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3.  Uterine fibroids are characterized by an impaired antioxidant cellular system: potential role of hypoxia in the pathophysiology of uterine fibroids 
Purpose
Fibroids are the most common smooth muscle overgrowth in women. This study determined the expression and the effect of hypoxia on two potent antioxidant enzymes, superoxide dismutase (SOD) and catalase (CAT) on human fibroid cells.
Methods
Immortalized human leiomyoma (fibroid) and myometrial cells were subjected to hypoxia (2 % O2, 24 h). Total RNA and cell homogenate were obtained from control and treated cells; CAT and SOD mRNA and activity levels were determined by real-time RT-PCR and ELISA, respectively.
Results
Fibroid cells have significantly lower antioxidant enzymes, SOD and CAT mRNA and activity levels than normal myometrial cells (p < 0.05). Hypoxia treatment significantly increased SOD activity in myometrial cells while significantly decreasing CAT activity in fibroid cells (p < 0.05). There was no significant difference in CAT mRNA levels or activity in response to hypoxia in myometrial cells. Also, there was no significant difference in SOD mRNA levels in response to hypoxia in myometrial cells.
Conclusion
This is the first report to show that uterine fibroids are characterized by an impaired antioxidant cellular enzymatic system. More importantly, our results indicate a role for hypoxia in the modulation of the balance of those enzymes in fibroid and myometrial cells. Collectively, these results shed light on the pathophysiology of fibroids thereby providing potential targets for novel fibroid treatment.
doi:10.1007/s10815-013-0029-7
PMCID: PMC3725220  PMID: 23812845
Uterine fibroid cells; Normal myometrial cells; Hypoxia; Catalase; Superoxide dismutase
4.  Raman spectroscopy-based approach to detect aging-related oxidative damage in the mouse oocyte 
Purpose
Detection of chemical modifications induced by aging-related oxidative damage in mouse metaphase II (MII) oocytes by Raman microspectroscopy.
Methods
CD-1 mice at the age of 4–8 weeks (young mice) and 48–52 weeks (old mice), were superovulated and oocytes at metaphase II stage were recovered from oviducts. MII oocytes from young animals were divided into three groups: A) young oocytes, processed immediately after collection; B) in vitro aged oocytes, cultured in vitro for 10 h before processing; C) oxidative-stressed oocytes, exposed to 10 mM hydrogen peroxide for 2 min before processing. Oocytes from reproductively old mice were referred to as old oocytes (D). All the oocytes were analyzed by confocal Raman microspectroscopy. The spectra were statistically analyzed using Principal Component Analysis (PCA).
Results
PCA evidenced that spectra from young oocytes (A) were clearly distinguishable from those obtained from in vitro-aged, oxidative-damaged and old oocytes (B, C, D) and presented significant differences in the bands attributable to lipid components (C = C stretching, 1,659 cm−1; CH2 bending, 1,450 cm−1; CH3 deformation,1,345 cm−1; OH bending, C-N stretching, 1,211 cm-1) and protein components (amide I band,1,659 cm−1; CH2 bending modes and CH3 deformation, 1,450 cm−1; C-N and C-C stretching vibrations, 1,132 cm−1; phenylalanine’s vibration, 1,035 cm−1)
Conclusions
Raman spectroscopy is a valuable non-invasive tool for the identification of biochemical markers of oxidative damage and could represent a highly informative method of investigation to evaluate the oocyte quality.
doi:10.1007/s10815-013-0046-6
PMCID: PMC3725222  PMID: 23860776
Raman spectroscopy; Aging; Oocyte; Oxidative stress
5.  Peroxisome proliferator activated receptor gamma gene variants influence susceptibility and insulin related traits in Indian women with polycystic ovary syndrome 
Purpose
Peroxisome proliferator activated receptor gamma (PPARγ), a transcription factor involved in glucose and lipid metabolism is one of the candidate genes associated with polycystic ovary syndrome (PCOS). We investigated individual and combined associations of Pro12Ala and His447His polymorphisms of PPARγ with PCOS susceptibility and its related traits (hyperinsulinemia, hyperandrogenemia and lipid parameters) in Indian women.
Method
Genotyping of PPARγ polymorphisms in this case–control study was performed in PCOS (n = 450) and age-matched controls (n = 300) by direct sequencing. Clinical, anthropometric, hormonal and metabolic parameters were estimated in 275 women with PCOS and 169 controls. Chi-square test was used to compare the categorical data while regression analysis was used to evaluate association of genotypes with PCOS as well as its related phenotypes.
