科学研发文摘与论文

Siew SC, Lee CSS, Lim YX, Leong WY. Clinical outcome of blastocysts derived from frozen donor oocytes versus fresh donor oocytes in fresh blastocyst transfer cycles.

Presented at 25th Congress of the Obstetrical & Gynaecological Society of Malaysia, 27- 30 of July 2017, Kuala Lumpur, Malaysia.

Objectives:

The Cryotec method has been employed in all frozen-warmed cycles at Alpha Fertility Centre (AFC) since July 2013. With the Cryotec method, we have consistently achieved 100% post-warmed survival rates of embryos (Lee et al, 2016), and a near 100% post-warmed survival of vitrified oocytes (Lui et al, 2016). This robust cryopreservation method has enable us to establish oocyte banking in AFC since 2014. In this study we compare the clinical outcome of vitrified-warmed oocyte and fresh oocytes in patients undergoing oocyte donation program.

Methods:

Forty-one women underwent fresh blastocyst transfer using anonymously donated oocytes at Alpha Fertility Centre, Malaysia from March 2014 until December 2016. Nineteen of these patients were allocated with vitrified-warmed donated oocytes (Group A) while 22 patients received donated oocytes from fresh retrievals (Group B). Oocytes from Group A were vitrified and warmed using the Cryotec method (Cryotech, Japan). All oocytes had Intra-Cytoplasmic Sperm Injection (ICSI) and resultant embryos were cultured to day 5 or 6. The mean age of oocyte donors in Group A and Group B was 23.2 and 24.4 respectively (p>0.05); and mean age of recipients was 41.0 and 39.8 respectively (p>0.05). All recipients underwent a medicated transfer cycle using down regulation with Intra-muscular Depot Leucrin 3.75mg, and endometrial priming with oral oestrogen (Progynova) in graduated doses. Progesterone pessaries were administered daily 7 days prior to the transfer. Blastocysts were transferred using standard embryo transfer (ET) protocols.

Results:

In Group A, a total of 284 oocytes were warmed. Two-hundred-and-seventy-two oocytes survived (Post-warmed Survival Rate: 95.8%). One patient in Group A failed to reach ET due to poor blastocyst quality obtained; while all patients in Group B progressed to ET. The mean number of blastocysts transferred was 1.8 and 2.0 for Group A and Group B respectively (p>0.05). Clinical Pregnancy Rate (CPR) for Group A was 66.7% and for Group B was 63.6%. Implantation Rates (IR) were 46.9% and 51.2% for Group A and B respectively. There was no statistical significance (p>0.05) found in CPR and IR between both groups.

Conclusion:

This study shows that vitrified-warmed donor oocytes using the Cryotec method yield clinical pregnancy and implantation rates comparable to fresh donor oocytes.

Chooi JMS, Lee CSS, Lim YX. The Cryotec Method yields 100% post-warmed survival rates in Day 3 cleavage stage embryos and its blastomeres.

Presented at 25th Congress of the Obstetrical & Gynaecological Society of Malaysia, 27 – 30 of July 2017, Kuala Lumpur, Malaysia.

Objective:

In our previous report in 2016, we achieved 100% post-warmed survival rate for all cleavage stage embryos vitrified using the Cyrotec Method. Cryotec is an innovative method of vitrification to preserve oocytes and embryos of any developmental stage. Here in this study, we further demonstrate the efficacy of the Cryotec Method through analysis of a greater sample size, establishing the validity of the earlier findings.

Materials and methods:

A total of 111 Day 3 cleavage stage embryos with 897 blastomeres underwent cryopreservation by vitrification and subsequently warmed for FET cycles using Cryotec Vitrification and Warming Media since we commenced Cryotec Method from July 2013 until now (April 2017) in Alpha Fertility Centre. The study consists of 57 cases with patients in the age range of 18 to 44 with a mean age of 35.3. Practitioner techniques for vitrification and warming were adhered to manufacturers outlined SOPs (Cryotech, Japan). All embryos ranged from 5 cells to 14 cells with <15% fragmentation and were derived from either intracytoplasmic sperm injection or in-vitro fertilization. The survivability of embryos and its blastomeres were assessed in terms of the number of intact or lysed cells upon warming.

Results:

After Cryotec warming, all 111 embryos survived with no degradation in quality, yielding 100% post-warmed embryo survival rate. Furthermore, the blastomere survival rate also achieved 100%, indicated by the 897 healthy and intact blastomeres and the absence of lysed cells upon observation. The survivability of embryos was not affected by the number of cells nor degree of fragmentation.

Conclusion:

This study validates that the use of Cryotec Method for embryo vitrification and warming consistently achieved 100% post-warmed survival rates in Day 3 cleavage stage embryos and blastomeres in Alpha Fertility Centre. The Cryotec method realizes the total potential of embryo cryopreservation and proves to be superior compared to other vitrification methods practiced in ART laboratories worldwide today.

Low SY, Lee CSS, Lim YX. 100% post-warmed survival rate for 1491 blastocysts in Alpha Fertility Centre.

Presented at 25th Congress of the Obstetrical & Gynaecological Society of Malaysia, 27- 30 of July 2017, Kuala Lumpur, Malaysia.

Introduction:

With the benefit of better endometrium receptivity in unstimulated cycles and supported by good post-warmed blastocysts survival rate, it is now clear that pregnancy rates for frozen blastocyst transfer is better than the transfer of fresh blastocysts. Alpha Fertility Centre has adopted the Cryotec Method for blastocyst vitrification and warming since July 2013. This study demonstrates the post-warmed survival rate for 1491 blastocysts in 1011 frozen blastocyst transfers (FBT).

Materials and Methods:

Since the commencement of the use of Cryotec Method in July 2013 till now (May 2017), Alpha Fertility Centre had vitrified and warmed 1491 blastocysts using the Cryotec Method for 1011 FBT patients. Only blastocysts which developed to at least expanding stage (quality of at least BG3BB according to Gardner’s Blastocyst Grading System) were vitrified and warmed. The blastocyst vitrification and warming protocols were conducted according to manufacturer’s protocols (Cryotech, Japan). The number of FBT cycles for each age group was 621 (<35 years old), 182 (35-37 years old), 111 (38-39 years old), 70 (40-41 years old) and 27 (≥42 years old). The number of blastocysts vitrified and warmed for each age group was 954, 258, 149, 91 and 39 respectively.

Results:

Of the 1491 blastocysts warmed, all blastocysts survived with morphologically intact inner cell mass and trophectoderm cells with no degradation in quality.

Conclusion:

This study shows that by using the Cryotec Method, we consistently achieved 100% post-warmed survival rate in blastocysts.

Lee, CSS, Lim MW, Lui WX. Administration of Canestan prior to Frozen Embryo Transfer (FET) of Euploid Blastocysts May Improve Clinical Outcome.

Presented at 16th International Conference on Preimplantation Genetics, 26 – 29 March 2017, Valencia, Spain.

Introduction:

Embryo transfer (ET) in IVF involves putting embryo(s) in transfer catheters which goes through the cervical os and into the uterine cavity. There is a risk of picking up bacteria and fungus from various vaginal-cervical microorganisms during the process. It has been reported that bacterial and Candida infection in women going through IVF can negatively affect embryonic implantation (Wittemer et al., 2004). To our knowledge, this is the only report which alludes to a possible relationship between vaginal yeast infection and implantation following ET. We hypothesize that a preventative treatment such as with clotrimazole (Canestan) to avoid candida infection may improve clinical outcomes. This is an empirical study comparing clinical outcomes between FET patients prescribed with Canesten pessaries (Group A) and those who were not prescribed with Canesten pessaries (Group B) prior to the transfer of euploid blastocysts. High vaginal swabs (HVS) were not routinely done in this study since the results were not sensitive and accurate enough based on our experience with the outsourced laboratories we work with.

Material and Methods:

A total of 100 patients with blastocysts screened using Preimplantation Genetic Screening (PGS) underwent FET cycles in Alpha Fertility Centre, Malaysia between June 2016 and October 2016. These patients were divided into 2 groups: 57 patients with 69 euploid blastocysts transferred in the treatment group (Group A), and 43 patients with 59 euploid blastocysts transferred in the control group (Group B). All patients were administered with oestrogen (Progynova) and progesterone (Cyclogest or Crinone 8%) for endometrium preparation. In Group A, patients were given Canesten 500 mg pessary 7 days prior to embryo transfer. The mean age of patients in Group A was 33.4 while the mean age of patients in Group B was 31.4 (p>0.05). All thawed blastocysts survived with morphologically intact inner cell masses and trophectoderm cells. The mean number of blastocysts transferred for Group A and B was 1.2 and 1.4 respectively. Clinical pregnancy and number of gestational sacs were determined by ultrasound.

Results:

Table 1: Clinical pregnancy rate and implantation rate between the treatment and control groups.

Overall, the implantation rate appears to be improved in patients treated with Canestan (63.8%) as opposed to those who were not (55.9%), despite the older age of patients in the treatment group. However, this difference was not significant (p>0.05). The clinical pregnancy rate in the treatment group (64.9%) is as good as the control group (62.8%), despite the treatment group being older and having less embryos transferred.

Conclusion:

This preliminary study suggests that the use of Canesten pessaries in FET cycles may improve implantation rate. Further studies which are controlled needs to be undertaken to confirm the above.

Lee CSS, Yap WY, Lim YX, Lim MW, Leong WY. Simultaneous Preimplantation Genetic Diagnosis (PGD) for Alpha-thalassemia and Preimplantation Genetic Screening (PGS) for aneuploidy on single biopsy using Ion-Torrent Next Generation Sequencing (NGS).

Presented at 7th Congress of the Asia Pacific Initiative on Reproduction, 30 March – 2 April 2017, Kuala Lumpur, Malaysia.

Introduction:

This report reviews the clinical outcome of embryo transfer following simultaneous PGD and PGS from a single biopsy using a single-NGS platform in Alpha Fertility Centre (AFC), Malaysia.

Materials and Methods:

There were 7 patients who underwent frozen embryo transfer (FET) following alpha-thalassemia PGD and PGS from the time AFC deployed the Ion-Torrent NGS platform from September 2015 till November 2016. Twenty-nine blastocysts with at least fair grade (Gardner, 1999) were biopsied and amplified using multiple displacement amplification. The amplification product from each blastocyst was separated into 2 aliquots: one for library construction using a customised thalassemia-Ampliseq panel; and another to construct PGS library. All libraries were pooled and sequenced simultaneously using a single-NGS platform according to the manufacturer’s specification (Ion Torrent, USA).

Results:

All 29 blastocysts were successfully amplified and analysed; 9 were unaffected, 12 were carriers and 8 were alpha-thalassemia major. Five out of the 9 unaffected blastocysts were euploid whereas 11 out of 12 carrier blastocysts were euploid. Two patients with unaffected-euploid blastocysts and five patients with carrier-euploid blastocysts underwent single blastocyst frozen embryo transfer. The clinical outcome of these patients are summarised in Figure 1.

Conclusions:

The Ion-Torrent NGS platform in AFC enabled both PGD for genetic disease diagnosis and PGS to be performed concurrently on a single platform. This eliminates the need of biopsy twice on the same blastocyst as required for tests performed on separate platforms, and prevents potential damage from a second biopsy. We believe this contributes to our high implantation rate of 71.4%.

