Decreased pregnancy and live birth rates after vitrification of in vitro matured oocytes

Purpose

To assess effects on fertilization rate, embryo quality, pregnancy, and live birth rates of vitrification and warming of oocytes that matured in vitro (vIVM) compared to fresh in vitro maturation (fIVM) cycles.

Methods

A retrospective cohort study conducted at a university hospital-affiliated IVF unit. Fifty-six cycles of vIVM cycles and 263 fIVM in women diagnosed with polycystic ovarian syndrome (PCOS) ovaries were included in the analysis. The study group included PCOS patients who failed ovulation induction with intrauterine insemination and were offered IVM cycle followed by oocyte vitrification and warming. The embryological aspects and clinical outcomes were compared to those of controls undergoing fresh IVM cycles during the same period. The main outcome measure was live birth rate.

Results

One thousand seventy oocytes were collected from 56 patients and underwent vitrification and warming. In the control group, 4781 oocytes were collected from 219 patients who had undergone a fresh IVM cycle. Oocyte maturation rates were similar between the groups (mean ± SD: 0.7?±?0.2 vs. 0.6?±?0.2, for vIVM and fIVM, respectively). Survival rate after warming was 59.8%. Fertilization and embryo cleavage rates per oocyte were significantly lower in the vIVM group. Clinical pregnancy (10.7 vs. 36.1%) and live birth rates (8.9 vs. 25.9%) per cycle were significantly lower in the vIVM group than those in the fIVM group (P?=?0.005 and P?<?0.001, respectively). Five healthy babies were born in the vIVM group.

Conclusions

The reproductive potential of vitrified IVM oocytes is impaired. This injury likely occurs through vitrification and warming.

     

The effect of vitrification on maturation and viability capacities of immature human oocytes

Background

15 % of oocytes collected from Assisted Reproductive Technology (ART) cycles are immature. These oocytes may be matured following in vitro maturation (IVM) program. It is possible to cryopreserve the immature oocytes for further use in ART after application of IVM.

Objective

The aim was to determine the maturation rate and viability of human oocytes that were matured in vitro after vitrification program.

Materials and methods

63 women (19–43 years old) who underwent controlled ovarian stimulation for ART were included in this study. 53 immature oocytes were used for fresh group (fIVM) and 50 immature oocytes for vitrification group (vIVM). The maturation medium was Ham’s F₁₀ supplemented with 0.75 IU FSH, 0.75 IU LH and 40 % human follicular fluid (HFF). After 36 h, maturation and morphology of all oocytes were assessed. Also, the oocyte viability was assessed using PI/Hoechst immunostaining technique.

Results

The maturation rates were reduced in vIVM group (56.0 %) in comparison to fIVM group (88.7 %; P < 0.001). Oocyte viability rate were also reduced in vIVM group (56.0 %) in comparison to fIVM (86.8 %, P < 0.007).

Conclusions

Cryopreservation via vitrification reduced both the maturation capacity and viability of human oocytes in IVM technology. It is, therefore, recommended to apply IVM on fresh immature oocytes, instead.     

The impact of vitrification on murine germinal vesicle oocyte In vitro maturation and aurora kinase A protein expression

Purpose

Investigate the effect of vitrification on in vitro maturation (IVM) and expression of Aurora kinases A, B, and C in germinal vesicle (GV)-stage oocytes.

Methods

GV-stage oocytes from B6D2F1 female mice 7–11 weeks of age were vitrified after collection, thawed, and matured in vitro for 0, 4, 8, and 12 h (hrs). The rate of germinal vesicle breakdown (GVBD), spindle apparatus assembly, and Aurora kinase mRNA and protein expression during IVM was measured.

Results

Oocyte vitrification was associated with significant delays in both GVBD and normal spindle apparatus assembly at 4 and 8 h of IVM (p < 0.05). There was no difference in mRNA levels between control and vitrified oocytes for any of the Aurora kinases. Aurora A protein levels were reduced in vitrified compared to control oocytes at 0 h (p = 0.008), and there was no difference at 4 and 8 h (p = 0.08 and 0.69, respectively) of IVM.

Conclusions

Vitrified oocytes have delayed GVBD and normal spindle assembly during in vitro maturation. Reduced levels of Aurora A protein immediately post-thaw may be associated with the impaired oocyte maturation manifested by the delayed progression through meiosis I and II, and the atypical timing of the formation of meiotic spindles in vitrified GV-stage oocytes.     

In-vitro maturation of human oocytes: before or after vitrification?