Results
The frequencies of CC and CG + GG genotypes of Pro12Ala (χ2 = 15.3, p < 0.0001) and CC and CT + TT genotypes of His447His (χ2 = 12.7, p = 0.0004) polymorphisms were significantly different between PCOS and controls. Logistic regression analysis revealed a significant association of PCOS with Pro12Ala but not the His447His polymorphism. Carriers of variant genotypes at both PPARγ loci showed significantly reduced 2 h glucose levels while carriers of variant His447His genotype showed lower fasting insulin and HOMA-IR levels in PCOS women.
Conclusions
Pro12Ala polymorphism of PPARγ showed significant association with decreased PCOS susceptibility. Both polymorphisms influenced insulin related traits (2 h glucose, fasting insulin and HOMA-IR) and improved glucose metabolism in these women. This is the first report to establish that variations in PPARγ gene influence the insulin resistance pathophysiology in Indian women with PCOS.
doi:10.1007/s10815-013-0025-y
PMCID: PMC3725223  PMID: 23748472
Peroxisome proliferator activated receptor gamma (PPARγ); Polycystic ovary syndrome (PCOS); Polymorphism; Insulin resistance; Genetic
7.  The influence of amifostine administration prior to cyclophosphamide on in vitro maturation of mouse oocytes 
Purpose
The protective effect of amifostine against cyclophosphamide (CP) was evaluated on mouse oocytes.
Materials and methods
Female mice were divided into four groups as follows: group1: cyclophosphamide (CP) (75 mg/kg, i.p) injection, group2: amifostine (250 mg/kg, i.p) injection, group3: amifostine (250 mg/kg, i.p) administered prior to CP (75 mg/kg, i.p) injection, Control group with injection of saline. About 21 days after injection, in vitro maturation (IVM) of oocytes was recorded. Furthermore the percentage of aneuploid oocytes was determined.
Results
In the CP group IVM rate was significantly decreased and aneploidy rate was significantly increased when compared to other groups (p < 0.05). With the administration of Amifostine prior to CP injection IVM rate was increased and aneploidy rate was decreased.
Discussion
Depletion in IVM rate with administration of CP is due its adverse effects on oocyte quality. Amifostine administration prior to CP injection appears to modulate deleterious effects of CP on oocytes.
doi:10.1007/s10815-013-0035-9
PMCID: PMC3725225  PMID: 23828370
Amifostine; Aneuploidy; Chromosome; Cyclophosphamide; Maturation; Oocyte
8.  Ubiquitin-specific protease (USP26) gene alterations associated with male infertility and recurrent pregnancy loss (RPL) in Iranian infertile patients 
Background and purpose
The human X chromosome is enriched with testis-specific genes that may be crucial for male fertility. Mutations in USP26 gene have been proposed to be associated with male infertility. Moreover, the importance of the ubiquitin pathway during different stages of mammalian fertilization and even embryo development has been addressed. Some mutations and haplotypes on this gene have been proposed to be associated with male infertility. In this study, five different mutations on USP26 were investigated: 1737 G > A, 1090 C > T, 370-371ins ACA, 494 T > C and 1423 C > T.
Methods
The study included 166 infertile men with non-obstructive azoospermia, 72 male partners of couples who had previously experienced ≥3 clinical first trimester spontaneous abortions and 60 fertile men. Besides family history of reproduction, hormonal evaluation and semen analysis were performed. DNA was extracted from blood samples. PCR-SSCP, PCR-RFLP and PCR Product Cloning methods were used and resumed by sequencing to insure about the mutations. Moreover, USP26 gene expression was studied by Real-Time PCR after RNA extraction followed by cDNA synthesis from 24 testis biopsies in obstructive and non-obstructive azoospermia patients.
Results
The results indicate that there is a haplotype between three observed mutations in Iranian population include: 370-371insACA, 1423C > T and 494 T > C. This haplotype was seen in control group as well. Surprisingly, total frequency of mutations in men with history of idiopathic RPL and azoospermic cases were significantly higher than that of in control groups (p < 0.05). Serum testosterone concentrations and testicular volume did not differ in the mutation positive group compared with the non-mutation group. About the USP26 gene expression, there is a significant difference between the expression levels of obstructive azoospermia, complete maturation arrest samples and SCO samples (P < 0.05).