Yap WY1; Lee CSS1; Lim YX1; Siew SC1. Euploidy rate of Day 7 blastocysts derived from In Vitro Fertilisation (IVF) .

Presented at 16th International Conference on Preimplantation Genetics, 26 – 29 March 2017, Valencia, Spain.

Introduction

Generally human embryos are cultured up to Day 6 and embryos that do not develop into utilisable blastocysts (assessed with Gardner’s grading system) are discarded at the end of Day 6. Occasionally, blastocyst culture is extended to Day 7 to further observe the development of slow growing blastocysts. These Day 7 blastocysts with at least fair graded inner cell mass and trophectoderm may be chromosomally normal and may result in pregnancy. In Alpha Fertility Centre (AFC), we practice blastocyst culture until Day 7 for patients who do not have utilisable blastocysts on Day 5 and/or Day 6. This is a retrospective study to assess the euploidy rate of Day 7 blastocysts at Alpha Fertility Centre, Malaysia from August 2015 to December 2016.

Materials and Methods

Twelve patients (age ranged 19 – 41, mean age 36.0) who underwent IVF had their embryos successfully cultured to Day 7. Of these, 2 patients had utilisable blastocysts on both Day 6 and 7 while the remaining 10 patients had only utilisable blastocysts on Day 7. Only Day 7 blastocysts were included in this study. All fifteen (15) Day 7 blastocysts were biopsied and proceeded with pre-implantation genetic screening (PGS) by either using Microarray Comperative Genomic Hybridisation (BlueGnome, UK) or Next Generation Sequencing (Ion Torrent, USA) according to manufacturer’s specifications. All Day 7 blastocysts were vitrified after biopsy.

Conclusions

Our preliminary results showed that Day 7 blastocysts can be chromosomally normal and should be considered for embryo transfer.

Lim YX, Lee CSS. Clinical outcomes of single euploid- versus double untested-blastocyst transfer in vitrified-warmed cycles.

Presented at 7th Congress of the Asia Pacific Initiative on Reproduction, 30 March – 2 April 2017, Kuala Lumpur, Malaysia.

Introduction

This is a retrospective study to examine whether transferring a single euploid-blastocyst (SeBT) following preimplantation genetic screening (PGS) can result in clinical outcome that is comparable to transferring 2 untested-blastocysts (DBT) while reducing the incidence of multiple pregnancies.

Materials and Methods

One-hundred and seventy-three euploid-blastocysts from 173 patients (Group 1) and 446 untested-blastocysts from 223 patients (Group 2) were thawed using the Cryotec Method between November-2013 and July-2016. One euploid-blastocyst was transferred in Group 1, and 2 untested-blastocysts were transferred in Group 2. Age was analysed as both a continuous and a categorical variable. The clinical pregnancy rate (CPR), implantation rate (IR) and multiple gestation rate (MGR) were analysed.

Results

All 619 blastocysts survived post-thaw (100% post-thaw survival), enabling transfer in all cases. The mean age of patients undergoing SeBT (33.1 years) was significantly higher than the mean age of patients undergoing DBT (31.9 years)(P=0.0415). The CPR in Group 1 and Group 2 was similar (61.8% versus 62.3%;p=1.0000) and there was no significant difference in CPR between Group 1 and 2 within each age group: <30, 30-34, 35–37, and 38-40 (except age group >40;p=0.0189). The IR in Group 1 was significantly higher than in Group 2 (61.8% versus 47.3%;p=0.0013) and within age group 35-37 (71.4% versus 44.3;p=0.0124) and >40 (72.7% versus 14.7%;p=0.0007). The incidence of multiple pregnancies was greatly reduced in Group 1 (51.8% to 0%; p<0.0001).

Conclusions

The clinical pregnancy rate of SeBT and DBT were equivalent, but SeBT had a higher implantation rate and a lower incidence of twins.

Lee CSS, Lim YX.Single frozen-thawed blastocyst transfer following Preimplantation Genetic Screening (PGS) results in high implantation rate and eliminates the risk of multiple birth

Presented at 7th Congress of the Asia Pacific Initiative on Reproduction, 30 March – 2 April 2017, Kuala Lumpur, Malaysia.

Introduction

This retrospective study compares the clinical outcome between frozen-thawed single blastocyst transfer (SBT) and frozen-thawed double blastocyst transfer (DBT) following PGS.

Materials and Methods

Three-hundred and seventeen euploid-blastocysts from 245 patients were thawed between November-2013 and July-2013. One euploid-blastocyst was transferred in 174 patients (Group 1) and 2 euploid-blastocysts were transferred in 72 patients (Group 2). Age was analysed as both a continuous and a categorical variable. The clinical pregnancy rate (CPR), implantation rate (IR) and multiple gestation rate (MGR) were analysed.

Results

All 317 euploid-blastocysts survived post-thaw (100% post-thaw survival). The mean age of patients undergoing SBT (33.1 years) was significantly higher than the mean age of patients undergoing DBT (30.1 years)(P<0.000.1). The CPR in Group 2 was significantly higher than in Group 1 (79.2% versus 61.8%;p=0.0109). However, there was no significant difference in CPR between Group 1 and 2 within each age group: <30, 30-34, 35–37, and >40 (except 38-40;p=0.0197). There was no significant difference in IR between Group 1 and 2 (61.8% versus 59.0%;p=0.6451) nor between Group 1 and 2 within each age group: <30, 30-34, 35–37, 38–40, and >40. The risk of multiple gestations was greatly increased between Group 1 and 2 (0% versus 49.1%;p<0.0001), and this difference did not vary across age groups (except >40;p=0.1111).

Conclusions

This study shows that frozen-thawed euploid SBT results in 100% singleton and a tendency towards higher implantation rate. These data should encourage clinicians to evaluate their embryo transfer policy and adopt SBT in euploid cryopreserved cycles as their routine practice.

Lim MW, Lee CSS, Keith J.GnRH Antagonist vs Agonist Protocol on Blastocyst Development and Blastocyst Utilisation in IVF Cycles

Presented at 7th Congress of the Asia Pacific Initiative on Reproduction, 30 March – 2 April 2017, Kuala Lumpur, Malaysia.

Objectives

Although the clinical outcome of controlled ovarian stimulation between GnRH-agonist and GnRH-antagonist protocols for IVF has been often analysed, the effect of these analogues on blastocyst development and blastocyst utilisation rate has not been reported. This is a retrospective study to determine the impact of both stimulation protocols on the blastulation and utilisation rate in patients of different age groups in Alpha Fertility Centre (AFC).

Methods

Oocyte donors were excluded from this study. Between June 2016 and September 2016, 71 patients and 91 patients were stimulated with GnRH agonist and GnRH antagonist respectively. Both GnRH analogue groups were subdivided into 3 age groups: <35, 35-41 and >41. Patients across all age groups had OPU and IVF with or without ICSI. The resultant embryos were cultured to blastocyst stage using Cook media and the blastocysts were subsequently transferred, biopsied or cryopreserved.

Results

Blastulation and Blastocyst Utilisation rates between GnRH-Agonist and GnRH-Antagonist Protocol in patients <35 years old and 35-40 years old

* Statistically significant

Blastulation and Blastocyst Utilisation rates between GnRH-Agonist and GnRH-Antagonist Protocol in patients >40 years old

The blastocyst utilisation rate is significantly higher in patients under 35 years old stimulated with GnRH-Antagonist (64.4%) compared to those stimulated with GnRH-Agonist (53.9%). Conversely, there was a trend towards higher blastulation and utilisation rates in >40 year old patients stimulated with GnRH-Agonist (71.7% and 67.6% respectively) compared to GnRH-Antagonist (57.9% and 47.8%).

Conclusion

We suggest that patients under the age of 35 who are undergoing IVF should be stimulated with a GnRH-Antagonist protocol to obtain higher blastulation and blastocyst utilisation rates. Further study will test whether patients above 41 years old would benefit from higher blastulation and utilisation rates when stimulated with a GnRH-agonist analogue rather than the GnRH-Antagonist protocol.

Lee, CSS.; Tee, ZQ.; Yap, WY.; LIM, MW.; Lim, YX. Euploidy Rates of Blastocysts Derived from Microsorted (MicroSort®) Vs Unsorted Sperm

Presented at 7th Congress of the Asia Pacific Initiative on Reproduction, 30 March – 2 April 2017, Kuala Lumpur, Malaysia.

Introduction

MicroSort® (MS) sperm sorting is a preconception method which separates X and Y sperm and is used for family balancing and the prevention of sex-linked diseases in offsprings. This study compares the euploidy rate of blastocysts derived from microsorted and unsorted sperm from January 2016 untill August 2016.

Materials and Methods

In this retrospective study, the chromosome copy number of blastocysts derived from microsorted sperm (study group) and unsorted sperm (control group) were assessed. In the study group, sperm was treated with fluorescence staining and were sorted into X or Y enriched sperm samples using flow cytometry (MicroSort®, USA). Sperm was processed by density gradient centrifugation with swim-up in the control group. Both groups had ICSI and embryos were cultured to Day 5 or 6 and blastocysts with at least an average grade (Gardner’s Grading) were biopsied and screened for aneuploidy.

Results

A total of 231 blastocysts from the study group and 566 blastocysts from the control group were biopsied. The mean age of patients from the study group and control group were 32.9 and 34.3 respectively (p> 0.05). There was no significant difference in euploidy rate between study group and control group (55.0% versus 58.7%; p= 0.3444) nor between both groups for each age group: ≤30, 31-35, 36-37, 38-39 and ≥40.

Conclusions

This study suggests that blastocysts derived from microsorted sperm have similar euploidy rate compared to blastocysts derived from non-sorted sperm.

Low SY, Lee CSS, Lim YX. Blastulation and blastocyst utilisation rates at Alpha Fertility Centre (AFC).

Presented at 7th Congress of the Asia Pacific Initiative on Reproduction, 30 March – 2 April 2017, Kuala Lumpur, Malaysia.

Introduction

We had selectively cultured embryos to blastocysts from year 2011 to April 2013. In that period of time, only 13.2% of the IVF cases were selected for blastocyst culture. We had then reported a blastulation rate of 72.5% and blastocyst utilisation rate of 62.8% (Lee et. al., 2014). Since then we increased the proportion of cases for blastocyst culture progressively. This study measures the blastulation and blastocyst utilization rates for all age groups in Alpha International Fertility Centre from June 2016 till now, during which time 95.7% of cases had blastocyst culture.

Materials and Methods

Since June 2016 till now, we adopted a policy of having all patients going for blastocyst culture with occasionally exception (95.7% of cases had blastocyst culture). In this study, blastulation rate was defined as total number of blastocysts formed per total number of 2PN zygotes. Blastocyst utilisation rate was defined as total number of blastocysts (2PN) utilised (transferred, cryopreserved or biopsied) per total number of blastocysts formed (2PN).

Results

As shown in Table 1, nearly all of the IVF cycles (95.7%) had blastocyst culture. The blastulation and blastocyst utilisation rates for patients aged <35 were 75.9% and 62.5% respectively. Similar rates were also achieved for age group: 35-37, 38-39, 40-41 and >41 (p>0.05). Overall, the blastulation rate and blastocyst utilisation rate for all age groups were 73.2% and 58.0% respectively.

Conclusions: Our study shows high blastulation and blastocyst utilisation rates among all age groups at Alpha Fertility Centre.