Purpose

This study aims to determine if in-vitro maturation (IVM) of human immature oocytes should be performed before or after vitrification.

Methods

A total of 184 immature oocytes were randomly divided into two different groups: 100 were vitrified at metaphase II (MII) stage 24 h-48 h after IVM (group 1) and 84 were immediately vitrified at germinal vesicle (GV) or metaphase I (MI) stages and in vitro matured after warming (group 2).

Results

Survival rate after warming was similar in both groups (86.9% versus 84.5%). However, oocyte maturation rate per collected oocyte was significantly higher for oocytes matured before vitrification (group 1, 46%) than for oocytes vitrified before IVM (group 2, 23.8%) (p < 0.01). Consequently, the number of MII oocytes inseminated per oocyte collected was significantly higher for group 1 (40%) than for group 2 (23.8%) (p < 0.05).

Conclusion

IVM procedure is more efficient when it is performed before oocyte vitrification.     

Prematuration of bovine oocytes with butyrolactone I reversibly arrests meiosis without increasing meiotic abnormalities after in vitro maturation

Objectives

Asynchrony between nuclear and cytoplasmic maturation, and possibly damage to the oocyte meiotic spindle, limits the application of in vitro maturation (IVM) in assisted reproduction. Several studies have suggested that Prematuration with meiosis blockers may improve oocyte quality after IVM, favoring early embryogenesis. Thus, we investigated the effect of Prematuration with the nuclear maturation inhibitor butyrolactone I (BLI) on the meiotic spindle and chromosomal configuration of bovine oocytes.

Study design

Immature oocytes obtained from cows slaughtered in a slaughterhouse (n = 840) were divided into the following groups: (1) control (n = 325), submitted only to IVM in TCM199 for 24 h; (2) BLI 18 h (n = 208) submitted to meiotic blockage with 100 μM BLI for 24 h (Prematuration) and then induction of IVM in TCM199 for 18 h; and (3) BLI 24 h (n = 307), pre-matured with 100 μM BLI for 24 h followed by 24 h of IVM in TCM199. The oocytes were then fixed, stained by immunofluorescence for morphological visualization of both microtubules and chromatin, and evaluated.

Results

Meiotic arrest occurred in 90.2% of the oocytes cultured with BLI. Maturation rates were similar for all groups (80.3%, 73.6% and 82.7% for the control, BLI 18 h and BLI 24 h groups, respectively). We observed 81.3% normal oocytes in metaphase II in the control group, and 80.0% and 81.2% in the BLI 18 h and BLI 24 h groups, respectively. The incidence of meiotic anomalies did not differ between groups (18.7%, 20.0% and 18.8% for the control, BLI 18 h and BLI 24 h, respectively).

Conclusion

Prematuration with butyrolactone I reversibly arrests meiosis without damaging the meiotic spindle or the chromosome distribution of bovine oocytes after in vitro maturation.     

Aberrant DNA methylation of imprinted loci in human in vitro matured oocytes after long agonist stimulation

Objective

To evaluate the epigenetic risk linked to assisted reproductive technology at oocyte level by analyzing methylation status of imprinted H19, PEG1 and KvDMR1 in human MII oocytes rescue-matured from MI/GV oocytes after long agonist stimulation.

Study design

580 MI/GV oocytes from 275 patients receiving an ICSI procedure were additionally cultured for 24 h, and 221 rescue-matured MII oocytes were obtained. Pyrosequencing with confirmatory routine bisulfite sequencing were used to determine the methylation status of H19 DMR in 35 oocytes, PEG1 DMR in 47 oocytes, and KvDMR1 in 34 oocytes.

Results

Abnormal methylation status were found in 22.9% oocytes at H19 DMR, 17.0% oocytes at PEG1 DMR and 8.8% oocytes at KvDMR1.

Conclusion

We hypothesize that the use of MII-rescue oocytes may increase the risk of imprinting defects because they might not have completed full imprinting programme.     

Cumulative live birth rates after GnRH-agonist triggering of final oocyte maturation in patients at risk of OHSS: A prospective,clinical cohort study

Objectives

To prospectively study the incidence of OHSS, live birth likelihood and neonatal outcome after GnRH-agonist triggering of final oocyte maturation and vitrification of all pronucleate (2PN) oocytes for later frozen–thawed embryo transfer (FRET) in an OHSS-risk population.

Study design

Prospective, clinical cohort study (12/2004–5/2009). Forty patients undergoing ovarian stimulation in a GnRH-antagonist protocol and at risk of developing severe OHSS underwent triggering with 0.2 mg triptorelin and elective vitrification of all 2PN-oocytes for later frozen–thawed embryo transfer.