Conclusions
According to our results, the USP26 gene may play an important role in male reproduction. The alterations of this gene may be involved in male infertility and RPL in Iranian population and may negatively affect testicular function.
doi:10.1007/s10815-013-0027-9
PMCID: PMC3725226  PMID: 23779098
Polymorphism; Male infertility; USP26
9.  Genetic variants associated with insulin signaling and glucose homeostasis in the pathogenesis of insulin resistance in polycystic ovary syndrome: a systematic review 
Background
Polycystic ovary syndrome must be recognized as a serious issue due to its implication on long term health regardless of an individual’s age. PCOS and insulin resistance are interlinked, as approximately 40 % of women with PCOS are insulin resistant. However, the detailed molecular basis for insulin resistance that is coupled with PCOS remains poorly understood.
Objective
To review the published evidence that polymorphisms in genes that are involved in insulin secretion and action are associated with an increased risk of PCOS.
Methods
We reviewed articles published through November 2012 which concerned polymorphisms of genes related to insulin signaling and glucose homeostasis as well as their associations with PCOS. The articles were identified via Medline searches.
Conclusions
No consistent evidence emerged of a strong association between the risk of PCOS and any known gene that is related to insulin signaling and glucose homeostasis. Moreover, recent genome-wide association studies are inconsistent in identifying the associations between PCOS and insulin metabolism genes. Many of the studies reviewed were limited by heterogeneity in the PCOS diagnosis and by not have having a sufficient number of study participants. Further studies are warranted to determine predisposing risk factors which could modify environmental factors and thus reduce the risk of PCOS. Large genome-wide association studies devoted solely to PCOS will be necessary to identify new candidate genes and proteins that are involved in PCOS risk.
doi:10.1007/s10815-013-0030-1
PMCID: PMC3725227  PMID: 23794114
Insulin resistance; PCOS; SNP; Genetics
10.  Impact of assisted reproduction treatments on Spanish newborns: report of 14,119 pregnancies 
Purpose
To investigate neonatal malformation, prematurity, and stillbirth in singleton and multiple pregnancies derived from different Assisted Reproductive Techniques (ART).
Methods
In this prospective cohort study data were collected, from private and public Spanish IVF units, during the years 2008 and 2009. During this period, 8,682 pregnancies were analysed from the initial 14,119 pregnancies reported. Pregnancies included in the study derived from IUI (n = 1,065), IVF (n = 838), ICSI (n = 5,080), FET (n = 1,404) and PGD (n = 295). This first analysis focuses primarily on neonatal malformation, prematurity, and stillbirth both in singleton and multiple pregnancies derived from different ART. Malformations were classified according to the WHO ICD 10 code.
Results
Malformations were found in 0.83 % of our newborns. No differences in malformations were observed between singletons or multiples independently of the ART used. There was a significant difference in prematurity rate among singletons depending on treatment but this association was not observed in multiple pregnancies. Stillbirth was significantly lower in singleton (0.72 %) than in multiple pregnancies (1.82 %).
Conclusions
The percentage of malformations observed in ART newborns was similar to the rate observed in the normally-conceived Spanish population. Multiplicity seems to be the most important factor associated with an increased incidence of newborn complications such as prematurity or stillbirth.
doi:10.1007/s10815-013-0023-0
PMCID: PMC3725228  PMID: 23779097
ART; Pregnancy outcome; Perinatal outcome; Malformations; Prematurity; Stillbirth
11.  Supplementation with low concentrations of melatonin improves nuclear maturation of human oocytes in vitro 
Purpose
Studies in bovine and porcine have indicated that melatonin (MT) could induce meiotic maturation of immature oocytes in vitro. The object of the current study was to investigate if MT could ameliorate human oocytes maturation during rescue in vitro maturation (IVM).
Methods
Two hundred seventy eight germinal vesicle (GV) oocytes and 451 (MI) metaphase I oocytes were vitrified, thawed and then matured in vitro. All the oocytes were randomly allocated into six groups in which the oocytes were cultured in medium supplemented with different concentrations of MT (0, 10−2, 1, 102, 104, 106 nM) and nuclear maturation was evaluated at 6 h, 12 h, 18 h, 24 h and 48 h of culture.