Lee CSS, Yap WY, Lim YX, Lui WX, Lim MW. Clinical outcome of preimplantation genetic diagnosis (PGD) for chromosomal translocation and inversion in Alpha Fertility Centre (AFC).

Presented at 24th Congress of the Obstetrical and Gynaecological Society of Malaysia, 2 – 5th June 2016, Kuala Lumpur, Malaysia.

Objectives:
Chromosomal rearrangement carriers (eg. translocation and inversion) are phenotypically normal, but have an increased risk of creating gametes with unbalanced chromosomal rearrangement which results in miscarriages. PGD can be used to screen for chromosomal rearrangement, hence helping such couples to select normal or balanced euploid embryos for transfer. This report reviews the clinical outcome of balanced chromosomal rearrangement carriers undergoing IVF-PGD using Microarray Comparative Genomic Hybridisation (MaCGH) or Next Generation Sequencing (NGS) in AFC.

Methods:
Eight patients from July 2011 to December 2015 with indication of a parental translocation/inversion as confirmed by karyotyping were included in this report. Of these, 7 patients had at least one miscarriage, (range 1 – 4 miscarriages, a total of 16 miscarriages), and one had primary infertility. Of all PGD cases, 76 embryos/blastocysts were obtained from 7 translocation carriers and 12 embryos were obtained from 1 inversion carrier. Biopsied blastomere/trophectoderm cells were analysed for chromosomal status according to manufacturer’s specifications (MaCGH from Illumina, USA and NGS from Ion Torrent, USA). Clinical pregnancy were determined using ultrasound. All pregnant patients were followed up until delivery.

Results:
Seventy-two (72) embryos/blastocysts from 7 translocation carriers and 10 embryos from an inversion case were successfully analysed. Of these, 16 were shown to be normal or balanced euploid embryos/blastocysts. Seven normal or balanced euploid embryos/blastocysts were transferred to 4 patients in fresh cycle and 2 had live births, of which one live birth resulting from a translocation case and another one live birth resulting from inversion case. The remaining 9 normal or balanced euploid blastocysts from 2 patients were frozen. These two patients each had one normal or balanced euploid blastocyst transferred in FET cycles and one patient resulted in live birth. All three pregnant patients had no miscarriages.

Conclusions:
Patients who have chromosomal rearrangement can have their embryos tested using PGD (MaCGH/NGS). This allows the selection of normal or balanced euploid embryos/blastocysts for transfer, thus, increasing the chance of a successful pregnancy, while significantly reducing miscarriage rate.

Lui WX, Lim YX, Low SY, Lim MW, Siew SC, Yap WY, Keith J, Lee CSS. Clinical outcome of single good-graded frozen blastocyst transfer in patients with and without preimplantation genetic screening (PGS).

Presented at 24th Congress of the Obstetrical and Gynaecological Society of Malaysia, 2 – 5th June 2016, Kuala Lumpur, Malaysia.

Objectives:
Screening of blastocysts using PGS has been implemented to improve clinical outcomes while reducing risk of multiple gestations by transferring a single euploid blastocyst. In this study, we compare the outcome of IVF patients who had PGS with those without PGS following single good-grade blastocyst transfer in FET cycles at Alpha Fertility Centre, Malaysia.

Methods:
Sixty-nine patients who had a single good-grade vitrified-thawed blastocyst transferred from December 2013 to December 2015 were analysed. Twenty-two (22) patients had unscreened blastocyst transfer (Group A) and 47 had euploid blastocyst transfer (Group B). In Group B, 3-5 trophectoderm cells had been biopsied and the blastocysts were vitrified on the day of biopsy. All blastocysts were vitrified and thawed using the Cryotec method (Cryotech, Japan). The mean age of patients in each group was similar: Group A was 32.6 (range 21-40) and Group B was 32.0 (range 18-42). A total of 22 unscreened good-grade blastocysts and 47 euploid good-grade blastocysts were thawed in Group A and B respectively. All 69 blastocysts survived with morphologically intact inner cell mass and trophectoderm cells (100% post-thaw survival rate). All 69 blastocysts that had been thawed were transferred.

Results:
Both Clinical Pregnancy Rate (CPR) and the Implantation Rate (IR) for Group A was 54.5% and for Group B was 70.2%. There was no statistical significance (p=0.2788) in CPR and IR between both groups, but shows a tendency for higher rates in Group B.

Conclusion:
This study suggests that single good-grade euploid frozen blastocyst transfer show a trend towards higher clinical pregnancy and implantation rates compared to non-PGS frozen blastocyst transfer. We believe that with a larger sample size, the clinical pregnancy and implantation rates between both groups may be statistically significant. Moreover, the clinical pregnancy and implantation rates of 70.2% for transferring single good-grade euploid frozen blastocyst supports the practice of transferring only one blastocyst.

Lui WX, Lim YX, Low SY, Lim MW, Siew SC, Yap WY, Keith J, Lee CSS. Clinical outcome of vitrified-thawed oocytes at Alpha International Fertility Centre (AFC).

Presented at 24th Congress of the Obstetrical and Gynaecological Society of Malaysia, 2 – 5th June 2016, Kuala Lumpur, Malaysia.

Objectives:
Recent studies have demonstrated post-thaw survival rates of oocytes ranging from 57.9% to 94.0% (Mashashige et al. 2005, Paffoni et al. 2011, Smith et al. 2010, Ghasem Salci et al. 2005, Yun-Xia Cao et al. 2009. Fadini et al. 2009, Machac et al. 2013, Panasco et al. 2012) using various vitrification methods other than the Cryotec method. This study presents the clinical outcome of vitrified-thawed oocytes using the Cryotec method at Alpha International Fertility Centre, Malaysia.

Methods:
Following vitrification, 20 patients had their oocytes thawed. The mean age of patients was 26.5 (range 19-42). All oocytes had Intra-Cytoplasmic Sperm Injection (Piezo-ICSI) and resultant zygotes were cultured to Day 3, 5 or 6. Post-thaw survival and fertilisation rate were measured. Clinical pregnancy and number of gestational sacs were determined using ultrasound.

Results:
A total of 231 oocytes were thawed. Two-hundred and twenty (220) oocytes survived (Post-thaw Survival Rate: 95.2%) and had Piezo-ICSI. One hundred and thirty-nine (139) oocytes were normally fertilised (Fertilisation rate: 63.2%). All 139 zygotes developed to embryos on Day 3, 2 of them were transferred into 1 patient, while the remaining 137 embryos were further cultured to Day 5/6. Of the 137 embryos, 90 developed to blastocysts on Day 5/6 whereby 31 were transferred into 17 patients, 20 were vitrified, and the remaining 39 blastocysts were poor quality. The mean number of embryos/blastocysts transferred was 1.8. Two (2) patients failed to reach ET due to poor blastocyst quality obtained. Of the 18 patients who had embryo transfer, 11 patients were clinically pregnant (Clinical Pregnancy Rate/ Embryo Transfer: 61.1%) with 14 embryos/blastocysts implanted (Implantation rate: 42.4%).

Conclusion:
Our experience with the Cryotec Method in the vitrification of oocytes showed 95.2% post-thaw survival rate with good clinical pregnancy and implantation rates. We believe Cryotec is the cryopreservation method of choice to maximise the freeze-thaw survival rate of oocytes.

Lim MW, Lim YX, Lee CSS. MicroSort® sperm sorting and euploidy in patients with advanced maternal age.

Presented at 24th Congress of the Obstetrical and Gynaecological Society of Malaysia, 2 – 5th June 2016, Kuala Lumpur, Malaysia.

Objective:
Microsort® sperm sorting technology is known to enrich either X- or Y-bearing sperm. Since the fluorescent signals in this technology is influenced by DNA content and the shape of the sperm head, theoretically, more morphologically normal sperm may be obtained. This increase in number of morphologically normal sperm may lead to a decrease in number of sperm with chromosomal abnormalities. Although the majority of embryonic aneuploidy is derived maternally especially in those with an advanced maternal age (AMA), some cases are of paternal origin. This is a prospective study in Alpha Fertility Centre (AFC) to investigate the use of Microsort® sperm sorting in decreasing embryonic aneuploidy in patients with AMA.

Methods:
Thirty (30) patients’ husband (Group 1) had Microsort® sperm sorting (MicroSort, Australasia) between December 2014 and December 2015. In the control group (Group 2), the semen sample of 70 patients’ husband were processed by discontinuous density gradient and washed. All patients are with an advanced maternal age (36-43 years old) with a comparable mean age between both groups (Group 1: 38.4; Group 2: 38.7). Both groups then had ICSI and blastocyst culture. Seventy-five (75) blastocysts from Group 1 and 159 blastocysts from Groups 2 were biopsied, screened for chromosome copy number and analysed.

Results:
The euploidy rate for blastocysts derived from MicroSort® sperm (Group 1) was 48.0% while the euploidy rate for blastocysts derived from unsorted sperm (Group 2) was 38.0%. Although the euploidy rates between both groups are not statistically significant, there is a tendency for higher euploidy rate in blastocysts derived from sorted sperm.

Conclusion:
Our preliminary findings demonstrated a tendency towards a higher euploidy rate in patients with advanced maternal age who had MicroSort® sperm sorting compared to those with unsorted sperm. The use of flow cytometry in ART may possibly reduce the number of morphologically abnormal sperm and perhaps sperm aneuploidy for use in ICSI. Nevertheless, further studies with a larger sample size should be carried out to ascertain the relationship between MicroSort® sperm sorting and sperm morphology/aneuploidy.

Lee C.S.S., Low S.Y., Lim Y.X., Keith J. Vitrified-warmed good graded euploid blastocysts aged less than 120hours at the time of transfer yield superior clinical pregnancy and implantation rates.

Presented at 24th Congress of the Obstetrical and Gynaecological Society of Malaysia, 2 – 5th June 2016, Kuala Lumpur, Malaysia.

Introduction:
With the advantage of adopting the Cryotec Vitrification and Warming Method in July 2013, our centre gradually switched from fresh transfer to elective frozen embryo transfer for cases selected for preimplantation genetic screening. In this retrospective study, we assessed the clinical pregnancy and implantation rates for vitrified-warmed good graded euploid blastocysts aged less than 120 hours at the time of transfer from July 2013 to December 2015 in Alpha Fertility Centre.

Methods:
Thirty-five (35) patients had one or two good graded (Gardner’s Grading) euploid blastocysts vitrified and warmed using the Cryotec Vitrification and Warming Method. The mean age was 32.1 (range: 18-41). The mean number of blastocysts transferred was 1.2. All 44 vitrified-warmed euploid blastocysts survived with morphologically intact inner cell mass and trophectoderm cells (100% post-thaw survival rate). All 35 patients had embryo transfer. Vitrification and warming of blastocysts was done by embryologists who were certified by Cryotech, Japan. Age of blastocysts at the time of transfer was defined as the duration from insemination to embryo transfer. Clinical pregnancy and number of gestational sacs were determined using ultrasound.

Results:
Twenty-nine (29) out of 35 patients became clinically pregnant (82.9%). Of these, 2 patients miscarried. The implantation rate was 79.5%. Of the on-going or delivered pregnancies, 23 were singleton (79.3%), 6 were twins (20.7%) and there are no higher order pregnancies.

