. Therefore, we recommend advising these women about the possibility of early decreasing ovarian function in order to prevent any delay in reproductive planning.

Development of PGD assays for molecular disorders is based on analysis of a familial mutation together with linked polymorphic STR markers; a process which is lengthy and requires the identification of multiple informative markers prior to PGD analysis. On the other hand, whole genome amplification (WGA), in conjunction with microarray platforms, allows the use of a universal assay for the analysis of a very large number of SNP markers at once. The aim of this study was to test high throughput pre-PGD familial haplotyping for in-case blastomere analysis in order to eliminate time-consuming pre-case preparations for each family.A PGD cycle was performed for a couple with paternal Charcot Marie Tooth 1A (CMT1A) using a classic multiplex nested PCR approach. Mutant embryos from the case were blindly reanalyzed, as single or multi-cell biopsies, using a multiple displacement amplification-based WGA protocol and microarray SNP analysis. In parallel, relevant genomic DNA samples from the family were also analyzed by SNP microarray.After applying a 'unique informative allele' selection algorithm to the data, this array-based assay reconfirmed the initial diagnosis in all samples.We describe a PGD method that is both accurate and feasible during the time-frame required for embryo transfer. This strategy greatly reduces the time for pre-case haplotype preparation.

Objective Dehydroepiandrosterone (DHEA) supplementation for poor responders may improve ovarian response and IVF treatment outcome. This study aimed to determine the mechanism of action of DHEA, and specifically, the stage of folliculogenesis influenced by DHEA. Study design This is a prospective, self-controlled study of poor responders to IVF treatment, comparing day 3 biochemical (anti-Mullerian hormone (AMH), inhibin B and FSH) and ultrasound (antral follicle count (AFC)) ovarian reserve markers and IVF treatment outcome before and after DHEA supplementation of at least 3 months duration. Results Thirty-two women were included. Following DHEA, there was a significant increase in AFC ( P =0.0003) without significant changes in the baseline biochemical parameters AMH, inhibin B, or FSH. The enhanced response comprised increased peak estradiol levels ( P =0.0005), number of follicles >15mm, oocytes, MII oocytes and embryos ( P =0.004, P =0.00001, P =0.0004 and P =0.0006, respectively) and oocytes number/total FSH dose ( P =0.0009). The proportion of cancelled cycles due to very poor response decreased significantly ( P =0.02). Conclusions DHEA does not appear to exert influence via recruitment of pre-antral or very small antral follicles (no change in AMH and inhibin B) but rather by rescue from atresia of small antral follicles (increased AFC).

Fertility treatments are responsible for the rise in high order pregnancies in recent decades and their associated complications. Reducing the number of embryos returned to the uterus will reduce the rate of high order pregnancies.To explore whether obstetric history and parity have a role in the clinician's decision making regarding the number of embryos transferred to the uterus during in vitro fertilization (IVF).In a retrospective study for the period August 2005 to March 2012, data of twin deliveries24 weeks were collected, including parity, mode of conception (IVF vs. spontaneous), gestational age at delivery, preeclampsia, birth weight, admission to the neonatal intensive care unit (NICU), and Apgar scores.A total of 1651 twin deliveries24 weeks were record- ed, of which 959 (58%) were at term (37 weeks). The early preterm delivery (PTD) rate (32 weeks) was significantly lower with increased parity (12.6%, 8.5%, and 5.6%, in women with 0, 1, and ≥ 2 previous term deliveries, respectively). Risks for PTD (37 weeks), preeclampsia and NICU admission were significantly higher in primiparous women compared to those who had one or more previous term deliveries. Primiparity and preeclampsia, but not IVF, were significant risk factors for PTD.The risk for PTD in twin pregnancies is significantly lower in women who had a previous term delivery and decreases further after two or more previous term deliveries. This finding should be considered when deciding on the number of embryos to be transferred in IV.

The aim of this study was to develop and perform a preimplantation genetic diagnosis (PGD) assay discriminating between wild-type and mutant alleles in two families with germline mosaicism. Family 1 had two children affected with severe myoclonic epilepsy (SCNA1A del exons 1-22). Family 2 had two children with tuberous sclerosis (TSC2 C1327T) and two healthy children. Neither mutation was detected in genomic DNA derived from the parents in either family. Informative microsatellite markers flanking SCNA1A and TSC2 along with the identified mutations were used to construct haplotypes. For tuberous sclerosis, single spermatozoa were analysed using a multiplex assay that included six informative markers and the TSC2 mutation. In family 1, deletion in the maternal allele was detected in the affected child. In family 2, both affected children and one healthy child shared the same paternal allele. To confirm mutant paternal transmission, single spermatozoa were analysed for the mutation along with six markers. Of 44 single spermatozoa, four showed the mutant T allele, allowing linkage between the mutation and the genetic markers. Both families delivered healthy children following IVF/PGD. In conclusion, germline mosaicism complicates allele assignment when constructing haplotypes for PGD. Sperm analysis is a useful tool for verifying allelic linkage.

Objective To develop a reliable preimplantation genetic diagnosis protocol for antihuman platelet antigen-1 incompatibility for a family in whom antenatal treatment was not possible because of the mother's hypersensitivity to intravenous immunoglobulin (IVIG). Methods Haplotypes were constructed from genomic DNA of the family members. A polymerase chain reaction protocol that included eight microsatellite polymorphic markers and the ITGB3-specific (T196C, rs5918) polymorphism were multiplexed to be used in a single cell protocol, and single blastomeres were analyzed. Results In one preimplantation genetic diagnosis cycle, out of 28 retrieved oocytes, 24 embryos fertilized and 12 underwent biopsy. Three embryos were found to be antihuman platelet antigen-1b/1b homozygotes and two were transferred. This cycle resulted in an uneventful pregnancy and birth of a healthy child. Conclusion In cases in which there is antihuman platelet antigen incompatibility and IVIG cannot be administered, preimplantation genetic diagnosis is a reliable alternative to enable birth of unaffected children.

Objectives Preimplantation genetic diagnosis (PGD) enables the identification of affected embryos prior to implantation. We present for the first time three families in which either the oocytes or embryos obtained from female carriers of mutations in the iduronate-2-sulfatase (IDS) gene underwent PGD for mucopolysaccharidosis type II (Hunter syndrome). Furthermore, we report the first ever derivation of a Hunter's syndrome (46, XX) human stem cell line from embryos (HESC) carrying the IDS and oculocutaneus albinism type 2 mutations. Methods Combined polar body (PB) 1 and 2 or a single cell of a six- to eight-cell embryo (blastomere) was used for genetic analysis by multiplex polymerase chain reaction assay using six microsatellite polymorphic markers flanking the gene and mutation. Results One couple underwent four PB-PGD cycles, with birth of a healthy girl; the second couple with one PB-PGD cycle had healthy twins; the third couple underwent seven cycles of double PGD for Hunter and Albinism syndrome with birth of healthy twins. One novel Hunter 46, XX HESC line was established displaying typical characteristics of HESC cells. Conclusions PGD is a reliable method to prevent pregnancy of children affected with Hunter syndrome. In addition, derived HESC can be further utilized for drug testing and better understanding of the pathogenesis of this syndrome. Copyright © 2011 John Wiley & Sons, Ltd.