Results
The optimal MT concentration for both GV and MI oocytes was 1 nM. At 24 h of culture, nuclear maturation rate of MI oocytes cultured in 1 nM MT medium was significantly higher than other groups (P < 0.05); Nuclear maturation rate of GV oocytes cultured in 1 nM MT medium was also significantly higher than the control group (P < 0.05). On the other hand, decreased nuclear maturation rate was observed in the high MT concentration group (106 nM).
Conclusions
The current study demonstrated that low concentration of exogenous MT could ameliorate nuclear maturation of human oocyte during rescue IVM, while high concentration of MT presented negative effects.
doi:10.1007/s10815-013-0021-2
PMCID: PMC3725229  PMID: 23737216
Melatonin; Human immature oocyte; In vitro maturation; Vitrification
12.  Derivation of human embryonic stem cell lines from single blastomeres of low-quality embryos by direct plating 
Purpose
To explore a simple method of establishing pluripotent human embryonic stem cell (hESC) lines from single blastomeres of low-quality (LQ) embryos.
Methods
Blastomeres were isolated from normally fertilized, day-3 pre-implantation LQ embryos by dissolving of the zona pellucida and were then plated directly onto inactivated human foreskin fibroblasts. The subsequent culture was identical to that used to derive a hESC line from the inner cell mass of a blastocyst. The established hESC lines were passaged and characterized.
Results
Two hESC lines were produced by culturing the blastomeres individually in a hESC culture system (hESC-CS). Both of the hESC lines maintained a normal 46-chromosome XY karyotype, expressed stemness markers, and showed a pluripotent phenotype, including the ability to differentiate into all three germ layers in vitro and in vivo.
Conclusions
The blastomeres of LQ embryos have a developmental capacity that necessitates prolonged culture. Plating of blastomeres from LQ embryos directly into the hESC-CS is a feasible method for deriving hESC lines.
doi:10.1007/s10815-013-0042-x
PMCID: PMC3725230  PMID: 23835723
Human embryonic stem cell; Single blastomere; Low-quality embryo; Human foreskin fibroblast
13.  In vitro propagation of male germline stem cells from piglets 
Purpose
To study the effects of serum and growth factors on propagation of porcine male germline stem cells (MGSCs) in vitro and develop a culture system for these stem cells.
Methods
Fresh testicular cells from neonatal piglets were obtained by mechanical dissociation and collagenase-trypsin digestion. After differential plating, non-adhering cells were cultured in media supplemented with different concentrations of serum (0, 1 %, 2 %, 5 %, 10 %). After 10 days of primary culture, the cells were maintained in media supplemented with different concentrations of growth factors (basic fibroblast growth factor and epidermal growth factor at 1, 5, 10 ng/ml). The number of MGSC-derived colonies with different sizes was determined in each treatment to assess the effects of serum concentrations and growth factors.
Results
The number of MGSC-derived colonies was significantly higher in the presence of 1 % rather than 10 % fetal bovine serum (FBS). Basic fibroblast growth factor (bFGF) at 1, 5 ng/ml and epidermal growth factor (EGF) at 5, 10 ng/ml significantly promoted colony formation. Immunocytochemistry, reverse transcriptase-polymerase chain reaction (RT-PCR) and xenotransplantation assays demonstrated the presence of functional stem cells in cultured cell population.
Conclusions
In vitro propagation of porcine MGSCs could be maintained in the presence of 1 % FBS and supplementation of growth factors for 1 month.
doi:10.1007/s10815-013-0031-0
PMCID: PMC3725231  PMID: 23779100
Pig; Male germline stem cell; Serum; Growth factor; Culture
14.  Oocyte cryopreservation: searching for novel improvement strategies 
Purpose
To highlight emerging techniques aimed at improving oocyte cryopreservation.
Methods
Review of available and relevant literature through Pubmed and Medline searches.
Results
Oocyte cryopreservation is an increasingly common procedure utilized for assisted reproduction and may benefit several patient populations. Therefore, improving efficiency is paramount in realizing the tremendous promise of this approach. However, in addition to numerous studies looking to improve oocyte cryopreservation efficacy via examination of variables involved with protocol methodology, such as type/concentration of cryoprotectant (CPA), type of storage device, or cooling/warming rates, there are more novel approaches for improvement. These alternate approaches include utilizing different the stages of oocytes, examining alteration of basal media and buffer composition, optimizing CPA exchange protocols and device loading through use of automated technology, as well as examination/manipulation of oocyte cellular composition to improve cryotolerance. Finally, elucidating more accurate or insightful indicators of “success” is crucial for continued improvement of oocyte cryopreservation.