Conclusion:
This study shows that vitrified-warmed good graded euploid blastocysts aged less than 120 hours (from insemination to the time of embryo transfer) not only achieved 100% post-thaw survival rate with intact inner cell mass and trophectoderm cells, but also high clinical pregnancy and implantation rate. This study also support the practice of transferring only 1 euploid blastocyst when its development from insemination to embryo transfer is less than 120 hours.

Yap WY; Lim YX; Lee CSS. Euploidy rates of Cleavage Stage Embryos versus Blastocysts following PGS in an Asian Population. To be publish on Reproductive Biomedicine Online.

Presented at the 15th International Conference on Preimplantation Genetic Diagnosis, 8-11th May 2016, Bologna, Italy.

Introduction:
In this study, we compare the euploidy rates of cleavage stage embryos versus blastocysts obtained from Asian women following PGS in Alpha International Fertility Centre, Malaysia.

Materials and Methods:
Three hundred and four (304) patients with 2027 cleavage stage embryos (Group A) and three hundred and eighteen (318) patients with 1397 blastocysts (Group B) were biopsied from July 2011 to December 2015. These patients were divided into 6 age groups, ≤30, 31 – 35, 36 – 37, 38 – 39 and 40 – 41 respectively. Of the biopsied samples, 3223 had Microarray Comparative Genetic Hybridisation (MaCGH) and 201 had Next Generation Sequencing (NGS). For MaCGH, the biopsied samples were amplified, labelled and hybridised on microarray slides according to manufacturer’s specification (Illumina, USA). Microarray slides were analysed using BlueFuse Software (Illumina, USA), identifying normals, gains or losses of chromosomes. For NGS, biopsied samples were amplified and had DNA libraries constructed for sequencing using Ion TorrentTM PGM benchtop sequencer (Thermo Fisher, USA). Data from MaCGH and NGS were combined for analysis. Cases with known altered karyotype of translocation and inversion; and indeterminate biopsies were excluded from this study. Blastocysts from non-Asian women were also excluded from this study.

Results:
The euploidy rates of cleavage stage embryos for patients aged ≤30, 31 – 35, 36 – 37, 38 – 39 and 40 – 41 were 33.2% (221/666), 30.3% (197/651), 26.3% (85/323), 13.4% (29/216) and 10.5% (18/171) respectively. The euploidy rates of blastocysts for patients aged ≤30, 31 – 35, 36 – 37, 38 – 39 and 40 – 41 were 61.7% (351/569), 55.4% (262/473), 51.8% (57/110), 34.0% (50/147) and 34.7% (34/98) respectively. The euploidy rates of each age group between Group A and B were statistically significant (p=0.0001 each). The euploidy rates for patients aged >35 years were significantly lower compared to patients aged <35 years in both Group A (p=0.0001) & Group B (p=0.0001) respectively.

Conclusions:
This study shows that blastocysts have significantly higher euploidy rate compared to cleavage stage embryos in all age groups in an Asian population. These rates are in keeping with studies done by others on predominantly non-Asian population (Kort et. al, 2015).

Lim Y.X., Yap W.Y., Keith J., Lee CSS. Case report: Live twin birth following transfer of a 2PN and a 0PN embryo after PGD-MaCGH. To be publish on Reproductive Biomedicine Online.

Presented at the 15th International Conference on Preimplantation Genetic Diagnosis, 8-11th May 2016, Bologna, Italy.

Introduction:
Normal fertilisation is routinely assessed 18-20 hours after oocyte insemination. The appearance of 2 pronuclei (2PN) indicates that normal fertilisation has taken place and they enter syngamy 20-22 hours post-insemination leading to subsequent cleavage. 2PN can be detected as early as 5 hours post-insemination – their absence does not necessarily indicate fertilisation failure. A study found that 20% of 0PN oocytes went on to cleave and it was suggested that the PN in these embryos may have been missed due to syngamy before fertilisation check or that PN only formed after fertilisation check (Feenan & Herbert 2006). We earlier reported the birth of a healthy baby resulting from a 0PN embryo following 5-probe FISH analysis and embryo transfer. The baby was confirmed chromosomally normal through amniocentesis (Khoo et al, 2007). We had also reported that 0PN-derived embryos have a euploidy rate similar to embryos resulting from 2PN (Lee et al, 2013). This case report describes a successful birth of twins following the transfer of one euploid-2PN embryo and one euploid-0PN embryo after microarray comparative genomic hybridisation (MaCGH) analysis.

Material and Methods:
A 44 year old patient who previously had a trisomy 18 baby which died after birth underwent a donor-IVF cycle with preimplantation genetic screening in September 2014. Twenty-three donor’s (aged 22) oocytes were retrieved, of which 15 were inseminated. Fertilisation check was conducted at 20 hours post-insemination. From these, 7 were normally fertilised exhibiting 2PN, one 3PN and seven 0PNs. Cleaved embryos with at least average grade (Gardner’s grading, 1999) were biopsied and biopsied cells were analysed using MaCGH (Illumina, UK). Three OPN oocytes went on to cleave normally producing good graded embryos. There were 9 embryos for biopsy on Day 3: 6 from 2PN and 3 from 0PN. The patient had fresh embryo transfer on Day 4.

Results:
Euploidy was confirmed in 3 embryos while the remaining 6 embryos displayed various chromosomal anomalies. Of the 3 euploid embryos, 2 were derived from 2PN and 1 derived from 0PN. The patient being 44 years old only wanted 1 final attempt at pregnancy and insisted on having 2 embryos for transfer. Two good graded euploid embryos (one 2PN and one 0PN) were transferred. The reason the euploid-0PN embryo was chosen for transfer rather than the other euploid-2PN embryo was because the 0PN embryo was of better quality. Both embryos implanted. An uneventful delivery at 36 weeks by caesarean section resulted in the birth of 2 normal healthy babies, weighing 2.8kg and 2.5kg respectively.

Conclusions:
This case report indicates that 0PN zygotes that go on to cleave may have been normally fertilised and these embryos can be chromosomally normal and result in live birth. These embryos should not be discarded but instead be treated as if they were 2PN embryos and be considered for transfer.

Low S.Y., Lim Y.X, Keith J., Lee C.S.S. Age of vitrified-warmed euploid blastocysts at the time of transfer vs their clinical outcomes. To be publish on Reproductive Biomedicine Online.

Presented at the 15th International Conference on Preimplantation Genetic Diagnosis, 8-11th May 2016, Bologna, Italy.

Introduction:
As a result of advances in vitrification and warming technology for blastocysts, vitrified-warmed blastocysts yield good post-thaw survival rates, and achieve high clinical pregnancy and implantation rates. Since initiating the use of the Cryotec Vitrification and Warming Method in July 2013, our centre has gradually switched from fresh transfer to elective frozen embryo transfer for IVF cases undergoing preimplantation genetic screening cases. In this retrospective study, we assessed the correlation between age of vitrified-warmed euploid blastocysts at the time of transfer and their clinical pregnancy and implantation rates.

Materials and Methods:
One hundred and thirty-three (133) women had 173 vitrified-warmed euploid blastocysts transferred from July 2013 to December 2015 in Alpha Fertility Centre. These 133 cases were divided into 4 blastocyst age groups at the time of embryo transfer, age <120, 120-130, 131-140 and >140 hours. The age of blastocysts at the time of transfer refers to the duration from insemination to embryo transfer. The number of cycles in each group was 42, 14, 8 and 69 respectively. Three to 5 trophectoderm cells were biopsied from each blastocyst on Day 5 or 6. All biopsied blastocysts were of at least average grade (Gardner et. al., 1999). Samples and reference DNAs were amplified, labelled and hybridized according to manufacturer’s (Illumina / Life Technologies) specifications. Biopsied blastocysts were frozen shortly after biopsy using Cryotec Method (Cryotech, Japan). When PGS results showed at least 1 euploid blastocysts available, the patient will be prepared for FET.

Results:
All 173 euploid blastocysts survived with morphologically intact inner cell mass and trophectoderm cells (100% post-thaw survival rate), enabling transfer in all 133 cases.

There was statistical significance in clinical pregnancy rates between blastocyst age <120 and 120-130 hours post-insemination. There were statistical significance in implantation rates for blastocyst age <120 compared to group 120-130, 131-140 and >140.

Conclusions:
Blastocysts aged less than 120 hours (from insemination to the time of embryo transfer procedure) demonstrated superior clinical pregnancy and implantation rates. When more than one euploid blastocysts are available, it is recommended to select blastocyst with an age of <120 hours.

Lee C.S.S., Low S.Y., Lim Y.X., Keith J. Maternal age does not affect clinical outcomes of vitrified-warmed euploid blastocysts: an Asian Study. To be publish on Reproductive Biomedicine Online.

Presented at the 15th International Conference on Preimplantation Genetic Diagnosis, 8-11th May 2016, Bologna, Italy.

Introduction:
Harton et. al. 2013 reported no statistical difference in implantation rates for women from different age groups (up to 42 years old) when euploid blastocysts were transferred after preimplantation genetic screening. This retrospective study analyses the clinical pregnancy and implantation rates for vitrified-warmed euploid blastocysts in an Asian population.

Materials and Methods:
This is a retrospective analysis of outcomes for 154 Asian women undergoing preimplantation genetic screening, who had their euploid blastocysts vitrified and warmed using the Cryotec Method in Alpha Fertility Centre from July 2013 to December 2015. Non-Asian women were excluded in this study. The patients were divided into 5 age groups, age <35, 35-37, 38-39 40-41 and 41-46. The number of cycles in each age group was 95, 26, 19, 12 and 2 respectively. Three to 5 trophectoderm cells were biopsied from each blastocyst on Day 5 or 6 and proceeded with PGS by microarray CGH or next generation sequencing (NGS). All biopsied blastocysts were of at least average grade (Gardner et. al., 1999). The biopsied blastocysts were vitrified shortly after biopsy using the Cryotec Method.

Results:
All 206 euploid blastocysts were warmed and survived with morphologically intact inner cell mass and trophectoderm cells (100% post-thaw survival rate), enabling transfer in all 154 cases.

There was no statistical significance in clinical pregnancy rates, implantation rates or on-going pregnancy rates between all age groups. Of the 96 on-going or delivered pregnancies, 77 were singleton (80.2%) and 19 were twins (19.8%). There are no higher order pregnancies.

Conclusions:
Blastocysts aged less than 120 hours (from insemination to the time of embryo transfer procedure) demonstrated superior clinical pregnancy and implantation rates. When more than one euploid blastocysts are available, it is recommended to select blastocyst with an age of <120 hours.

Siew SC; Lim YX; Low SY; Keith J; Lui WX; Lim MW; Yap WY; Lee, CSS. Clinical Outcome of Assisted Reproductive Technology Treatments In Relation to Oocyte Retrieval Day. Reproductive Biomedicine Online, Vol.32, Supplement 1, Page S21.

Presented at 11th Alpha Biennial Congress, 5 – 8th May 2016, Copenhagen, Denmark.

Introduction:
A retrospective study comparing the outcomes of IVF cycles with different oocyte retrieval (OR) days.