Background Preimplantation genetic diagnosis (PGD) is a technique that enables identification of unaffected embryos prior to in vitro fertilization (IVF) transfer in couples at risk for a Mendelian disorder. Most cases involve severe genetic diseases with neurological features and/or major malformations. We present two couples in which PGD was performed for prevention of type 1 Gaucher disease, a non-neuronopathic, non-lethal disorder. Materials and methods We developed a multiplex fluorescent PCR protocol, simultaneously amplifying the familial mutations and eight closely spaced, highly polymorphic informative microsatellite markers surrounding the gene, to be used for PGD analysis. Results Couple #1 mother was homozygous for the N370S mutation and the father carried the 84GG mutation; their first daughter receives specific Gaucher therapy. One PGD cycle resulted in seven embryos of which four had the paternal wild type allele; two were transferred resulting in a healthy baby boy born at term. Couple #2, each a carrier (N370S and R359Q), whose first-born child had died (age 5 years) of Gaucher disease, underwent 7 PGD cycles. Only one cycle resulted in a clinical pregnancy but a miscarriage was followed at 10 weeks. Conclusions PGD is an effective and accurate method for preventing Gaucher disease type I in carrier couples. Since this disease is treatable, special ethical considerations and careful selection of couples should be performed.

Objective To report the first case series of ovarian hemorrhage after transvaginal ultrasonographically guided oocyte aspiration (TVOA). Design Retrospective analysis. Setting In vitro fertilization unit of a tertiary university hospital. Patient(s) Patients who underwent TVOA during a 6-year period. Intervention(s) Surgical intervention due to active bleeding from the ovary. Main Outcome Measure(s) Prevalence and risk factors. Result(s) Among 3,241 patients undergoing TVOA, 7 were diagnosed as having ovarian hemorrhage afterward. All patients were thin, with a body mass index of 19–21 kg/m 2 , and 4 had polycystic ovary syndrome (PCOS). The prevalence of ovarian bleeding among lean patients with PCOS was 4.5%. The odds ratio for bleeding in lean patients with PCOS vs. all other patients was 50 (95% confidence interval 11–250). The interval between the TVOA and surgical intervention ranged from 5 to 18 hours (mean ± SD, 11.4 ± 5 hours). The Δ decrease in hemoglobin levels was 3.2–9 g/dL (mean 6.1 ± 1.8). In 6 of the 7 patients, laparoscopically guided electrocoagulation was sufficient to achieve hemorrhagic control. Conclusion(s) Although acute hemorrhage is a rare event after TVOA, lean patients with PCOS specifically are at much higher risk for this complication.

Background Single cell diagnosis for preimplantation genetic diagnosis (PGD) requires simultaneous analysis of multiple linked polymorphic markers in addition to mutation analysis in order to reduce misdiagnosis. This type of analysis requires building family haplotypes spanning at least two generations. We present three childless couples in whom the female was a de novo mutation carrier in the Duchenne Muscular Dystrophy (DMD), incontinentia pigmenti (IKBKG) or Neurofibromatosis type 2 (NF2) genes, precluding linkage prior to the PGD cycle. We constructed haplotypes based on linked polymorphic markers in these families and performed concurrent diagnosis enabling embryo transfer from the first PGD cycle. Methods Informative markers flanking the DMD, IKBKG and NF2 genes were used to construct non-linked haplotypes. Polar bodies 1 (PB1) and 2 (PB2) were biopsied and analyzed to determine allelic association between the mutation and markers in multiplex PCR reactions. Results For each family, the first PGD cycle allowed the establishment of linked haplotypes based on homozygous PB1 and PB2 analysis; however, no embryos were available for transfer. Subsequent cycles, when performed, confirmed this linkage. A mutation-free child was born to the family affected with DMD and an ongoing pregnancy (32 weeks) was achieved with the carrier of the IKBKG deletion. Conclusions PB analysis for reverse linkage in real-time coupled with the PGD cycle is a powerful tool for diagnosis and linkage between markers and de novo mutations for maternal autosomal dominant or X-linked disorders. Simultaneous amplification of multiple informative markers in conjunction with the mutation allows the building of familial haplotypes and accurate PGD analysis.

Development of an efficient and reliable PGD protocol for nonsyndromic deafness, by polar body (PB) and blastomere PGD.The GJB2/GJB6 mutations along with 12 polymorphic markers were used in PGD analysis of blastomeres or polar bodies in 14 couples for 35 cycles. Marker informativity, diagnosis rates, Allele Drop Out (ADO) rates and PB1 heterozygosity rates were assessed.Six cycles were performed by PB biopsy, 27 by blastomere and two combined cycles, resulting in delivery of three unaffected children and five ongoing pregnancies. Diagnosis rates for PB and blastomeres were similar. Only 17% PB1s were heterozygote. ADO rates of 19% were observed in both groups.We have developed a single cell multiplex PGD protocol for nonsyndromic deafness with a high efficiency of diagnosis. Most PB1 are homozygous, and similar ADO rates were observed; therefore, blastomere biopsy appears to be the method of choice for this autosomal recessive disease.

A high prevalence of low levels of cobalamin had been found in a survey of multi-ethnic normal individuals in Israel. The purpose of this study was to investigate the incidence of cobalamin deficiency among Israeli couples suffering from infertility. All couples seen at the in vitro fertilization clinic at an urban hospital (Shaare Zedek Medical Center) in Jerusalem for a 6-month period were invited. Mean cobalamin levels were 259.2 pg ml(-1) in males and 275.1 pg ml(-1) in females (normal >200 pg ml(-1)), 35.5% of 172 men and 23.3% of 223 females had cobalamin deficiency (P = 0.01). There were 171 couples with complete demographic questionnaires and cobalamin values for each partner. In 74 couples (43.3%), one partner was cobalamin deficient, with no significant difference between those with unexplained infertility versus those with explained infertility; and in 13 couples, both partners were cobalamin deficient. Thirty-nine per cent of all men with an abnormal semen analysis had cobalamin deficiency, a finding that requires further investigation. This study questions whether higher rates of male infertility in Israel are partially ascribable to cobalamin deficiency. Recommendation for supplementation in both males and females to achieve high-normal levels of cobalamin would be prudent.