Conclusion
Oocyte cryopreservation has improved dramatically in recent years and is receiving widespread clinical use. Novel approaches to further improve success, as well as improved methods to assess this success will aid in continued improvement.
doi:10.1007/s10815-013-0028-8
PMCID: PMC3725232  PMID: 23779099
Oocyte; Vitrification; Cryopreservation; Meiotic stages; Microfluidics; Composition; Membrane; Buffers
15.  Effect of seminal plasma application to the vaginal vault in in vitro fertilization or intracytoplasmic sperm injection treatment cycles—a double-blind, placebo-controlled, randomized study 
Purpose
To study whether intravaginal application of seminal plasma after follicle aspiration has the potential to increase implantation and clinical pregnancy rates after IVF-ET.
Methods
We conducted a prospective, double-blind, placebo-controlled randomized study of 230 patients undergoing IVF-ET cycles. 500 μL of Fresh seminal plasma from the patient’s partner or culture medium (placebo) were injected in the vaginal vault just after follicle aspiration. The main outcome measured was ongoing clinical-pregnancy rate.
Results
After ET cancellation in ten patients due to lack of fertilization or embryo cleavage, 220 embryo transfers (103 and 117 in the study and control groups) resulted in a clinical pregnancy rate of 36.9 % and 29.1 % for the study and control groups, corresponding to a relative increase of 26.8 %. After an early pregnancy loss of 13.1 % (5/38) and 23.5 % (8/34) in the study and control groups respectively an ongoing pregnancy rate of 32.0 % (33/103) and 22.2 % (26/117) was achieved corresponding to a relative increase of 44.1 %. Multivariate logistic regression analysis adjusted for study group, age, infertility, and cycle characteristics did not demonstrate any parameter that could predict occurrence of clinical pregnancy rates after IVF-ET.
Conclusions
Patients who underwent SP intravaginal insemination after oocyte pick-up reached higher implantation and clinical pregnancy rates following ET compared to controls, although the difference did not reach statistical significance. More studies and variable methodologies may clarify the potential clinical effect of SP in improving live birth rates after ART.
doi:10.1007/s10815-013-0033-y
PMCID: PMC3725233  PMID: 23812801
Seminal plasma; IVF; Intercourse; Implantation; Pregnancy rate
16.  Legal termination of a pregnancy resulting from transplanted cryopreserved ovarian tissue due to cancer recurrence 
Purpose
To report on a woman who conceived naturally and had a normal intrauterine pregnancy following transplantation of frozen/thawed ovarian tissue but decided to have an early abortion due to recurrence of breast cancer.
Methods
The patient was diagnosed breast cancer and received antineoplastic treatment that forced her into premature ovarian insufficiency and infertility. Ovarian tissue cryopreserved prior to chemotherapy was transplanted following cancer treatment restoring fertility and regular menstrual cycles.
Results
The patient conceived 6 month after transplantation. However, she experienced recurrence of breast cancer and decided on legal termination of the pregnancy in the first trimester.
Discussion
The obtained pregnancy only 6 month following transplantation underlines the ability of the procedure. The recurrence occurred near the original site of the tumor and was most unlikely related to the transplantation. The activity of the transplanted tissue is likely to be destroyed by the renewed antineoplastic treatment she will receive. However, she still has the majority of one ovary cryostored and may later want to undergo additional transplantation to regain fertility or to have menstrual cycles back.
doi:10.1007/s10815-013-0026-x
PMCID: PMC3725234  PMID: 23748473
Transplantation; Fertility preservation; Cancer; Ovarian cryopreservation; Premature ovarian insufficiency
17.  What is the optimal threshold of serum Anti-Müllerian hormone (AMH) necessary for IVM treatments? 
Purpose
To assesse circulating levels of Anti-Müllerian hormone (AMH) as a predictor of oocyte number and their potential to mature in vitro in both normo-ovulatory (NO) women and in women with Polycystic Ovary Syndrome (PCOS) undergoing in vitro maturation (IVM) treatments.