Materials and Methods:
One thousand one hundred and thirty four (1134) patients aged less than 38 years old (mean age: 31.8, range 18-37) had OR in Alpha International Fertility Centre (AFC), Malaysia from July 2011 to December 2015. Clinical outcomes were analysed retrospectively according to day of OR as follows: 8 (n=1), 9 (n=10), 10 (n=54), 11 (n=102), 12 (n=287), 13 (n=360), 14 (n=173), 15 (n=104), 16 (n=30), 17 (n=9), 18 (n=3), and 21 (n=1); where Day 1 is the start of gonadotropin administration. All cases were intended for fresh embryo transfer at either cleavage stage or blastocyst.

Results:
There was no statistical significance in number of embryo(s) transferred in all groups. Of the 1134 OR’s, 966 cases (85%) had embryo transfer (ET). Cases that failed to reach ET were due to no chromosomally suitable embryos (n=70), poor embryo quality (n=50), failed fertilisation (n=37) and others (n=11).

Clinical pregnancy rates (CPR) per OR were 0.0%, 60.0%, 57.4%, 54.9%, 48.8%, 48.3%, 53.2%, 51.0%, 36.7%, 33.3%, 0.0% and 100.0% for Day 8 to Day 21 respectively (P>0.05). We observed a tendency towards lower CPR/OR for patients who had their OR on Day 16 and Day 17. CPR/ET were 0.0%, 66.7%, 70.5%, 61.5%, 56.2%, 57.0%, 60.9%, 61.6%, 47.8%, 60.0%, 0.0% and 100.0% for Day 8 to Day 21 respectively (P>0.05). Implantation rates (IR) according to day of OR were 0.0%, 44.4%, 49.4%, 41.6%, 41.4%, 39.5%, 46.1%, 39.7%, 31.8%, 33.3%, 0.0% and 100.0% for Day 8 to Day 21 respectively, with no statistical significance in all groups (P>0.05). Again, we observed a tendency towards lower IR for patients who had their OR on Day 16 and Day 17. Outcomes of Day 8, Day 18 and Day 21 may not be reflective due to small numbers of data.

Conclusions:
This study suggests that prolonged ovarian stimulation beyond Day 15 tends to have lower pregnancy rates and implantation rates.

WY Yap, YX Lim, CSS Lee. Relative Amount of Mitochondria DNA (mtDNA) in Euploid and Aneuploid Blastocysts. Reproductive Biomedicine Online, Vol.32, Supplement 1, Page S22.

Presented at 11th Alpha Biennial Congress, 5 – 8th May 2016, Copenhagen, Denmark.

Introduction:
Mitochondria play a vital role in preimplantation embryo development where initial stages such as spindle formation, chromatid separation and cell division are supported by mitochondria derived from the oocyte. Elevated levels of mtDNA in human blastocysts were shown to be associated with aneuploidy, advanced maternal age and implantation failure (Fragouli et al, 2015). This is a retrospective study to analyse the relative amount of mtDNA in euploid and aneuploid blastocysts using a single aneuploidy screening platform. This study was performed at Alpha Fertility Centre (AFC), Malaysia from September to December 2015.

Materials and Methods:
Forty-five patients (age range 23 – 43) underwent IVF cycles and their embryos were further cultured to blastocyst stage. Blastocysts with at least fair grade (using Gardner blastocyst grading system) were biopsied and screened for chromosome copy number using next generation sequencing (NGS) according to an optimised manufacturer’s protocol in AFC (Thermo Fisher, USA). The relative amounts of mtDNA of all euploid (Group A) and aneuploid (Group B) blastocysts were simultaneously assessed using NGS being determined as number of reads for mtDNA / number of reads for genome DNA ratio. The mean age of patients from Group A and B were 33.7 and 35.6 respectively (p>0.05).

Results:
One hundred and sixteen blastocysts were assessed, of which 60 were euploid (Group A) and 56 were aneuploid (Group B). The relative amount of mtDNA for Group A ranged from 2.0 x 10⁻⁴ – 1.4 x 10⁻³ and the relative amount of mtDNA for Group B ranged from 1.0 x 10⁻⁴ – 8.9 x 10⁻³. The mean relative amounts of mtDNA for Group A and Group B were 4.0 x 10⁻⁴ – 1.0 x 10⁻³ respectively (p=0.0061).

Conclusions:
Our preliminary results demonstrated a significantly higher relative amount of mtDNA in aneuploid blastocysts. This supports the findings which indicate increased mtDNA may be correlated to aneuploidy genesis. Since aneuploidy is largely responsible for implantation failure and miscarriage, relative mtDNA might be used as a biomarker to assess implantation potential. However, further validation is required to confirm this proposal.

Low SY; Lee, CSS; Lim YX. Euploid blastocysts show a trend of higher implantation and clinical pregnancy rates compared to untested blastocysts in FET cycles. Reproductive Biomedicine Online, Vol.32, Supplement 1, Page S21.

Presented at 11th Alpha Biennial Congress, 5 – 8th May 2016, Copenhagen, Denmark.

Introduction:
This is a retrospective study comparing the implantation rates (IR) and clinical pregnancy rates (CPR) in IVF patients who had PGS with those who did not have PGS (non-PGS) in frozen embryo transfer (FET) cycles.

Materials and Methods:
154 FET cycles had frozen euploid blastocyst(s) transferred (PGS group) and 249 FET cycles with unscreened blastocyst(s) (non-PGS) transferred at Alpha International Fertility Centre (AFC), Malaysia from July 2013 to December 2015. All 639 blastocysts were thawed with a 100% survival rate.

Results:
The mean age was comparable in all the age groups. The mean number of embryos transferred were 1.4 and 1.7 for PGS group and non-PGS group respectively (p<0.0001). There was no statistical significance in clinical pregnancy rates between the 2 groups for all ages. However there is a trend of higher CPR in the PGS group. The IR for PGS group was significantly higher than the non-PGS group for age groups 38-39 (58.3% vs 25.0%; p<0.009) and 40-41 (61.5% vs 24.3%; p=0.0210). IR for age groups <35, 35-37 and >41 were not statistically different but there is a trend of higher IR in PGS group. There was no significant difference in the IR between all age groups in the PGS group.

Conclusions:
This study showed euploid blastocysts have a trend of higher CPR compared to untested blastocysts in FET cycles despite higher mean number of blastocysts transferred in non-PGS (1.7 vs 1.4). The IR for the PGS group was significantly higher in age groups 38-39 and 40-41.

Colin SS, Lee, WX Lui, YX Lim, WY Yap, SY Low, MW Lim, WY Leong. Mixed Day 5 and Day 6 Frozen Blastocysts In Frozen Embryo Transfer (FET).

Presented at the 6th Congress of the Asia Pacific Initiative on Reproduction, 8 – 10th April 2016, Jakarta, Indonesia.

Introduction:
In standard clinical practice where 2 blastocysts are opted for, either 2 frozen Day 5 or 2 frozen Day 6 blastocysts are usually transferred. However, there may be situations of double embryo transfer where one does not have 2 of either Day 5 or Day 6 blastocysts available. The aim of this study is to investigate the efficacy of transferring together a frozen Day 5 and a frozen Day 6 blastocyst.

Materials and Methods:
From 2014 till August 2015, 14 patients had both Day 5 and Day 6 vitrified-blastocysts thawed and transferred. All blastocysts were vitrified and warmed using Cryotec Method (Cryotech, Japan). All 14 patients (mean age: 32.8, ranged 24 to 41) had gonadotropin-releasing hormone (Leucrin depot) and estradiol (Progynova) followed by progesterone (Cyclogest) supplementation. Despite being counselled for single embryo transfer, these 14 patients opted for a double embryo transfer. Each patient had a Day 5 and a Day 6 blastocyst transferred on the 7th day of progesterone administration.

Results:
All 14 patients each had one Day 5 and one Day 6 frozen blastocyst thawed. All survived with morphologically intact inner cell mass and trophectoderm cells. Ten patients were clinically pregnant (CPR: 71.4%) with 13 intra-uterine gestational sacs (IUGS) (implantation rate: 46.4%). From these 10 patients, 2 patients delivered twins, 2 patients delivered singletons and the rest are on-going pregnancies.

Conclusion:
This study shows Day 5 and Day 6 blastocysts transferred together yields good implantation and pregnancy rates.

Lee CSS.; Tee ZQ.; Lim YX. Microsort® sperm sorting may result in higher implantation rate.

Presented at the 6th Congress of the Asia Pacific Initiative on Reproduction, 8 – 10th April 2016, Jakarta, Indonesia.

Introduction:
Since December 2014, Alpha Fertility Centre started using MicroSort® (USA), a scientifically proven preconception technology, to increase the likelihood of having a baby of the specified gender. This study describes our initial experience using micro-sorted sperm in ICSI and the clinical outcome following Micro-Array Comparative Genomic Hybridisation (MaCGH) with subsequent frozen embryo transfer (FET).

Material and Methods:
During this period, nine men had MicroSort® sperm sorting. On the day of oocyte pick-up, freshly collected semen samples were processed to recover motile sperm. Washed sperm cells were then subjected to staining, and sorted using a flow cytometer based on fluorescence intensity of X- and Y-bearing sperm. Sorted sperm were then injected into the oocytes by ICSI method. All blastocysts were frozen (Cryotec, Japan) after biopsy on Day 5 and/or Day 6 for elective FET. The biopsied cells were analysed using MaCGH.

Results:
The maternal mean age was 35.4 (ranged 30-40). Twelve euploid blastocysts were thawed and all survived intact (post-thaw survival rate = 100%). The mean number of blastocysts transferred was 1.3. Of the 9 patients, 8 were clinically pregnant (Clinical pregnancy rate = 88.9%) with a total of 11 intrauterine gestational sacs (Implantation rate = 91.7%). Of the on-going pregnancies, 5 were singleton, 3 were twins and there is no higher order pregnancies.

Conclusion:
This preliminary study which shows a high clinical pregnancy rate and implantation rate suggests that Microsort® sorting may perhaps select sperm that result in embryos capable of higher implantation potential.

Lim YX.; Lee CSS.; Tee ZQ.; Lim MW. MicroSort® Sperm Sorting: An Asian Study.

Presented at the 6th Congress of the Asia Pacific Initiative on Reproduction, 8 – 10th April 2016, Jakarta, Indonesia.

Introduction:
Human semen samples usually contain equal amounts of sperm carrying the Y- and X-chromosome. Flow cytometric sorting (MicroSort®, USA) can be used to separate sperm based on the chromosome they are carrying. The difference in DNA content of the X- and Y-bearing sperm is made evident by the intensity of the fluorescent signal emitted by the stained sperm. This enables sorting and collection of samples enriched in either X- or Y-bearing sperm for use in ART. This is a collaborative retrospective study by MicroSort Australasia and Alpha Fertility Centre to determine the effectiveness of flow cytometric sorting of human Y-bearing sperm.

Material and Methods:
This study was divided into 2 groups – Group 1: blastocysts derived from sperm sorted for Y-chromosome; and Group 2: blastocysts derived from non-sorted sperm from December 2014 to August 2015. Seventy-nine blastocysts from Group 1 and 314 blastocysts from Group 2 were biopsied on Day 5 and/or Day 6. The biopsied cells were analysed (Illumina, UK) for comprehensive chromosome screening. The sex of the blastocysts were also noted.

Results:
In Group 1, 59 out of 79 blastocysts (74.6%) were male displaying Y chromosome. In Group 2, 164 out of 314 blastocysts (52.2%) were male displaying Y chromosome. The number of male blastocysts were significantly higher (p=0.0003) in Group 1 compared to Group 2.