The tendency to postpone childbearing in developed countries and the relatively high rate of infertility in older women contribute to an increase in the portion of women aged 40 years and older opting for infertility treatments. The main factor for infertility in this group is oocyte senescence, but since this process does not have a specific diagnosis many of those will be classified as having 'unexplained infertility'. The efficacy of the traditional clinical approach for 'unexplained infertility' in older women is questionable. Reviewing the current literature, clomiphene citrate seems to be inefficient in this group of patients, while delivery rates of gonadotrophins and intrauterine insemination cycles are less than 5%. Although low in absolute terms, IVF is more efficient. The chance for delivery, however, diminishes with each year of age above 40. Therefore, after a short trial of gonadotrophins and intrauterine insemination, women aged 40-41 years should be quickly referred to IVF. At an older age, IVF is the primary treatment option.

Achondroplasia, the most common form of dwarfism, is a candidate for preimplantation genetic diagnosis (PGD) because a single mutation accounts for almost all cases. Multiplex fluorescent assay including the common G380R mutation in the FGFR3 gene and eight close polymorphic markers was developed. First and second polar bodies (PB) were used for PGD analysis. An affected woman was treated with routine long-protocol ovarian stimulation and puncture. In the first PGD cycle, out of four fertilized oocytes, PB analysis revealed two mutant oocytes, one with total amplification failure of the maternal allele and one with inconclusive results. In the second PGD cycle, 14 oocytes were retrieved following a higher FSH dose and by performing oocyte retrieval and by placing the patient in the anti-Trendelenburg position using abdominal pressure to allow all follicles to be drained. Following PB analysis, two embryos containing the wild-type FGFR3 allele were transferred. This led to an uncomplicated pregnancy and delivery by Caesarean section at week 38 of a healthy boy, carrying the FGFR3 wild-type maternal allele. In conclusion, oocyte retrieval, while difficult in patients with achondroplasia, can be successfully performed. PB analysis is a reliable and sensitive method for PGD for maternal achondroplasia.

Objective The development of a preimplantation genetic diagnosis (PGD) protocol for Alagille syndrome (AGS), a rare autosomal dominant disorder with hepatic, cardiac and ophthalmologic involvement. Methods We developed a polar body (PB)-based multiplex fluorescent PCR reaction for a female affected with AGS. The protocol included analysis of the Jagged 1 (JAG1) familial mutation and five closely linked highly polymorphic markers (D20S162, D20S901, D20S894, and D20S186). Results In two cycles of PGD 9 of ten embryos were accurately diagnosed by assessment of first and second PBs, one embryo required additional blastomere biopsy. Conclusions This protocol takes advantage of the larger window of opportunity for transfer and the increased accuracy of diagnosis afforded by the combination of PB biopsy and multiple marker analysis. Two cycles resulted in the transfer of two and three mutation-free embryos and a subsequent pregnancy as measured by the rising hCG levels. Copyright © 2007 John Wiley & Sons, Ltd.

Preimplantation genetic diagnosis (PGD) for single gene defects is described for a family in which each parent is a carrier of both Tay-Sachs (TS) and Gaucher disease (GD). A multiplex fluorescent polymerase chain reaction protocol was developed that simultaneously amplified all four familial mutations and 10 informative microsatellite markers. In one PGD cycle, seven blastomeres were analysed, reaching a conclusive diagnosis in six out of seven embryos for TS and in five out of seven embryos for GD. Of the six diagnosed embryos, one was wild type for both TS and GD, and three were wild type for GD and carriers of TS. Two remaining embryos were compound heterozygotes for TS. Two transferable embryos developed into blastocysts (wt/wt and wt GD/carrier TS) and both were transferred on day 5. This single cycle of PGD resulted in a healthy live child. Allele drop-out (ADO) was observed in three of 34 reactions, yielding an 8% ADO rate. The occurrence of ADO in single cell analysis and undetected recombination events are primary causes of misdiagnosis in PGD and emphasize the need to use multiple polymorphic markers. So far as is known, this is the first report of concomitant PGD for two frequent Ashkenazi Jewish recessive disorders.

The aim of our study was to assess the prevalence of subclinical hypothyroidism (SH) after administering a thyrotropin-releasing hormone (TRH) stimulation test among women with normal serum thyroid-stimulating hormone (TSH) levels and various causes of infertility. Eighty-seven infertile women (39 with ovulation disorders and 48 with other causes of infertility) had a TRH stimulation test on day 3 - 7 of their cycle. Exaggerated TSH response (>30 mIU/l at 20, 40 or 60 min) following intravenous injection of 400 microg TRH was defined as SH. The TRH test was performed 2 - 4 months after the first visit to the clinic. We found that the prevalence of SH was significantly higher among women with ovulation disorders (20.5%) than among women with normal ovulation (8.3%). In addition, we found that although basal TSH levels were normal at recruitment, 2 - 4 months later these levels were abnormally high in 8% of the women. All these women had an abnormal TRH test. We recommend performing TRH stimulation testing in women suffering from ovulation disorders who have normal basal TSH levels, followed by repeat assessments of thyroid function to enable treatment with thyroxine in cases with abnormal results.

Pregnancy rate following one cycle of IVF and ET can be as high as 60%. But even in the very successful units, some couples fail repeatedly. The causes for repeated implantation failure (RIF) may be because of reduced endometrial receptivity, embryonic defects or multifactorial causes. Various uterine pathologies, such as thin endometrium, altered expression of adhesive molecules and immunological factors, may decrease endometrial receptivity, whereas genetic abnormalities of the male or female, sperm defects, embryonic aneuploidy or zona hardening are among the embryonic reasons for failure of implantation. Endometriosis and hydrosalpinges may adversely influence both. In this mini review, we discuss the suggested methods for evaluation and treatment of RIF: repeated hysteroscopy, myomectomy, endometrial stimulation, immunotherapy, preimplantation genetic screening (PGS), assisted hatching, zygote intra-Fallopian transfer (ZIFT), co-culture, blastocyst transfer, cytoplasmic transfer, tailoring stimulation protocols and salpingectomy for hydrosalpinges.

BACKGROUND We aimed to examine the serum levels of inhibin A, vascular endothelial growth factor (VEGF), tumour necrosis factor alpha (TNFalpha), estradiol (E2) and progesterone levels after triggering of final oocyte maturation with GnRH agonist compared with HCG in patients with polycystic ovaries (PCO) and to investigate the relationship between these markers and ovarian hyperstimulation syndrome (OHSS). METHODS Twenty-eight patients with PCO, undergoing controlled ovarian hyperstimulation with FSH and GnRH antagonist for IVF-embryo transfer treatment, were randomized for triggering of final oocyte maturation with GnRH agonist (GnRH agonist group, n = 15) or HCG (HCG group, n = 13). Blood samples were obtained on the day of randomization and thereafter every 2-7 days. Serum levels of inhibin A, VEGF, TNFalpha, E2 and progesterone, the incidence of OHSS, ovarian size and pelvic fluid accumulation were evaluated. RESULTS Serum inhibin A, E2 and progesterone levels were significantly lower in the GnRH agonist group compared with the HCG group, particularly on the day of embryo transfer (P < 0.0001). Serum VEGF and TNFalpha levels were similar between the two groups. Four patients in the HCG group developed severe OHSS, whereas no patient had any symptoms or signs of OHSS in the GnRH-agonist group (P < 0.05). CONCLUSIONS In patients with PCO treated with FSH/GnRH antagonist, final oocyte maturation with GnRH agonist instead of HCG reduces significantly inhibin A, E2 and progesterone levels during the luteal phase. This phenomenon reflects the inhibition of the corpus luteum function and may explain, at least in part, the mechanism of OHSS prevention in high-risk patients. Our results do not support a crucial role for VEGF or TNFalpha in OHSS.