Methods
We prospectively studied NO women and women diagnosed with PCOS, (age range 21–39 years) underwent IVM treatments at our center. Serum AMH levels were quantified before each cycle and correlated to oocytes number, maturation and fertilization during in vitro maturation.
Results
104 NO and 30 PCOS IVM cycles were followed with retrieval of a total of 672 and 491 oocytes, respectively. In NO women, the serum AMH level positively correlated with the number of oocytes retrieved, (R = 0.6; P <0.0001) the number of M2 oocytes at 24 and 48 h (R = 0.4; P <0.01; R = 0.26 p < 0.007, respectively) and with the total number of M2 oocytes (R = 0.47; P < 0.0001). In the PCOS group, the serum AMH level positively correlated only with the number of oocytes retrieved (R = 0.43; P <0.03). Receiver operating characteristic (ROC) analyses showed that a cutoff AMH level of 1.56 (ng/ml) could identify patients with 5 or more oocytes at OPU with a sensitivity of 83 % and a specificity of 75 %. An AMH level of 1.63 (ng/ml) was the threshold for 5 or more matured oocytes (sensitivity = 81 %, specificity = 53 %).
Conclusions
Serum AMH may be used as a marker to identify candidates for IVM treatment in both NO and PCOS women.
doi:10.1007/s10815-013-9996-y
PMCID: PMC3696446  PMID: 23608780
Anti-Müllerian hormone; In vitro maturation; Oocyte maturation; PCOS
18.  Comparison of clinical outcomes between single and double vitrified-warmed blastocyst embryo transfer according to the day of vitrification 
Purpose
To compare the efficacy of single vitrified-warmed blastocyst embryo transfer (SVBT) versus double vitrified-warmed blastocyst embryo transfer (DVBT) according to the day of vitrification.
Methods
This retrospective study included a total of 1,051 cycles in women less than 37 years of age with their autologous SVBT cryopreserved on day 5 (5d-SVBT, n = 737) or day 6 (6d-SVBT, n = 154) and DVBT on day 5 (5d-DVBT, n = 129) or day 6 (6d-DVBT, n = 31) from January 2009 to December 2011.
Results
The clinical pregnancy rate (41.8 % vs. 48.1 %, p = 0.184) and ongoing pregnancy rate (36.6 % vs. 45.0 %, p = 0.072) were not significantly different between the 5d-SVBT group and the 5d-DVBT group. However, the clinical pregnancy (29.9 % vs. 58.1 %, p = 0.003) and ongoing pregnancy rates (23.4 % vs. 51.6 %, p = 0.001) were significantly lower in the 6d-SVBT group compared with those in the 6d-DVBT group. The implantation rate (42.2 % vs. 34.5 %, p = 0.03) of the 5d-SVBT group was significantly higher than that of the 5d-DVBT group, while the implantation rate (29.9 % vs. 37.1 %, p = 0.303) of the 6d-SVBT group was not statistically different compared with that in the 6d-DVBT group. The multiple pregnancy rates (1.0 % in the 5d-SVBT group vs. 38.7 % in the 5d-DVBT group, p < 0.001 and 0 % in the 6d-SVBT group vs. 22.2 % in the 6d-DVBT group, p = 0.001) were statistically significantly lower in the SVBT group compared with those in the DVBT group regardless of the day of vitrification.
Conclusions
This study showed that the 5d-SVBT resulted in comparable clinical outcomes compared to the 5d-DVBT while the 6d-SVBT yielded significantly lower clinical outcomes compared to the 6d-DVBT.
doi:10.1007/s10815-013-0017-y
PMCID: PMC3696441  PMID: 23715875
Single embryo transfer; Single vitrified-warmed blastocyst embryo transfer; Double vitrified-warmed blastocyst embryo transfer; Vitrification; Multiple pregnancies
19.  Rescue in vitro fertilization using a GnRH antagonist in hyper-responders from gonadotropin intrauterine insemination (IUI) cycles 
Objective
To evaluate the outcomes in the conversion of high-response gonadotropin intrauterine insemination (IUI) cycles to “rescue” in vitro fertilization (IVF) using a Gonadotropin-Releasing Hormone (GnRH) antagonist, with regards to implantation rates, pregnancy rates, cost, and ovarian hyperstimulation syndrome (OHSS) as compared to matched, hyper-responder, IVF controls.