Conclusion:
Flow cytometric sorting of human sperm shifted the X:Y ratio of 48:52 to 25:75 and this result supports the effectiveness of the use of MicroSort® in ART for sperm sorting.

Lee CSS; Lim YX. Blastocysts derived from 0PN and 1PN may be considered for transfer.

Oral presentation at the 6th Congress of the Asia Pacific Initiative on Reproduction, 8 – 10th April 2016, Jakarta, Indonesia.

Introduction:
We had previously reported on the birth of a healthy baby following frozen embryo transfer of a unipronuclear (1PN) embryo after microarray comparative genomic hybridisation (MaCGH) (Lim et al, 2015). We had subsequently demonstrated that apronuclear (0PN) and 1PN zygotes that cleaved have euploidy rates similar to embryos resulting from 2PN (Lee et al, 2013). The aim of this retrospective study was to assess the euploidy rates of blastocysts derived from 0PN and 1PN IVF-zygotes at Alpha Fertility Centre from May-2013 to August-2015.

Materials and Methods:
Fifty-patients who underwent IVF with preimplantation genetic screening had 941 zygotes of which 624 were 2PNs (66.3%), 179 were 0PNs (19.0%), 59 were 1PNs (6.3%) and 79 were ≥3PNs (8.4%). Fertilisation assessment was done 18-20 hours post-insemination. The blastulation rates for 2PN, 0PN and 1PN were 77.7%, 23.5% and 50.8% respectively. There were 330 (61.6% of 2PN’s; 38.1% of 0PN’s; 50% of 1PN’s) morphologically normal blastocysts biopsied. The biopsied cells were analysed using MaCGH.

Results:
The mean age was 31.9 (range 21-43). Euploidy rates of blastocysts derived from 2PN, 0PN and 1PN were 53.8%, 62.5% and 46.7% respectively. No significant differences (p>0.05) were found between the 3 groups.

Conclusion:
0PN and 1PN IVF-zygotes that are capable of developing past early embryonic development and form morphologically normal blastocyst may had been normally fertilised and these blastocysts would be expected to show similar euploidy rates to those blastocysts arising from 2PN. These blastocysts may be considered for transfer when no suitable 2PN-blastocysts are available.

Lee CSS, Low SY. Outcome of vitrified-thawed oocytes at Alpha International Fertility Centre (AFC).

Oral presentation at the 24th Asian & Oceanic Congress of Obstetrics & Gynaecology, 3-6 June 2015, Kuching, Sarawak, Malaysia.

Introduction:
Until recently, cryopreservation and thawing of oocytes yielded unsatisfactory post-thaw survival rates. In 2013, Dr. Masashige Kuwayama introduced Cryotec Method of vitrification and warming of oocytes, claiming a post-thaw survival rate of 100%. AFC adopted the Cryotec Method from August 2013. We hereby present the outcome of vitrified-thawed oocytes using this method in AFC.

Method:
Following cryopreservation using the Cryotec Method, 5 patients had their oocytes thawed. The mean age of patients was 27.2 (ranged 21 -37). All oocytes had Intra-Cytoplasmic Sperm Injection (ICSI) and resultant embryos were cultured to day 5 or 6. All 5 cases had blastocyst transfer. The mean number of blastocysts transferred was 1.8. Clinical pregnancy and number of gestational sacs were determined using ultrasound.

Results:
A total of 65 oocytes were thawed. Sixty three oocytes survived (Post-thaw Survival Rate: 96.9%) and had ICSI. Fourty six (46) oocytes were normally fertilised (2PN) while 2 were 3PN (Fertilisation rate: 76.2%). Of these, 26 embryos formed blastocysts (Blastulation rate: 56.5%). Nine blastocysts were transferred into these 5 patients while 4 were frozen (Blastocyst Utilisation rate: 50%). Four patients were clinically pregnant (4/5 pregnant) with 6 blastocysts implanted (Implantation rate: 66.6%). Of the clinical pregnancies, 2 were singleton and 2 were twins.

Conclusion:
Our experience with cryopreservation of oocytes using the Cryotec Method showed a near 100% post-thaw survival rate; with fertilisation rate, blastulation rate, implantation rate and clinical pregnancy rate comparable to cycles using fresh oocytes.

Lee CSS, Low SY. Review of Fresh Blastocyst Culture and Transfer Program at Alpha International Fertility Centre (AFC).

Oral presentation at the 24th Asian & Oceanic Congress of Obstetrics & Gynaecology, 3-6 June 2015, Kuching, Sarawak, Malaysia.

Introduction:
It is well known that blastocysts are more robust and it is also more physiological to transfer embryos on Day 5. It is also known that blastocysts have higher euploidy rates and thus higher implantation rate (IR) and clinical pregnancy rate (CPR) compared to cleavage stage embryos. However, to enable blastocyst culture and transfer as a routine, the IVF laboratory must first achieve optimum culture conditions to enable high blastulation rates. From the commencement of AFC, we took great pains to ensure such conditions are achieved. Once we have achieved sustained blastulation rates of about 70%, we decided from May 2013 to encourage most of our patients to go for blastocyst culture and transfer. We embarked on blastocyst culture and transfer as a matter of preference since then. In this study, the main outcome measured were blastulation rate, blastocyst utilization rate, IR, CPR, multiple pregnancy rate, and number of babies born or expected to be born per embryo transfer (NBB-EtbB/ET).

Methods:
This is a retrospective analysis of patients who had fresh blastocyst culture and transfer (but without PGD) since we started AFC (July 2011) till now. The percentage of patients who had fresh blastocyst culture in year 2011, 2012, 2013 and 2014 were 7.5% (8/107), 4.9% (8/162), 46.9% (107/228) and 70.5% (203/288) respectively. The mean age of patients for blastocyst culture and transfer were 32.4, 29.4, 30.7, and 31.4 (ranged 20-37). The mean number of blastocysts transferred were 2.7, 2.1, 1.9 and 1.8 respectively. Clinical pregnancy and number of gestational-sacs were determined by ultrasound.

Results:
Over this period of time, the blastulation rate was fair consistent at about 71% and the blastocyst utilisation rate was 64.6%. The IR ranged from 47.4% to 73.3% over the period of study with no significant differences between the years. The CPR were 85.7%, 100%, 65.9% and 63.6% and were not statistically significant. Singleton pregnancy rates were 50%, 42.9%, 44.8% and 63.6% respectively. Twin pregnancy rates showed a steady decline from 50% in year 2011 to 36.4% in year 2014. There were no higher-order multiple pregnancies over the years. The NBB-EtbB/ET was 91.9%.

Conclusion:
With consistent blastulation rates over the years (around 71%), we confidently increased the percentage of patients going for blastocyst culture while maintaining the CPR, IR and NBB-EtbB/ET despite progressively reducing the mean number of blastocysts transferred year-on-year. Twin pregnancy rates were also reduced progressively. There were no higher-order multiple pregnancies.

Lee CSS, Lim YX. Clinical Outcome following Transfer of Frozen Euploid Blastocysts after Micro-Array Comparative Genomic Hybridisation (MaCGH) at Alpha Fertility Centre (AFC).

Oral presentation at the 24th Asian & Oceanic Congress of Obstetrics & Gynaecology, 3-6 June 2015, Kuching, Sarawak, Malaysia.

Introduction:
Perhaps the main cause of implantation failure is aneuploidy which increases with age. MaCGH is thus expected to improve clinical outcome by selecting only euploid blastocysts to be transferred. Lower pregnancy rates in older women could therefore be avoided. Good clinical outcome was also observed following embryos vitrified using Cryotec Method which achieves 100% post-thaw survival rate. This study describes our experience of combining these 2 recent IVF technologies in elective frozen blastocyst transfer for patients less than 38 years old. It also looks at the clinical outcome at different age groups following transfer of euploid blastocysts.

Method:
This is a retrospective analysis of outcome from 32 frozen euploid blastocyst transfers in AFC since the introduction of Cryotec Method (November 2013 to November 2014). The cases were divided into 3 groups, age ≤30 (Group A), 31-34 (Group B) and 35-37 (Group C). All blastocysts were frozen after biopsy on Day 5 and/or Day 6 for elective FET. The biopsied cells were analysed using MaCGH. Clinical pregnancy and number of gestational sacs were determined using ultrasound.

Results:
The mean age was comparable between Group A, B and C (26.0, 32.5 and 36.0 respectively). Fourty-six frozen euploid blastocysts were thawed and all survived intact (Post-thaw survival rate = 100%). All 46 thawed blastocysts were transferred. The mean number of blastocysts transferred was 1.6 in Group A and 1.3 in Group B and C. Clinical pregnancy rates (CPR), implantation rates (IR) and live birth/on-going pregnancy rates for Group A, B and C were 66.7% vs 50.0% vs 85.7%; 54.2% vs 38.5% vs 66.7%; and 60.0% vs 50.0% vs 71.4% respectively. There was no statistical significance in CPR, IR and live birth/on-going pregnancy rates between all groups. For all the 32 patients as a whole, the CPR, IR and live birth/on-going pregnancy rates were 65.6%, 52.2% and 59.4% respectively. Of the on-going or delivered pregnancies, 16 were singleton, 3 sets were twins and there is no higher order pregnancies.

Conclusion:
This study shows that elective FET of euploid blastocysts leads to good clinical pregnancy, implantation and live birth/on-going pregnancy rates. The risk of multiple pregnancies are also reduced. By transferring only euploid blastocysts, the clinical pregnancy and implantation rates were similar and did not show a decline with advancing age.

Lee CSS, Lim YX, Lui WX. Clinical Outcome of Fresh versus Frozen Day 6 blastocyst transfer.

Oral presentation at the 24th Asian & Oceanic Congress of Obstetrics & Gynaecology, 3-6 June 2015, Kuching, Sarawak, Malaysia.

Introduction:
Impaired clinical outcome from fresh Day 6 blastocyst transfers due to poor endometrial receptivity after ovarian hyperstimulation were extensively reported recently. To our knowledge, a few reported studies demonstrated that slow-growing Day 6 blastocysts using freeze-thaw technique contributes to better clinical outcome compared to fresh Day 6 blastocysts. The aim of this retrospective study is to investigate the efficacy of fresh and frozen Day 6 blastocyst transfers at Alpha International Fertility Centre (AFC), Malaysia in patients aged 40 and below.

Methods:
All 30 patients who had Day 6 blastocysts transferred from the commencement of AFC (July 2011) till now were analysed. Twelve (12) had fresh Day 6 blastocyst transfer (Group A) and 18 had frozen Day 6 blastocyst transfer (Group B). These were all relatively slow-growing blastocysts and were not suitable for transfer or freezing on Day 5. Patients who had pre-implantation genetic screening were excluded in this study. The mean age of patients in each group was similar: Group A was 31.4 (ranged 21-40) and Group B was 32.3 (ranged 21-39). A total of 22 blastocysts were transferred in Group A. In Group B, 29 blastocysts were thawed. All 29 survived with morphologically intact inner cell mass and trophectoderm cells (100% post-thaw survival rate). All 29 blastocysts thawed were transferred. The mean number of blastocysts transferred was 1.8 in Group A and 1.6 in Group B. Clinical pregnancy and number of gestational sacs were determined by ultrasound.