BACKGROUND: We evaluated basal and dynamic hormonal markers [(FSH, inhibin B, estradiol and anti-Mullerian hormone (AMH)] during the follicular phase and luteal phase of the menstrual cycle and ultrasonic ovarian morphology as predictors of IVF outcome. METHODS: Fifty-six women, aged 20 oocytes) had significantly higher AFC, AMH and luteal stimulated inhibin B and estradiol than normal responders. Multivariate regression analyses showed that the best models for predicting oocyte number included AFC, follicular phase AMH and stimulated inhibin B. Only AMH showed a significant difference between pregnant and non-pregnant women at both cycle phases. CONCLUSIONS: In young women (

BACKGROUND: Anti-Mullerian hormone (AMH) is expressed in pre- and small-antral follicles. High serum levels are found in women with polycystic ovaries (PCO), accordant with their increased content of small follicles. To evaluate the relationship between AMH, folliculogenesis and hyperandrogenism, we compared serum AMH levels between women with PCO with and without hyperandrogenism and normal controls during controlled ovarian hyperstimulation (COH). METHODS: Nineteen women with PCO and hyperandrogenism (group A), 10 women with PCO but no hyperandrogenism (group B) and 23 ovulatory women with normal ovarian morphology (group C, controls) underwent COH with the long protocol. Serum levels of AMH, estradiol, androstenedione and follicular tracking were determined before gonadotropins treatment (day 0) and every 2 –4 days up to the day of HCG administration. RESULTS: AMH levels declined gradually throughout COH in the three groups, but remained higher in groups A and B compared with the controls. Significantly higher levels were found in group A compared with group B, despite comparable numbers of small follicles. Multiple regression analysis revealed that both the number of small follicles and serum androgens were correlated to AMH. CONCLUSIONS: Women with PCO have higher serum AMH levels during COH than controls. Hyperandrogenism is associated with an additional increase in AMH. It is conceivable that hyperandrogenism may reflect more severe disruption of folliculogenesis in women with PCO or may affect AMH secretion.

BACKGROUND: The number of (trinucleotide) CAG repeats within the androgen receptor (AR) gene is inversely correlated with transcriptional activity of testosterone-target genes. Although abnormally long CAG repeats are strongly associated with male infertility, it is unclear whether CAG repeat length polymorphism can affect androgen receptor activity and sperm parameters. To explore the previously suggested association between CAG repeats and male fertility, we conducted this prospective cohort study. METHODS: We enrolled 172 men attending the IVF unit in Shaare-Zedek Medical Center. Sperm concentration, motility and morphology and the number of CAG repeats in the AR gene were measured. RESULTS: Mean CAG repeat length was greater in teratozoospermia (

In a previous study we have found that in normal ovulatory women, serum inhibin B levels on days 4-6 of FSH administration correlated with the number of oocytes retrieved. In the current study we examined the significance of earlier inhibin B measurements in predicting the oocyte number, in both normal and low responders.Study A consisted of 19 patients undergoing their first IVF cycle (n = 10) or had a normal response ( vertical line 6 oocytes retrieved, n = 9), while study B consisted of 15 patients with a previous low ovarian response (or=5 oocytes retrieved). All patients had day 3 FSH levels10 IU/l. After pituitary suppression, 300 (study A) or 600 IU (study B) of pure FSH was administered daily. Serum FSH, inhibin A, inhibin B and estradiol (E(2)) were determined prior to and every 1-2 days throughout FSH treatment.Study A: oocyte number between 4 and 14 correlated significantly with serum inhibin B levels on all days of FSH treatment, and with inhibin A and E(2) late during treatment. No correlation was found between inhibin B and when oocyte number was16. Study B: oocyte number correlated significantly with inhibin B and inhibin A on all days of FSH treatment, even on day 2 (r = 0.90, P0.001 and r = 0.65, P0.05 for inhibin B and A respectively). No significant correlation was found with E(2) levels. In both studies, all patients with inhibin B100 pg/ml on treatment day 2 had6 oocytes.Our data suggest that serum inhibin B measured early during FSH stimulation may indicate whether sufficient oocytes will be retrieved, in both normal and low responders. Serum inhibin B measured during early FSH treatment may be of predictive value in monitoring ovarian stimulation treatment for IVF.

Background Recurrent pregnancy loss (RPL) has been associated with impaired maternal–fetal communication. Protease-activated-receptor 1 (PAR1) is critical for trophoblast invasion and establishment unrelated to its role in vascular biology. Objectives To analyze whether polymorphisms of PAR1 [-1426C/T], [-506I/D], and/or IVS[-14A/T] are associated with unexplained RPL. Patients/methods A case-control pilot study conducted in 39 healthy women with history of unexplained RPL and 98 women with a full-term, uncomplicated deliveries and no history of RPL. Results Women with RPL were significantly more likely to be heterozygous for [-1426C/T] (12.8% versus 3.2%; p = 0.049); the heterozygous state for IVS[-14A/T] was also more common (15.4% versus 4.4%; p = 0.064). There was no difference between groups for [-506I/D] genotypes. The functional consequence for [-1426C/T] and IVS[-14A/T] polymorphisms is underscored by the markedly low PAR1 mRNA levels in those women. Bioinformatics indicate generation of a new consensus motif for repressor Kruppel-like factor 3 (KLF3) in [-1426T]. Moreover, chromatin immunoprecipitation (ChIP) analysis confirmed a physical association between KLF3 protein and the hPar1 DNA obtained from women with the [-1426C/T] polymorphism. Conclusions We hypothesize that the significantly low PAR1 levels impact placenta establishment and consequently pregnancy outcome, thereby profiling a novel risk factor for unexplained RPL.