Methods
This prospective cohort study was conducted between January 2007 and December 2009 at our institution. In order to decrease high-order multiple pregnancy, minimize the incidence of OHSS, and avoid cycle cancellation, high-response stimulated-IUI patients opted to convert to “rescue” IVF using the GnRH antagonist cetrorelix acetate. We then compared their clinical outcomes with matched patients from high-response IVF cycles of the standard long mid-luteal GnRH agonist protocol (14 or more collected oocytes). Only cases of conventional IVF without intra-cytoplasmic sperm injection (ICSI) were included in the control group.
Results
Out of 184 patients undergoing stimulated-IUI cycles with gonadotropins, 87 patients developed a hyper-response, and 20 opted to convert to “rescue” IVF. These patients were compared with 157 matched, hyper responder IVF controls from our registry. The implantation rate was 25.6 % in the “rescue” IVF group and 20.7 % in the control IVF group (p < 0.0047). The ongoing clinical pregnancy rate per embryo transfer was 45.0 % and 33.6 % in the “rescue” IVF and the control IVF groups, respectively (p < 0.0001). The mean duration of stimulation was comparable between cohorts (10.0 vs.10.4 days, p = 0.6324). The mean dose of gonadotropin used per cycle was higher in the control group, 2664 international units (IU) of follicle stimulation hormone (FSH) compared to 1450 IU of FSH in the “rescue” IVF group (p < 0.0001). The incidence of severe OHSS is also higher in the control group, 5.1 % versus no cases in the “rescue” IVF group (p < 0.0001).
Conclusion
Our study demonstrates that conversion of high-response gonadotropin-IUI cycles to “rescue” IVF using a GnRH antagonist is a cost-effective strategy that produces better results than regular IVF with relatively minimal morbidity, and shorter duration to achieve pregnancy. Implantation and ongoing clinical pregnancy rates tend to be higher than those from hyper-responder regular IVF patients.
doi:10.1007/s10815-013-0016-z
PMCID: PMC3696442  PMID: 23715874
Stimulated IUI; Hyper-responders; Rescue IVF; GnRH antagonist
20.  Partial molar pregnancy after intracytoplasmic sperm injection occurring as a result of diploid sperm usage 
Purpose
Partial molar pregnancies are rare conceptions characterized by having 69 rather than 46 chromosomes, the additional chromosome complement usually occurring as a result of fertilization of the ovum by two sperm. Although assisted conception with intracytoplasmic sperm injection (ICSI) should prevent the development of a partial molar pregnancy, occasional cases have been described after assisted conception using ICSI. The objective of this study was to investigate the cause of partial molar pregnancy in a couple who had undertaken assisted conception with ICSI.
Methods
Fluorescent microsatellite genotyping of DNA from the couple and tissue from their partial molar pregnancy was performed in order to confirm diagnosis and investigate the origin of the additional chromosome set.
Results
Genotyping confirmed that the partial molar tissue was triploid with an additional chromosome complement from the father. Genotyping of additional loci proximal to the centromere demonstrated that the two paternal sets of chromosomes originated in a single sperm with a double complement of paternal DNA resulting from non-reduction at the second meiotic division.
Conclusions
This study confirms that partial molar pregnancy may occur after assisted conception with ICSI and that this occurs as a result of fertilization with a diploid sperm.
doi:10.1007/s10815-013-0002-5
PMCID: PMC3696443  PMID: 23640373
Assisted conception; ICSI; Molar pregnancy; Trophoblast; Genetic analysis
21.  Combination of cabergoline and embryo cryopreservation after GnRH agonist triggering prevents OHSS in patients with extremely high estradiol levels—a retrospective study 
Purpose
Embryo cryopreservation after triggering oocyte maturation with GnRH agonist (GnRHa) in GnRH antagonist protocols has been proposed to prevent ovarian hyperstimulation syndrome (OHSS). However, a small percentage of patients still developed severe OHSS. The purpose of the study was to investigate the efficacy of preventing OHSS in patients at very high risk when cabergoline was given in addition to elective cryopreservation after GnRHa triggering.
Methods
This is a retrospective observational study. The patients were stimulated with GnRH antagonist protocol. When serum E2 concentration was >6,000 pg/ml and there were more than 20 follicles ≥11 mm on the day of final oocyte maturation, GnRHa was used to trigger oocyte maturation. Cabergoline was given to augment the effect of preventing OHSS. The embryos were electively cryopreserved by vitrification and thawed in subsequent cycles. The primary outcome measure was the incidence of severe OHSS. The secondary outcome measure was the clinical pregnancy rate in the first frozen-thawed embryo transfer cycle.