Results:
Clinical pregnancy rates per embryo transfer (ET) for Group A and Group B were 58.3% and 61.1% respectively. The implantation rates for Group A and Group B were 36.4% and 55.2% respectively. Number of babies born or expected to be born per ET (NBB-EtbB/ET) for Group A was 50.0% and for Group B was 72.2%. There were no significant differences found in clinical pregnancy rate, implantation rate and NBB-EtbB/ET between both groups, but shows a tendency for higher rates in the frozen group.

Conclusion:
Our preliminary findings demonstrated that transferring frozen Day 6 blastocysts yielded a tendency towards higher clinical pregnancy rate, implantation rate and number of babies born or expected to be born per ET compared to transferring Day 6 blastocysts in fresh cycles.

Low SY, Lee CSS, Lim MW, Yap WY, Lui WX, Lim YX. Euploidy rates of day 3 apronuclear (0PN) and unipronuclear (1PN) cleaved embryo arising from IVF. Reproductive Biomedicine Online 2015; 31(2):294.

Presented at the 14th International Conference on Preimplantation Genetic Diagnosis, 11-13 May 2015, Chicago, Illinois, USA.

Introduction:
We had previously reported on: i) Euploidy Rates of Day-3 apronuclear (0PN) and unipronuclear (1PN) embryos arising from Intra-Cytoplasmic Sperm Injection (ICSI) (CSS Lee et. al., PGDIS 2013) and ii) Blastomere biopsy results in IVF is not affected by sperm DNA (Yap WY et. al., 2014). With these studies in mind, we proceeded to perform IVF (whenever semen parameters are met) for cases where Micro-array Comparative Hybridisation (MaCGH) was planned for. All resulting Day-3 embryos even those arising from 0PN and 1PN had MaCGH when they are of at least average quality. We now report the euploidy rates of these 0PN and 1PN cleaved embryos (Alpha Fertility Centre (AFC): July 2011 to December 2014).

Material & Methods:
This is a retrospective study to assess the chromosome copy number of cleaved embryos arising from 0PN & 1PN zygotes following IVF. Fertilisation checks were done between 18 to 20 hours after insemination. There were 25 patients (mean age: 33.2 ranged 20-40) had cleaved embryos arising from 0PN & 1PN zygotes. Between them, they had a total of 28 0PN, 81 1PN, 196 2PN and 14 3PN oocytes/zygotes during fertilisation check. Out of these oocytes/zygotes, 7 0PN (25%), 9 1PN (11.1%) and 163 2PN (83.1%) zygotes cleaved and were biopsied on the morning of Day-3. All biopsied embryos were of at least average grade. MaCGH was performed according to the manufacturer’s specifications (24sureV3, BlueGnome, United Kingdom) and analysed using Bluefuse Multi Version 3.1 Software (BlueGnome, United Kingdom). Cases with known altered karyotype of inversion and translocation; and indeterminate biopsies were excluded from this study.

Results:
Euploidy rates of Day-3 0PN, 1PN and 2PN cleaved embryos arising from IVF were 28.6%, 77.8% and 31.3% respectively. Aneuploidy rates of 0PN, 1PN and 2PN cleaved embryos arising from IVF were 71.4%, 22.2% and 68.7% respectively. All 3 groups showed no significant differences in euploidy and aneuploidy rates. We have previously reported a live birth following frozen embryo transfer of a 1PN embryo subjected to MaCGH analysis (submitted as another abstract for PGDIS 2015). Another patient whom we transferred both a 0PN and a 2PN embryo (double embryo transfer) is now in her third trimester with a twin pregnancy.

Conclusions:
0PN and 1PN oocytes/zygotes are much less likely to reach cleavage stage of at least average quality. However if they do reach that stage, then they have similar euploidy rate compared to 2PN cleaved embryos. This study shows that there are no significant differences in euploidy rates in embryos arising from Day-3 0PN, 1PN and 2PN cleaved embryos resulting from IVF. Therefore, 0PN and 1PN cleaved embryos of at least average grade should be screened via PGD rather than discarded. In IVF centers with no PGD facility and for those embryos without PGD analysis, 0PN and 1PN cleaved embryos should be considered for transfer but any resultant pregnancy subjected to full antenatal surveillance.

Lim YX, Lee CSS, Yap WY, Low SY, Lui WX, Lim MW. Live birth following frozen embryo transfer of a 1PN embryo after PGD-MaCGH analysis. Reproductive Biomedicine Online 2015; 31(2):293.

Presented at the 14th International Conference on Preimplantation Genetic Diagnosis, 11-13 May 2015, Chicago, Illinois, USA.

Introduction:
1PN oocytes are often observed during fertilisation check at 18-20 hours post-insemination. This may be due to oocyte parthenogenetic activation, possible fusion of male and female pronuclei or asynchronous pronuclear formation. The development of parthenogenetic oocytes usually become impaired after activation, thus leading to early embryo arrest. A small proportion however, exhibit further cleavage and at this stage are indistinguishable from 2PN embryos. Chromosomal studies revealed that 45-48% of diploid 1PN embryos are in fact fertilised displaying Y signals and were not due to parthenogenetic activation (Staessen and Steirteghem, 1997). In our previous study, we had demonstrated that 1PN embryos that develop to the cleavage stage have euploidy rates similar to embryos resulting from 2PN (Lee et al, 2013). This case report describes a successful birth following frozen embryo transfer (FET) of a 1PN embryo after microarray comparative genomic hybridisation (MaCGH) analysis.

Method:
The patient, aged 37 years, who presented with polycystic ovarian syndrome underwent IVF treatment with preimplantation genetic screening at Alpha Fertility Centre in November 2013. Following oocyte retrieval, her oocytes were inseminated and were cultured in G-1TM PLUS medium and subsequently in G-2TM PLUS medium (Vitrolife, Sweden). Fertilisation check was performed 18 hours 10 minutes post insemination. All embryos arising from 2PN or 1PN zygotes that were at least average grade were subjected to biopsy on Day 3. Biopsied cells were analysed using MaCGH (Illumina, UK). Euploid embryo was cultured to Day 5 and vitrified using Cryotec Method (Cryotech, Japan) for elective FET. No fresh transfer was carried out in view of the risk of ovarian hyperstimulation syndrome.

Results:
Eighteen oocytes were retrieved, of which, 17 oocytes were inseminated. Thirteen out of 17 (13/17) were normally fertilised (2PN), one was 1PN and three were 3PNs. There were 5 embryos (4 from 2PNs and 1 from 1PN) suitable for biopsy on Day 3. Euploidy was confirmed in the embryo resulting from 1PN zygote, while all four 2PN embryos were aneuploid. The euploid-1PN embryo displayed Y hybridisation signal, thus affirming that fertilisation had occurred. The euploid-1PN embryo survived post-thawed, was morphologically intact, and transferred. An uneventful delivery at 39 weeks resulted in birth of a normal healthy baby boy.

Conclusion:
This report shows that the transfer of a frozen-thawed euploid embryo derived from 1PN “embryo” resulted in the birth of a normal healthy baby. It appears that the initial fertilisation observation of 1PN at the normal time of fertilisation assessment cannot be the absolute indicator of development incompetence. MaCGH can be used to ascertain the chromosomal constitution of 1PN “embryos” to confirm whether fertilisation had occurred, particularly those that display Y signals. Such embryos can be selected for transfer when no 2PN euploid embryos are available. To the best of our knowledge, this is the first case report of birth following transfer of 1PN embryo after MaCGH analysis.

Lee CSS, Yap WY, Lim MW, Lim YX, Low SY, Lui WX. Euploidy rates of apronuclear (0PN) and unipronuclear (1PN) blastocysts fertilised using in vitro fertilization (IVF). Reproductive Biomedicine Online 2015; 31(2):292.

Presented at the 14th International Conference on Preimplantation Genetic Diagnosis, 11-13 May 2015, Chicago, Illinois, USA.

Introduction:
We had previously reported on the birth of a healthy baby resulting from a 0PN embryo transfer (fertilised using IVF) screened via 5-probe FISH, and was confirmed chromosomally normal using amniocentesis (Khoo, G. et al, PGDIS 2007). We have subsequently demonstrated that fertilisation via IVF does not predispose to sperm DNA contamination for cleavage stage embryos subjected to Microarray Comparative Genomic Hybridisation (MaCGH) with SurePlex DNA amplification kit (Yap, WY. et al, 2014). With this understanding, we decided since November 2013 to perform Preimplantation Genetic Diagnosis (PGD) using MaCGH on 0PN and 1PN blastocysts resulting from IVF. This retrospective study reports the euploidy and aneuploidy rates of IVF-fertilised 0PN and 1PN blastocysts. To the best of our knowledge, this is the first such report on the euploidy and aneuploidy rates of IVF-fertilised 0PN and 1PN blastocysts using MaCGH.

Material & Methods:
For the period November 2013 till December 2014, all IVF cases had fertilisation checks done 18 – 20 hours post insemination. There were 811 zygotes of which 161 was 0PN (19.9%), 44 was 1PN (5.4%), 535 was 2PN (66.0%) and 71 was 3PN (8.7%). All zygotes were cultured to blastocysts. There were 273 blastocysts formed with at least a BG3BB grade (Gardner’s grading) from 9.9% of 0PN’s (16/161), 38.6% of 1PN’s (17/44) and 44.9% of 2PN’s (240/535). No blastocyst with at least a BG3BB grade were formed from 3PN zygotes. These blastocysts had trophectoderm cells biopsied and screened using MaCGH. The biopsied cells were amplified using SurePlex DNA Amplification System (Bluegnome), labelled and hybridised together with a set of reference DNA according to the manufacturer’s protocols (BlueGnome). The microarray slides were analysed using BlueFuse Multi Software Version 3.1 (BlueGnome) to determine the chromosomal status of the blastocysts. Cases with known altered karyotype of inversion and translocation; and indeterminate biopsies were excluded from this study.

Results:
Euploidy rates of blastocysts arising from 0PN, 1PN and 2PN were 68.8%, 52.9% and 51.7% (p>0.05) respectively. Conversely, aneuploidy rates of blastocysts resulting from 0PN, 1PN and 2PN were 31.3%, 47.1% and 48.3% (p>0.05) respectively. No significant differences were found between the 3 groups in euploidy and aneuploidy rates.

Conclusions:
It is known that 0PN and 1PN zygotes are less likely to become blastocysts with at least a BG3BB grade compared to 2PN zygotes. However, this study indicates that if 0PN and 1PN zygotes developed into at least a BG3BB blastocyst, they have euploidy rates similar to blastocysts derived from 2PN zygotes. Therefore, blastocysts arising from 0PN and 1PN zygotes should not be discarded but should be screened for euploidy. Euploid blastocysts arising from 0PN and 1PN zygotes should be considered for transfer especially when 2PN-derived blastocysts are unavailable since the former could result in pregnancies and live births of normal babies.

Lee, CSS.; Low SY.; Leong WY; Lui WX; Yap WY; Lim YX

Objective: This is a retrospective analysis comparing outcome of IVF patients on agonist protocol who had Norethisterone (NE) to adjust the timing of the next period (Group A) versus those who had natural menses (Group B).