Results

The incidence of OHSS was 0% (0/40; 95% confidence interval: 0.0–6.4%). Thirty-nine patients underwent 87 FRETs (mean number of FRETs per patient: 2.2 ± 1.6; range: 1–7). The cumulative live birth rate per patient was 35.0% (14/40; 95% confidence interval: 23.9–48.0%). Mean time-to-conception resulting in live birth after agonist triggering was 24.2 (±17.1; range: 9–67) weeks. Nine healthy singletons and five twins were born.

Conclusions

A treatment algorithm combining agonist trigger with vitrification of all 2PN-oocytes is feasible and safe, and provides patients with a good cumulative chance of live birth.     

DHEA supplementation may improve IVF outcome in poor responders: a proposed mechanism

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  > 15 mm, 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).     

Effect of female nargile smoking on in vitro fertilization outcome

Objective

Smoking is a significant health hazard that has been associated with poor reproductive outcome and reduced fertility in reproductive age women. The aim of this study was to assess the effect of nargile smoking on intra-cytoplasmic sperm injection (ICSI) outcome.

Study design

A prospective analysis of the outcomes of 297 women who underwent ICSI treatment at the ART Unit at the American University of Beirut Medical Center between January 1, and December 31, 2006 was done. The patients were divided into 3 groups based on their smoking status: cigarette smokers (n = 42), nargile smokers (n = 51) and non-smokers (n = 204).

Results

The mean age of nargile smokers was significantly lower than the other groups; however, the 3 groups were similar with respect to the cause of infertility, total dose of follicular stimulating hormone (FSH), number of oocytes and embryos obtained, and number and quality of embryos transferred. There was no significant difference in the clinical pregnancy rate between nargile smokers and non-smokers (51.0% vs 43.6%). However, cigarette smokers had a significantly lower clinical pregnancy rate compared to non-smokers (23.8% vs 43.6%, p = 0.0238). On multiple logistic regression analysis, factors that decreased the clinical pregnancy rates were cigarette smoking and maternal age.

Conclusion

Although this study did not find a deleterious effect of nargile smoking on ICSI outcome, the results need to be confirmed in prospective studies that would include larger number of women with more objective measures of nargile smoke exposure.     

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.     

PCOS patients can benefit from in vitro maturation (IVM) of oocytes

Objective

Our aim was to compare treatment outcome following in vitro maturation (IVM) compared with IVF in patients with polycystic ovarian syndrome (PCOS).

Study design

Retrospective evaluation of treatment in women with PCOS who underwent IVM (108) and IVF (108).

Results

We found a significant difference in outcome between IVM and IVF, with an increase in the number of mature oocytes derived (10.5 ± 6.5 vs. 15.3 ± 8.8, p < 0.0001) and the cleavage rate (92.4 ± 13.0 vs. 95.2 ± 11.7, p = 0.03) in IVM cycles. Due to the lower implantation rate (16.1% vs. 21.6%, p = 0.07) we tend to transfer more embryos in the IVM group (3.4 ± 0.8 vs. 2.8 ± 1.0, p < 0.0001), but the multiple pregnancy rate in that group was not higher. Importantly, the delivery rate was similar in both groups (26.8% vs. 25%). We also report a yearly change in the success rate of IVM during this period.

Conclusions

IVM treatment for PCOS patients may be a valid alternative treatment to IVF with the advantage of eliminating the risk of OHSS and reducing the cost of medication, whilst maintaining high clinical pregnancy rate.     

A randomized pilot study of acupuncture treatment for primary dysmenorrhea

Objective

To compare the therapeutic effect of acupuncture and non-steroidal anti-inflammatory drug (NSAID) therapy in primary dysmenorrhea patients.

Study design

Thirty-five young women with a diagnosis of primary dysmenorrhea were recruited for the study. Their dysmenorrhea severity was rated by visual analog scale (VAS) immediately prior to entry into the study. They were randomly divided into two groups; and the following month they were given NSAID (group 1, n = 24) or acupuncture treatment (group 2, n = 11). Pain was rated again using VAS during menstruation in both groups.

Results

After one month's treatment, pain scores were significantly lower in both groups (p < 0.05). Mean pain scores decreased by 52.2% and 69.5% in the NSAID and acupuncture groups, respectively.

Conclusion

Acupuncture was as effective as NSAID therapy for patients with primary dysmenorrhea. Since this was a pilot study with a small sample size and short follow-up period, larger studies are needed to clarify the effect of acupuncture in the treatment of primary dysmenorrhea.     