Introduction: Preimplantation genetic diagnosis (PGD) of balanced translocations is usually performed using Fluorescence-in-Situ-Hybridization (FISH), and more rarely by Polymerase-Chain-Reaction (PCR). These methods assess only chromosomes involved in the translocation, as opposed to array comparative-genomic-hybridization (aCGH) which screens all chromosomes. Material and Methods: In this report, we compared the diagnosis and pregnancy rates for balanced translocations using all three techniques in a retrospective study of 75 PGD cycles. Results: Of 331 embryos analyzed by FISH, 225 (68%) were successfully diagnosed, 34 (15%) were transferable yielding a pregnancy rate of 13% per Ovum-Pick-Up (OPU). With PCR, 80 out of 124 embryos (65%) were diagnosed, 21 (26%) were transferable, and a pregnancy rate of 14.3% per OPU was achieved. Using aCGH, 60 of 71 embryos (85%) were successfully diagnosed, 26 of which (43%) presented with chromosomal aneuploidies not associated with the parental translocation. Nine embryos diagnosed by aCGH were transferable yielding a pregnancy rate of 40% per OPU. The proportion of embryos successfully diagnosed and pregnancy rates per OPU were significantly higher using aCGH compared to FISH or PCR (p= 0.036; p=0.017), respectively. Conclusion: Altogether, these data indicate that the use of aCGH for chromosomal translocation PGD significantly increases diagnosis and pregnancy rates.

Objective: To investigate the clinical efficacy of mild inhibition of ovarian steroidogenesis by very low-dose ketoconazole during induction of ovulation in patients with polycystic ovary syndrome (PCOS). Design: Prospective, randomized, cross-controlled study in consecutive cycles. Setting: Large tertiary care center. Patient(s): Eighteen patients with PCOS undergoing hMG superovulation with or without ketoconazole. Intervention(s): A fixed hMG dosage was initiated on cycle days 5–9 in both of the study cycles. Further hMG adjustment was done according to serum E 2 levels and follicular measurements. Ketoconazole was administered in one of the cycles by two protocols. Main Outcome Measure(s): Serum E 2 and P levels, lead follicles, pregnancy rate, and development of ovarian hyperstimulation syndrome. Result(s): Although higher daily hMG doses were needed in cycles with ketoconazole compared with cycles without the drug, the peak E 2 levels were substantially lower in the ketoconazole cycles. Although the number of lead follicles did not differ between treatments, the addition of ketoconazole significantly reduced the number of hyperstimulated cycles. Consequently, the cancellation rate dropped dramatically, thus yielding a higher pregnancy rate per patient in the ketoconazole protocols. Conclusion(s): Use of a very low dose of ketoconazole during ovulation induction effectively attenuates ovarian steroidogenesis in patients with PCOS. This effect may serve as an adjunct to better control the ovarian response in women who are prone to hyperstimulated cycles.

Objective : To determine and compare the transvaginal ultrasonographic (US) endometrial changes immediately after IUI using the Edwards Wallace (H.G. Wallace, Limited, Colchester, Essex, UK) and Tom-Cat (Sherwood Medical, St. Louis, MO) catheters. Design : Prospective study. Setting : IVF unit. Patient(s) and Intervention(s) : Eighty-two infertile patients underwent 112 cycles of ovulation induction with IUI. Either the Edwards Wallace catheter (group 1, n = 32) or the TomCat catheter (group 2, n = 80) was used for sperm insemination. The presence of an endometrial three-layer pattern before IUI was a prerequisite for inclusion in the study. After each IUI, the endometrium was reassessed by transvaginal US. Main Outcome Measure(s) : Ultrasonographic endometrial changes, clinical pregnancy rates (PRs), complications, and patients' complaints were compared between the two groups. Result(s) : Total destruction of the endometrial three-layer pattern was observed in 12.5% of the cycles in group 1, compared with 50% of the cycles in group 2. Clinical pregnancies occurred in 14 (12.5%) of the 112 IUI cycles. A higher PR was achieved when the endometrial three-layer pattern was preserved after IUI. The patients in group 2 had more complaints of bleeding and pain during the procedure. Conclusion(s) : Ultrasonographic changes after IUI suggest that the Edwards Wallace catheter is significantly less traumatic to the endometrium than the Tom-Cat catheter. Although both catheters yielded the same overall PR, there was a trend indicating that sparing the endometrial three-layer pattern from damage increases the chance of conception.

Objective To determine whether the decrease in AMH levels during ovarian hyperstimulation for IVF occurs in patients with polycystic ovary syndrome (PCOS) and patients with low ovarian reserve (LOR), as in normal cycling women. Study design A cohort of 22 infertile patients treated in a single tertiary center with a GnRH-antagonist short protocol for IVF were prospectively included and divided into three groups: PCOS with hyperandrogenism ( n =7), LOR ( n =8) and control ( n =7). Serum AMH levels were measured before and during FSH treatment, on the day of HCG administration, at the mid-luteal phase, and 14 days after embryo transfer. The three groups were compared using an ANOVA model in the case of continuous data and with Fisher's exact test when the data were discrete. Results In the PCOS group, AMH levels increased at the beginning of the stimulation, but later decreased, until the mid-luteal stage. In the other two groups, AMH levels decreased throughout ovarian stimulation until the mid-luteal stage. In all groups, AMH levels returned to baseline levels two weeks after HCG administration, regardless of treatment outcome (pregnancy or not). Conclusions AMH levels decline during controlled ovarian hyperstimulation with a GnRH-antagonist short protocol in women with low and normal ovarian reserves. In contrast, in women with PCOS, an increase in AMH levels precedes this decline. These findings may support the hypothesis that androgens may play a role in AMH regulation in women.

Background Among children conceived by assisted reproductive technology (ART), increased risk of adverse birth outcomes has been observed, including multiple births, preterm births, and congenital malformations. Regarding cancer among ART-conceived children, findings are discrepant. Methods This is a historical cohort of 9,042 ART-conceived children and 211,763 spontaneously conceived (SC) children born from 1997 through 2004. The median duration of follow-up was 10.6 years (interquartile range 9.0–12.3) in the ART group and 9.3 years (interquartile range 8.0–10.6) in the SC group. The cohort database was linked with the Israel National Cancer Registry updated until December 31, 2011 using each child's personal identification number. Results Twenty-one cases of cancer were identified in the ART group (2.2 per 10,000 person-years), as compared to 361 cancer cases in the SC group (1.8 per 10,000 person-years). The relative risk (RR) for overall cancer in the ART group compared to the SC group adjusted for maternal characteristics was 1.18 (95% confidence interval [CI] 0.80–1.75). ART children had a significantly increased risk for specific cancers, although based on small number of cases, including two cases of retinoblastoma (RR 6.18, 95% CI 1.22–31.2), as well as four cases of renal tumors (RR 3.25, 95% CI 1.67–6.32). Conclusion A statistically significant increased risk for two pediatric cancers was found. However, for overall types of cancer the risk estimate was elevated but not statistically significant. Further studies with larger sample size and longer follow-up time are warranted in order to either confirm or refute these findings.