Results
One hundred and ten patients underwent 110 stimulated cycles were included for analysis. No patients developed moderate/severe OHSS. Mean E2 concentration on the day of final oocyte maturation was 7,873 pg/ml, and an average of 22.7 oocytes was obtained from each patient. One hundred and ten thawing cycles were performed, resulting in 69 clinical pregnancies (62.7 %).
Conclusions
Combining cabergoline and embryo cryopreservation after GnRHa triggering in GnRH antagonist protocol could prevent OHSS in patients at very high risk.
doi:10.1007/s10815-013-9997-x
PMCID: PMC3696444  PMID: 23606010
Cabergoline; Ovarian hyperstimulation syndrome; GnRH agonist triggering; Cryopreservation
22.  High prevalence of isolated sperm DNA damage in infertile men with advanced paternal age 
Background
Sperm DNA damage is associated with male infertility, lower pregnancy rates and pregnancy loss.
Objective
The primary aim of our study was to evaluate the prevalence of sperm DNA damage in younger and older men with normozoospermia.
Design, Setting and Participants
We obtained semen from 277 consecutive non-azoospermic men presenting for sperm DNA testing.
Outcome Measurements and Statistical Analysis
The main outcome measures included sperm % DNA fragmentation index (%DFI, using sperm chromatin structure assay), sperm concentration, motility and morphology, and, paternal age.
Results and Limitations
Sperm % DFI was positively correlated with paternal age (r = 0.20, P < 0.001) and inversely correlated % progressive motility (r = −0.16, P = 0.01). Sperm %DFI was significantly higher in older (≥40 years) compared to younger (<40 years) normozoospermic men (17 ± 13 vs. 12 ± 8, respectively P = 0.008), whereas, sperm concentration, progressive motility and morphology were not significantly different in these two groups. Moreover, the prevalence of high levels of sperm DNA damage (>30 % DFI) was significantly higher in older compared to younger normozoospermic men (17 % vs. 3 %, respectively, P < 0.001).
Conclusion
The data indicate that a conventional semen analysis can often fail to detect a defect in spermatogenesis (high %DFI) in older men and suggest that infertile couples with advanced paternal age, including those with normal semen parameters, should consider sperm DNA testing as part of the couple evaluation.
doi:10.1007/s10815-013-0015-0
PMCID: PMC3696445  PMID: 23722935
Sperm; DNA fragmentation; Age; Normozoospermia; Infertility
25.  Analysis of telomere length in couples experiencing idiopathic recurrent pregnancy loss 
Purpose
Telomere length plays a significant role in various disorders; however, its role in idiopathic recurrent pregnancy loss (iRPL) is not known. The objective of this study was to assess telomere length in peripheral blood leukocytes in couples experiencing unexplained recurrent pregnancy loss (iRPL).
Methods
The study included 25 couples experiencing iRPL and 20 controls. The mean relative telomere length was measured by quantitative Real Time PCR (Q-PCR) based assay, which measures the average ratio of telomere repeat copy number to a single copy gene (36B4) copy number (T/S ratio) in each sample.
Results
The relative leukocyte mean telomere length (T/S) in both men and women from iRPL group was significantly lower (p < 0.05) when compared to controls. A significant (P < 0.05) negative correlation was found between age and leukocyte telomere length (T/S ratio). Among the sperm parameters seminal volume was found to be negatively (r = −0.4679) associated with the telomere T/S ratio. The DNA fragmentation index of sperm showed positive correlation (r = 0.4744) with telomere length. In this preliminary study, we found that shorter telomere length in both men and women may be associated with early pregnancy loss.
Conclusion
In conclusion, shorter telomere length in both male and female partners appears to play a role in the idiopathic recurrent pregnancy loss. Loss of telomeric DNA due to oxidative stress needs further analysis. Analysis of telomere length in germ cells are needed to further substantiate the findings of this study.
doi:10.1007/s10815-013-9993-1
PMCID: PMC3696449  PMID: 23608779
Telomere length; Recurrent pregnancy loss; Q-PCR, Sperm chromatin structure assay; Reactive oxygen species

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