Plasminogen activator inhibitor-1 4G/5G polymorphism and susceptibility to endometriosis in the Italian population

Objectives

Some controversy exists for the potential association of the plasminogen activator inhibitor-1 (PAI-1) gene polymorphism 4G/5G and susceptibility to endometriosis. To clarify this issue, we have examined the prevalence of this polymorphism in a case–control study in the Italian population.

Study design

The PAI-1 4G/5G polymorphism was evaluated in n = 368 reproductive year aged Caucasian women who underwent gynaecological laparoscopy for chronic pelvic pain, infertility, ovarian cysts and myomas. A second group of controls included n = 329 normal subjects.

Results

The 697 women enrolled were divided as follows: the endometriosis group (n = 204), the gynaecological control group (n = 164) and the general population control group (n = 329). No statistical significant differences emerged between endometriosis patients and gynaecological controls with regard to the allele frequencies and co-dominant and dominant models of genotype distribution. A borderline statistical difference was only observed for the recessive model of inheritance in which, contrary to previous findings, the PAI-1 4G/4G genotype seems to be less linked to the disease development.

Conclusion

The findings reported herein do not support the previously reported data indicating a greater susceptibility to endometriosis in patients harbouring the PAI-1 4G/5G and 4G/4G genotypes and exclude a significant role of polymorphism in endometriosis development.     

The effect of tobacco exposure on maternal and fetal thyroid function

Objective

To determine the effect of smoking on maternal and fetal thyroid function.

Study design

This prospective cohort study involved healthy women undergoing elective cesarean section for term singleton infants. Maternal and fetal thyroid indices were compared between smokers (n = 21) and non-smokers (n = 83).

Results

Maternal thyrotropin (TSH) concentrations were significantly lower in smokers than non-smokers (1.75 mIU/L versus 2.15 mIU/L, respectively, p = 0.007), with similar free thyroxine (FT4) concentrations (9.59 pmol/L versus 9.56 pmol/L, p = 0.755). For women who smoked, the correlation between the average number of cigarettes smoked per day and maternal TSH concentrations was −0.427, p = 0.054. Infants of smokers and non-smokers had similar umbilical TSH (5.43 mIU/L versus 5.82 mIU/L, p = 0.124) and FT4 concentrations (13.06 pmol/L versus 13.57 pmol/L, p = 0.049).

Conclusion

We demonstrated for the first time that women who smoke during pregnancy have significantly lower TSH concentrations than non-smokers.     

Increased levels of oxidative stress markers in the peritoneal fluid of women with endometriosis

Objective

To evaluate 8-hydroxy-2-deoxyguanosine (8-OHdG) and 8-isoprostane levels in the peritoneal fluid (PF) of women with endometriosis.

Study design

One hundred and ten women with laparoscopically and histopathologically confirmed endometriosis and, as reference groups, 119 patients with simple serous (n = 78) and dermoid (n = 41) ovarian cysts were studied. Peritoneal fluid 8-OHdG and 8-isoprostane concentrations were evaluated by enzyme-linked immunosorbent assays.

Results

8-OHdG and 8-isoprostane levels in peritoneal fluid were significantly higher in patients with endometriosis compared with the reference groups. Higher PF 8-OHdG and 8-isoprostane concentrations were observed in patients with advanced stages of endometriosis. A statistically significant positive correlation was found between 8-OHdG and 8-isoprostane levels in peritoneal fluid.

Conclusion

Endometriosis induces greater oxidative stress and frequent DNA mutations in peritoneal fluid than nonendometriotic ovarian cysts. The most severe oxidative stress occurs in the peritoneal cavity of women with more advanced stages of the disease.     

Association between a single nucleotide polymorphism in MTHFR gene and polycystic ovary syndrome

Objective

The aim of the present study was to investigate whether there is an association between the C677T polymorphism in MTHFR and PCOS in a Korean population.

Study design

The prevalence of MTHFR gene was compared between women with PCOS (n = 227) and normal patients (n = 115) using restriction fragment length polymorphism (RFLP) analysis. The HapAnalyzer was used to analyze the genotype of MTHFR polymorphism in PCOS and control subjects. We considered a p-value less than 0.05 as statistically significant.

Results

The frequency of C/C, C/T, and T/T genotype showed similar proportion between PCOS and control subjects. In addition, the frequencies of co-dominant (p-value = 0.8334, odds ratio (OR) = 1.04), dominant (p-value = 0.8749, OR = 0.96) and recessive alleles (p-value = 0.5574, OR = 1.22) did not show any association between PCOS and control subjects.