Long-range PCR is generally employed for the analysis of disease-causing mutations in genes with homologous pseudogene copies. However, long-range PCR is challenging when performed on single cells, as in preimplantation genetic diagnosis (PGD) of monogenic disorders. PGD on single cells requires concurrent analysis of a mutation together with multiple linked polymorphic markers from closely related family members to prevent misdiagnosis. In PGD cases involving childless de novo mutation carriers, linkage cannot be performed based on family members but rather must first be identified in single gametes. This can be an especially difficult task if the mutation to be assayed lies in a duplicated genomic region because gene-specific long-range PCR must be coupled with short-range PCR analysis of genetic markers on single cells. Here, we describe a novel method by which accurate PGD of pseudogene-homologous mutations can be achieved. Essentially, we performed whole genome amplification on single sperm or blastomeres followed by haplotype construction and long-range PCR-based mutation analysis. This original and universal strategy was used to establish allelic association for two different mutations in genes with one or more pseudogene copies (IKBKG and PKD1). The method was also sensitive enough to detect unexpected germline mosaicism in one mutation carrier.

Preimplantation genetic diagnosis (PGD) allows birth of unaffected children for couples at risk for a genetic disorder. We present the strategy and outcome of PGD for four lysosomal storage disorders (LSD): Tay-Sachs disease (TSD), Gaucher disease (GD), Fabry disease (FD), and Hunter syndrome (HS), and subsequent development of stem cell lines. For each disease, we developed a family-specific fluorescent multiplex single-cell PCR protocol that included the familial mutation and informative markers surrounding the mutation. Embryo biopsy and PGD analysis were performed on either oocytes (polar bodies one and two) or on single blastomeres from a six-cell embryo. We treated twenty families carrying mutations in these lysosomal storage disorders, including 3 couples requiring simultaneous analysis for two disorders (TSD/GD, TSD/balanced Robertsonian translocation 45XYder(21;14), and HS/oculocutaneus albinism). These analyses led to an overall pregnancy rate/embryo transfer of 38% and the birth of 20 unaffected children from 17 families. We have found that PGD for lysosomal disorders is a safe and effective method to prevent birth of affected children. In addition, by using mutant embryos for the derivation of stem cell lines, we have successfully established GD and HS hESC lines for use as valuable models in LSD research.

OBJECTIVE To develop a reliable and accurate preimplantation genetic diagnosis (PGD) method in six families with endocrine diseases: persistent hyperinsulinemic hypoglycemia of infancy (PHHI), congenital adrenal hyperplasia (CAH) salt-wasting form, Sanjat-Sakati syndrome and multiple endocrine neoplasia 2A (MEN 2A). METHODS For each disease a battery of at least four informative markers surrounding the tested gene were identified and for each family a protocol of multiplex fluorescent markers was developed and performed on single cells. RESULTS PGD for PHHI was performed in three families. In family 1 two healthy children were born from different cycles, in family 2 three healthy children were born from two cycles, and in family 3 a healthy boy was born. For CAH in one family a healthy girl was born. One PGD cycle for Sanjat-Sakati resulted in a clinical pregnancy that was terminated due to high nuccal translucency (46X0). For one family with MEN 2A disease, the eighth PGD cycle resulted in birth of healthy twins. In all children genetic confirmation of the healthy status was performed. CONCLUSIONS PGD is an effective method for preventing birth of affected children with endocrine disorders. Increasing the awareness of clinicians to the availability of these methods is most important.

Objective To produce age-related normograms for serum antimullerian hormone (AMH) level in infertile women without polycystic ovaries (non-PCO). Design Retrospective cohort analysis. Setting Fifteen academic reproductive centers. Patient(s) A total of 3,871 infertile women. Intervention(s) Blood sampling for AMH level. Main Outcome Measure(s) Serum AMH levels and correlation between age and different percentiles of AMH. Result(s) Age-related normograms for the 3rd, 10th, 25th, 50th, 75th, 90th, and 97th percentiles of AMH were produced. We found that the curves of AMH by age for the 3rd to 50th percentiles fit the model and appearance of linear relation, whereas the curves of >75th percentiles fit cubic relation. There were significant differences in AMH and FSH levels and in antral follicle count (AFC) among women aged 24–33 years, 34–38 years, and ≥39 years. Multivariate stepwise linear regression analysis of FSH, age, AFC, and the type of AMH kit as predictors of AMH level shows that all variables are independently associated with AMH level, in the following order: AFC, FSH, type of AMH kit, and age. Conclusion(s) Age-related normograms in non-PCO infertile women for the 3rd to 97th percentiles were produced. These normograms could provide a reference guide for the clinician to consult women with infertility. However, future validation with longitudinal data is still needed.

background: PGD for fragile X syndrome (FRAX) is inefficient, probably owing to fewer oocytes, poor embryo quality and difficulties in genetic analysis. We investigated IVF –PGD in FRAX mutation carriers compared with controls, looking at the effects of oocyte and embryo number/quality on live birth outcome. methods: We performed IVF –PGD in 27 patients with the FRAX mutation and 33 controls with other genetic diseases. Genetic testing was by multiplex PCR. results: Seventy-nine and 108 IVF –PGD cycles were started in FRAX mutation carriers and controls, respectively. Twenty-two patients had a premutation (CGG repeat number 60 –200) and five had a full mutation (300–2000 CGG repeats). FRAX patients required higher doses of gonadotrophins (6788+ 2379 versus 4360+ 2330, P , 0.001) but had lower peak serum estradiol levels (8166+ 5880 versus 10 211+ 4673, P ¼ 0.03) and fewer oocytes retrieved (9.8+ 6 versus 14+ 8, P ¼ 0.01). The cancelation rate (unsatisfactory ovarian response) was higher in the FRAX group than in the control group (13 versus 1%, P , 0.001). When embryos were transferred, ongoing pregnancy/live birth rates per transfer were similar (29 versus 36%, P ¼ 0.54). conclusions: Ovarian dysfunction in FRAX carriers is more prevalent and profound than previously appreciated, with a high cancelation rate and reduced efficiency of PGD. The main determinant for successful PGD for FRAX is ovarian dysfunction. When embryo transfer is possible, the results are comparable to PGD for other monogenic diseases.

To determine if sera of some women have antibodies against capacitated but not freshly ejaculated sperm.The sera of 66 women undergoing in vitro fertilization (IVF) were tested for sperm antibodies after 1 hour and 18 hours of sperm incubation in the maternal sera. Subsequently, 5 sera were tested with capacitated versus noncapacitated sperm cells.The study was carried out in a university hospital department.The patients were 66 consecutive couples undergoing IVF.Sera and semen that were taken for routine tests as part of the IVF procedures were used.A case with IVF failure associated with late appearance of sperm antibodies prompted us to study the detection of sperm antibodies after 1 hour and 18 hours incubation.Of 37 cases negative for sperm antibodies after 1 hour incubation, 7 demonstrated high levels of antibodies after 18 hours incubation. In 21 of 23 cases with low or intermediate levels of antibodies after 1 hour incubation, significantly higher levels (P less than 0.05) of antibodies were found after 18 hours. Different and higher levels of sperm antibodies were observed in five sera after incubation of 1 hour with capacitated sperm as compared with noncapacitated controls.Major antigenic differences may exist between capacitated and noncapacitated sperm. In some women sperm antibodies are reactive against capacitated sperm only. This has no certain clinical significance but may explain certain cases of IVF failure, unexplained infertility, and part of the variation in sperm antibodies testing methods.

Preimplantation genetic diagnosis (PGD) is usually used to establish a non-affected pregnancy for those couples facing a genetic risk of having an affected child. However, an invasive test is still recommended to all PGD patients due to the risk of misdiagnosis. The discovery of cell-free fetal DNA in maternal plasma provides the possibility for noninvasive prenatal diagnosis. Studies have shown that fetal single-gene disorders can be detected in cell-free fetal DNA by the matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) assay with single-allele base extension reaction (SABER) approach or by the size-fractionation approach, whereby cell-free fetal DNA is enriched on the basis of its smaller size compared with maternal DNA fragments. Recent studies have indicated that a combination of the two approaches increases the accuracy of detection. This study combined the two methods and examined fetal paternally inherited gene mutations in maternal plasma obtained from four PGD-conducted pregnancies. The presence or absence of mutations was correctly detected in all cases. This combined method could be used for risk-free prenatal diagnosis of diseases caused by single-gene mutations, and in particular for couples who undergo PGD who opt not to perform invasive prenatal confirmation due to the risk of abortion.

ObjectivesMedical castration with long-acting GnRH-agonist (GnRHa) is a well-established treatment for metastatic prostate cancer. Our aim was to explore the relationships between FSH, inhibin B, anti-Mullerian hormone (AMH), and testosterone during treatment with an implant releasing GnRHa.DesignAnalysis of hormone levels in frozen serum samples.MethodsTen patients aged 77±7 (means±s.e.m.) years with prostate cancer were treated with the GnRHa histrelin for at least a year. Two weeks prior to insertion and for 3–4 months following removal the patients were treated with the antiandrogen flutamide. Serum inhibin B, FSH, testosterone, and AMH levels were measured retrospectively.ResultsFSH, inhibin B, and testosterone increased during antiandrogen administration and levels fell after implant insertion. Four weeks post insertion, FSH gradually increased while inhibin B and testosterone remained fully suppressed. AMH levels did not change during antiandrogen treatment, but increased following implant insertion and remained elevated for the duration of implant use. Following removal, FSH and testosterone increased, inhibin B remained low, while AMH decreased.ConclusionsThe secondary increase in FSH following initial suppression with the implant is probably related to impaired inhibin B secretion. The lack of inhibin B response to the secondary increase in FSH suggests that long-term exposure of Sertoli-cells to GnRHa impairs their function. This effect appears to be selective since unlike inhibin B, AMH increased. In the absence of testosterone, FSH has a role in AMH regulation.

Objective To investigate whether estrogen may modulate anti-mullerian hormone (AMH) expression in women. Design Prospective analysis. Setting Fertility clinic of tertiary university hospital. Patient(s) Cycling infertile women. Intervention(s) Blood samples were taken at the early, middle, and late follicular phase in five groups: spontaneous cycle (n = 10), ovulation induction with clomiphene-citrate (n = 15) or gonadotropins (n = 9), controlled ovarian hyperstimulation for IVF (COH-IVF; n=10) and in women who were treated with exogenous E 2 for frozen-thawed embryo-transfer (FET) with no follicular development (n = 20). Main Outcome Measure(s) AMH and E 2 serum levels. Result(s) Basal serum AMH and E 2 levels were similar in all groups. AMH levels were stable in all women during the follicular phase except for significant reduction in the COH-IVF group. In women in the FET group with high E 2 levels, comparable to the COH-IVF group, AMH levels remained stable. Conclusion(s) In women, estrogen does not appear to have a direct role in AMH regulation.

Objective Accounting for possible recombinations in developing an accurate preimplantation genetic diagnosis (PGD) protocol based on familial haplotypes. Methods Haplotypes were constructed from genomic DNA in a family where the male was affected with tuberous sclerosis complex (TSC). Embryos were biopsied at day 3, and single blastomeres were analyzed by multiplex polymerase chain reactions (PCRs) including multiple microsatellite markers and single-cell sequencing. Results Informative markers used in the initial haplotype analysis, based on the genomic DNA of the parents and affected child, were analyzed in the first PGD cycle. All embryos appeared to show recombination and none were transferred. Prior to the next cycle, the parents of the affected male were included in the haplotype analysis, demonstrating that the affected child had a recombinant allele. In the second PGD cycle, two nonrecombinant intragenic single nucleotide polymorphisms (SNPs) were used with the TSC2 mutation and four microsatellite markers for a second PGD cycle. In this cycle two wild-type embryos were transferred, resulting in the birth of a healthy girl carrying the wild-type TSC2 allele. Conclusions PGD analysis requires the use of more than one meiosis for constructing accurate haplotypes. Testing for multiple closely linked markers and the familial mutation are necessary to detect recombination events and enable precise diagnosis. Copyright © 2008 John Wiley & Sons, Ltd.

The pregnancy rates after triggering of final oocyte maturation with gonadotrophin-releasing hormone (GnRH) agonist in GnRH-antagonist ovarian stimulation protocols are lower than those following triggering with human chorionic gonadotrophin (HCG). Furthermore, lower pregnancy rates following GnRH-antagonist protocols compared with long GnRH-agonist protocols have been reported. The differences might be due to an impact on oocyte number and quality or on the endometrium. If any stimulation protocol had a negative impact on oocyte quality, then further evidence of this effect would be observed following frozen-thawed embryo transfer originating from that stimulation cycle. The outcome of frozen-thawed embryo transfer was retrospectively analysed using the long protocol with triptorelin depot 3.75 mg (n = 215) or 0.1 mg/day (n = 83), or GnRH-antagonist protocol with either HCG (n = 69) or GnRH-agonist (n = 25) for final oocyte maturation. The outcomes measured were implantation rate, clinical pregnancy rate, ongoing pregnancy rate and embryo survival rate. All outcomes were similar in the four groups. It is concluded that the potential for frozen-thawed embryos to implant and develop following transfer is independent of the GnRH-analogue and the final oocyte maturation protocol used in the collection cycle. Lower IVF embryo transfer success using GnRH-antagonist/GnRH-agonist protocol does not appear to be related to an adverse effect on oocyte quality.