Conclusion

Our data demonstrate that the C677T polymorphism of MTHFR gene is not associated with PCOS in a Korean population, suggesting that the C677T polymorphism in MTHFR may have different influences in various ethnic groups and diseases.     

Offspring birth weight and maternal fasting lipids in women screened for gestational diabetes mellitus (GDM)

Objectives

Maternal lipid metabolism is altered during pregnancy but little is known about the influence of these alterations on either intrauterine fetal development or maternal wellbeing. The purpose of this study was to examine the relationship between both fasting cholesterol and triglycerides and offspring birth weight in women screened selectively for gestational diabetes mellitus (GDM).

Study design

In a prospective observational study in a University Maternity Hospital, women were recruited at their convenience when they were screened for GDM with a diagnostic 75 g oral glucose tolerance test (OGTT). An additional sample was taken for a lipid profile at the time the fasting glucose was obtained. Clinical and socio-demographic details were recorded.

Results

Of the 189 women recruited, the mean age was 32 years, 35.4% (n = 67) were primigravidas, 44.1% (n = 82) were obese and 11.6% (n = 22) had an abnormal OGTT. On univariate analysis, increasing birth weight was correlated positively with multiparity, first trimester body mass index (BMI), GDM and hypertriglyceridaemia but not with cholesterol levels. On multivariate analysis, increased birth weight correlated positively only with hypertriglyceridaemia.

Conclusions

This study provides further evidence that maternal hypertriglyceridaemia is important in programming intrauterine fetal growth and raises questions about whether women should be screened selectively for dyslipidaemia before, during and after pregnancy.     

Role of phyto-oestrogens in ovulation induction in women with polycystic ovarian syndrome

Objective

To study the role of a phyto-oestrogen, Cimicifuga racimosa extract (Klimadynon^®, Bionorica, Neumarkt i.d.OBf., Germany), in ovulation induction in women with polycystic ovarian syndrome (PCOS).

Study design

Prospective randomized controlled trial in Minia University Hospital, Minia, Egypt. One hundred women with PCOS were allocated into one of two groups: one group (n = 50) received clomiphene citrate 100 mg daily for 5 days, and the other group (n = 50) received C. racimosa 20 mg daily for 10 days. Both groups received medication starting from the second day of the cycle for three consecutive cycles, during which changes in follicle-stimulating hormone (FSH), luteinizing hormone (LH), FSH/LH ratio, progesterone, endometrial thickness and pregnancy rate were measured.

Results

The groups were similar in terms of age, clinical presentation and hormonal levels before treatment. Following treatment, significant favourable changes in LH level and FSH/LH ratio (p = 0.007 and 0.06, respectively) were seen in the Klimadynon group. In this group the progesterone level was higher from the first treatment cycle, indicating better ovulation (p = 0.0001), and endometrial thickness was greater (p = 0.0004). The pregnancy rate was higher in the Klimadynon group but the difference between the groups was not significant (p = 0.1).

Conclusion

Phyto-oestrogen can be used as an alternative to clomiphene citrate for ovulation induction in women with polycystic ovarian syndrome.     

Preoperative MRI criteria for trials on less radical surgery in Stage IB1 cervical cancer

Objective

The aim of this study is to identify a patient group with a low-risk of parametrial involvement (PMI) in Stage IB1 cervical cancer using preoperative magnetic resonance imaging (MRI) parameters.

Methods

In total, 190 Stage IB1 cervical cancer patients with clinically visible lesions who had undergone Type C2 radical hysterectomy and preoperative MRI were included in this study. Clinical records, pathology reports, and preoperative MRI findings were reviewed retrospectively.

Results

Of the 190 patients, 19 (10%) had pathologic PMI. The largest tumor diameter identified by MRI ranged from zero (no definite mass on the cervix) to 60 mm, with a median of 21 mm. Patients were identified as being either low-risk (tumor size ≤ 25 mm and no evidence of PMI, n = 127) or high-risk (tumor size > 25 mm and/or findings indicating PMI, n = 63) based on MRI parameters. The rate of pathologic PMI in low- and high-risk patients was 0.0% and 30.2%, respectively (P < 0.001). Five-year progression-free survival in low-risk patients was 95.9%, which is significantly better than the rate of 85.6% for patients in the high-risk group (P = 0.039).

Conclusions

Preoperative MRI parameters can help identify patients with a low-risk of PMI and, therefore, possible candidates for trials on less radical surgery.