BACKGROUND The aim of our study is to evaluate the efficacy of applying lidocaine 25 mg-prilocaine-25 mg/G cream (EMLA 5%) on the uterine cervix for pain relief when performing hysterosalpingography (HSG). METHODS Eighty-two patients undergoing HSG as part of infertility evaluation were randomized into groups receiving EMLA (42) or placebo cream (40) in a double-blinded prospective study from which four women were later excluded. The cream was applied to the uterine cervix by means of a cervical cup 30 min before the HSG. Pain perception related to the HSG procedure was scored by visual analogue scale (VAS) at five predefined steps: after speculum application, after cervical instrumentation of the tenaculum and cannula, at the end of uterine filling, at completion of tubal spillage, and immediately following instrument removal. In addition, the patients were asked to retrospectively rate the pain during the entire procedure in a telephone interview the following day. RESULTS Cervical instrumentation was found to be the most painful step of HSG (P < 0.001). When comparing the VAS pain scores, cervical instrumentation in the EMLA-treated patients was associated with significantly less pain than the control group: 3.3 +/- 2.9 versus 4.9 +/- 2.7, respectively (P = 0.02). CONCLUSIONS Topical application of EMLA 5% cream on the uterine cervix before performing HSG significantly reduced the pain during this procedure.

Aim: To determine the effect of a copper-medicated intrauterine device (IUD) on ovarian, uterine, arcuate, radial and subendometrial Doppler-derived indices of blood flow. Method: 23 regularly menstruating patients requested insertion of an IUD. All patients had a copper T (Nova T) IUD inserted between days 8 and 11 of the menstrual cycle. Ovarian, uterine, arcuate, radial and subendometrial artery pulsatility indices (PIs) were assessed by transvaginal color Doppler prior to insertion between days 8 and 11 of the menstrual cycle, and after 2 months in the same period of the cycle. Ovarian, uterine, arcuate, radial and subendometrial artery PIs were considered prior to and following IUD insertion. Results: No differences were recorded in any of the blood vessels sampled between pre- and post-insertion PIs. Conclusion: No significant change in ovarian or in uterine system vascular impedance is associated with the presence of a copper-medicated IUD.

BACKGROUND: Neurofibromatosis type 1 (NF1) is an autosomal dominant disorder caused by mutations in the neurofibromin gene. Approximately, 50% of cases are caused by de-novo mutations. Even when the NF1 mutation is known, accuracy of PGD is highly enhanced by simultaneous analysis of linked markers. In a childless couple referred to PGD, the male carried a de-novo mutation, precluding the possibility of typing relatives to establish the mutation-associated haplotype. We developed a single-sperm haplotype analysis strategy to establish the haplotype linked to the NF1 mutation. METHODS: Spermatozoa from freshly ejaculated semen were used as a substrate for multiplex PCR on single sperm. RESULTS: In addition to the NF1 mutation, six informative polymorphic markers flanking the NF1 gene (D17S1294, D17S1849, D17S841, D17S975, NF1TG2 and NF1AC5) were linked to individual alleles in single sperm from the affected male. CONCLUSIONS: Single-sperm analysis established the haplotypes of both mutant and wild-type NF1 alleles and enabled the implementation of a PGD protocol using polymorphic marker analysis. This method is generally applicable to PGD for any disease in which the haplotype of paternal mutations cannot be determined by typing relatives.

To describe a successful pregnancy and delivery after calcium ionophore oocyte activation in a normozoospermic patient with previous repeated failed fertilization after intracytoplasmic sperm injection (ICSI).Case report. In vitro fertilization unit in a university affiliated medical center.A couple with 5 years of unexplained primary infertility who had repeated failed fertilization after ICSI. Controlled ovarian hyperstimulation, oocyte pick-up, ICSI, assisted oocyte activation with calcium ionophore, embryo culture, and ET. Fertilization rate, implantation, pregnancy, and delivery. Assisted oocyte activation with calcium ionophore A23187 after ICSI resulted in reasonable fertilization rates in three cycles (4/6, 5/16 and 7/20 oocytes). Two pregnancies were achieved; the first ended with second trimester miscarriage due to fetal anomaly and the second with a delivery of three healthy babies.Calcium ionophore oocyte activation seems to be a useful method in cases of repeated failed fertilization after ICSI.

A retrospective study was designed to assess the presence of antizona pellucida autoantibodies in in-vitro fertilization (IVF) patients in relation to low ovarian response, multiple IVF attempts and unexplained infertility. Antizona pellucida and antisperm antibodies were determined in serum samples obtained from 37 women undergoing IVF-embryo transfer and 20 fertile women. Antizona pellucida antibodies were measured using enzyme immunoabsorbent assay. Antisperm antibodies were evaluated by the immunobead binding method. Three of 10 patients with low response to ovarian stimulation had antizona pellucida antibodies in serum and one patient had antisperm antibodies. None of the other participants in the study and the control groups demonstrated measurable levels of serum antigamete antibodies. The results in this small group suggest an association between antizona pellucida antibodies and suboptimal response to gonadotrophins. It is indicated that repeated stimulation and puncture of ovaries in IVF procedures do not elicit autoimmunity to gametes.

The number of embryos transferred, ease of embryo transfer, and rate of embryo cleavage are significant factors affecting the success of IVF. High levels of oestradiol occurring when HCG is given may reduce implantation and pregnancy rates. A discrepancy between the stage of embryonic development and the phase of endometrial maturation at embryo transfer may be responsible for IVF failure in some cases.

A prospective study was conducted on the incidence of intra-uterine pathology diagnosed by hysteroscopy in 147 women who underwent dilatation and sharp curettage due to spontaneous first trimester abortion. The diagnostic hysteroscopies were performed 4-6 weeks after the miscarriage, using CO2 as medium for uterine distention. In 38 patients (25.9%), uterine pathology was found, of whom 28 patients (19%) had intra-uterine adhesions (IUA). The incidence of IUA following only one abortion was 16.3% (16 out of 98 cases), all were of mild extent and filmy consistency, occupying less than one-quarter of the uterine cavity. The incidence of IUA after two abortions was 14% (3/21) but the incidence after three or more spontaneous abortions was significantly elevated (32%, 9/28), (P < 0.05, chi-square test). In addition, 58% of IUA diagnosed in the latter two groups were of a more severe extent. After a first miscarriage, occurrence of post-abortion IUA was not related to the serum oestrogen concentration on the day of curettage, nor was it related to any of the oestrogen treatment modalities used in this study. Although in most of these cases normal recovery of the endometrium was the rule, the high incidence of IUA diagnosed in this prospective study stresses the need for preventive treatment and close follow-up of patients after sharp curettage due to spontaneous abortion. Obviously oestrogen treatment alone did not prove to be effective and other treatment methods should be evaluated. The high incidence and severity of IUA in habitual aborters clearly justifies the performance of a diagnostic hysteroscopy as a standard evaluation.