Materials and Methods: 57 patients in Group A and 106 in Group B aged 36 from slow were analyzed from July 2011 to June 2013 in Alpha International Fertility Centre. Oocyte donation and PGD cases were excluded. The mean age of patients for Group A vs Group B were 31.9 vs 32.4 (p>0.05). The indications for IVF and the mean number of embryos transferred were similar in both groups. In Group A, patients were given 10mg NE 3 times per day until they are ready to start their IVF cycle. Buserelin injections were started about 1 week before menses in Group A, and on day 21 or 22 in Group B. Puregon or Gonal-F was used. Final maturation of oocytes was triggered with Ovidrel 36.5 hours before the planned OPU time. Clinical pregnancy and number of gestational sacs were determined by ultrasound.

Results: Clinical pregnancy rates per embryo transferred in Group A and Group B were 75.9% and 64.4% respectively (p>0.05). Implantation rate for Group A was statistically higher (44.1% versus 32.9%) than Group B (p=0.03).
Conclusion: This study shows that the use of NE in IVF agonist cycles did not adversely affect clinical pregnancy and implantation rates. Unexpectedly, NE cycles had better implantation rates compared to those who had menstrual cycles.

Presented at The 5th Congress of Asia Pacific Initiative on Reproduction (ASPIRE), 4-6 April 2014, Brisbane, Australia

Lee, CSS.; Yap WY.; Low SY.; Lim YX.

Introduction: In this study, we compared the euploidy and aneuploidy rates of PGD cases in Alpha International Fertility Centre, Malaysia following biopsy at the cleavage versus the blastocyst stage using Micro-array Comparative Genomic Hybridisation (MaCGH) for patients less than 37 years old.

Materials and Methods: 130 patients had cleavage stage biopsy, while 18 patients had blastocyst stage biopsy MaCGH from July 2011 to August 2013. The mean maternal age of patients from the cleavage stage and blastocyst stage biopsy MaCGH cases are 30.5 and 29.8 respectively (p=0.1). 1151 cleavage stage embryos had a blastomere cell biopsied to perform MaCGH. Whereas for the blastocyst stage biopsy MaCGH, 108 blastocysts had 3 – 7 trophectoderm cells biopsied. The biopsied sample(s) and reference DNAs were amplified, labelled and hybridised according to manufacturer’s (BlueGnome) specifications. Microarray slides were analysed using BlueFuse Software Version 3.1 (BlueGnome), revealing normal or whole and partial chromosome losses and gains. Cases excluded from the study were those with known altered karyotype of translocation and inversion; and indeterminate biopsies.

Results: The euploidy rates of cleavage stage embryos and blastocysts were 31.0% and 41.9% (p=0.01) respectively. The aneuploidy rates of cleavage stage embryos and blastocysts were 63.5% and 50.4% (p=0.01) respectively.

Conclusions: Blastocysts have significantly higher euploidy rate compared to cleavage stage embryos. We believe this is the primary reason for the higher implantation rate for blastocysts compared to cleavage stage embryos, and supports the practice of blastocyst culture and transfer.

Presented at The 5th Congress of Asia Pacific Initiative on Reproduction (ASPIRE), 4-6 April 2014, Brisbane, Australia

Lee, CSS.; Yap WY.; Low SY.; Lim YX.

Introduction: In this study, we compared the euploidy and aneuploidy rates of PGD cases in Alpha International Fertility Centre, Malaysia following biopsy at the cleavage versus the blastocyst stage using Micro-array Comparative Genomic Hybridisation (MaCGH) for patients less than 37 years old.

Materials and Methods: 130 patients had cleavage stage biopsy, while 18 patients had blastocyst stage biopsy MaCGH from July 2011 to August 2013. The mean maternal age of patients from the cleavage stage and blastocyst stage biopsy MaCGH cases are 30.5 and 29.8 respectively (p=0.1). 1151 cleavage stage embryos had a blastomere cell biopsied to perform MaCGH. Whereas for the blastocyst stage biopsy MaCGH, 108 blastocysts had 3 – 7 trophectoderm cells biopsied. The biopsied sample(s) and reference DNAs were amplified, labelled and hybridised according to manufacturer’s (BlueGnome) specifications. Microarray slides were analysed using BlueFuse Software Version 3.1 (BlueGnome), revealing normal or whole and partial chromosome losses and gains. Cases excluded from the study were those with known altered karyotype of translocation and inversion; and indeterminate biopsies.

Results: The euploidy rates of cleavage stage embryos and blastocysts were 31.0% and 41.9% (p=0.01) respectively. The aneuploidy rates of cleavage stage embryos and blastocysts were 63.5% and 50.4% (p=0.01) respectively.

Conclusions: Blastocysts have significantly higher euploidy rate compared to cleavage stage embryos. We believe this is the primary reason for the higher implantation rate for blastocysts compared to cleavage stage embryos, and supports the practice of blastocyst culture and transfer.

Presented at The 5th Congress of Asia Pacific Initiative on Reproduction (ASPIRE), 4-6 April 2014, Brisbane, Australia

Yap WY, Lim YX, Low SY, Lui WX, Lee CSS.

Introduction: Our in-house analysis of the outcome of embryos fertilised by Intra-Cytoplasmic Sperm Injection (ICSI) or In Vitro Fertilisation (IVF) suggests that IVF embryos tended to have higher pregnancy and implantation rates. This led us to analyse the euploidy and aneuploidy rates of cleavage stage embryos resulting from each method of fertilisation. Our preliminary results suggest that cleavage stage embryos resulting from IVF has lower aneuploidy rates. However, we were concerned of the prospect of amplifying the deoxyribonucleic acid (DNA) of potential sperm attached to the oocyte leading to erroneous results from Micro-array Comparative Genomic Hybridisation (MaCGH). In this study, we assess whether sperm DNA can be amplified using SurePlex (BlueGnome, UK).

Material & Methods: Semen sample from a normozoospermic man was processed as if it was prepared for IVF. The semen sample was collected into a sterile wide-mouth container by masturbation and kept at ambient temperature to allow liquefaction. The sperm was prepared by discontinuous density gradient (SpermGrad, Vitrolife) to separate the motile fraction of sperm free from debris, leucocytes, non-germ cells, degenerating germ cells and dead or immotile sperm. The sperm was resuspended for washing in culture medium (Vitrolife) followed by a swim up procedure to further purify the sperm. A serial dilution was done on the post swim up sperm. The stock concentration was 1 x 106/ml and was diluted into 6 different concentrations of 1 x 105/ml, 1 x 104/ml, 1 x 103/ml, 1 x 102/ml, 1 x 101/ml, and 1 x 100/ml using the same culture medium under aseptic condition. 0.5 μl from each concentration of sperm sample (including stock concentration) was transferred into PCR tubes containing 2.5 μl of 1xPBS buffer under aseptic condition. The samples were then amplified using SurePlex DNA amplification system according to the manufacturer’s protocols (BlueGnome). The ability of SurePlex DNA amplification system to amplify the sperm DNA was confirmed by gel electrophoresis and imaging system.

Results: None of the sperm concentrations show any discreet smears or bands in the gel electrophoresis.
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Conclusion: SurePlex DNA amplification system did not amplify sperm DNA at all the various sperm concentrations used. Thus, we conclude that fertilisation of oocytes via IVF can be safely done without fear of contaminating the biopsy specimen from cleavage stage embryos obtained for SurePlex DNA amplification in MaCGH (BlueGnome).

Yap WY, Lim YX, Low SY, Lui WX, Lee CSS. Sperm DNA does not contaminate blastomere biopsy results in IVF. Presented at the 13th Preimplantation Genetic Diagnosis International Society Annual Meeting, 29th April – 2 May 2014, Canterbury, United Kingdom.

Lee, CSS.; Lim YX; Low SY

Introduction: Extending the duration of embryo culture to the blastocyst stage in assisted reproduction offers several advantages over the transfer of cleavage stage embryos. Advantages include higher implantation rate, better selection of most viable embryo(s) for transfer, reducing the risk of multiple pregnancies and a better embryo-endometrium synchronization at the time of embryo transfer. In this study, the main outcomes measured were clinical pregnancy rate, implantation rate, number of babies born or expected to be born per embryo transfer (NBB-EtbB/ET) and multiple pregnancy rate.

Material & Methods: This is a retrospective analysis of all 91 fresh blastocyst transfer cycles performed at Alpha International Fertility Centre (AFC) from 2011-2013. These patients did not undergo Pre-implantation Genetic Diagnosis (PGD). The mean age of the patients was 30.1 years old (range 23-35). Vitrolife sequential media was used for culture from Day 0 to Day 5 or 6. The mean number of embryos transferred was 2.0. Embryo transfer was carried out on Day 5 or Day 6 after oocyte collection and beta hCG was accessed 10-14 days after the day of embryo transfer.

Results: Sixty-six out of 91 patients became clinically pregnant (72.5%). Of these, 3 patients miscarried. The implantation rate was 55.4%. Of the on-going or delivered pregnancies, 30 were singleton, 36 sets were twins and there were no triplets. number of babies born or expected to be born per embryo transfer (NBB-EtbB/ET) was 112.1% (102/91).

Conclusion: There is a trend to freeze all blastocysts for all cases in some centres nowadays. Our data shows fresh blastocyst transfer leads to high clinical pregnancy rate, implantation rate, NBB-EtbB/ET and no higher order pregnancies. Fresh blastocyst transfer is cost effective and logistic effective, and enables a patient to achieve a successful pregnancy earlier. Thus, fresh blastocyst transfer should be performed for all patients who do not have a contra-indication such as intrauterine polyps or ovarian hyper-stimulation syndrome.

Lee, CSS.; Lim YX; Low SY. Fresh blastocyst transfer yields high clinical pregnancy and implantation rates and avoids higher order multiple pregnancies. Presented at the 23rd Congress of the Obstetrical & Gynaecological Society of Malaysia, 5-8 June 2014, Malaysia

Lee, CSS.; Lui WX; Low SY; Lim YX

Introduction: Culture of embryos to the blastocyst stage has been widely employed in IVF treatment during the last decade. High blastulation rates of 63.4% and 68.0% were reported by leading IVF centres such as, Colorado Center for Reproductive Medicine, USA (Yang et al, 2012) and Genea, Australia (Krisman E, Leigh D et al, 2013) respectively. Skilled embryologists and optimized culture conditions are required in order to attain high blastulation rates. In the early years, we tend to transfer cleavage stage embryos back to the uterus. With improvements in culture techniques, freeze-thaw techniques and optimal laboratory environment, we now prefer to culture embryos to the blastocyst stage. In this study, the main outcomes measured were blastulation rate and blastocyst utilization rate in Alpha International Fertility Centre from July 2011 to December 2013.

Material & Methods: This is a retrospective study to assess the outcome from all 91 fresh blastocyst transfer cycles performed during that period of time. The mean age of the patients was 30.1 years old (range 23-35). Vitrolife sequential culture media was used. Manipulation of oocytes and embryos were done in a temperature-CO2-humidity controlled IVF chamber (Origio, Denmark) and ICSI chamber (HDS, Australia). Thus, the warm chain was preserved as much as possible. All embryos were cultured in MINC incubators and one chamber was dedicated to only one patient. Furthermore, the chambers and incubators were well positioned to minimize exposure of oocytes/embryos to outer environment. Embryology laboratory’s temperature, humidity and fresh air change per hour were well controlled by Heating, Ventilation and Air Conditioning (HVAC) System. The mean number of blastocysts transferred was 2.0. Surplus blastocysts were cryopreserved using Medicult or Cryotech Vitrification Kit. Blastulation rate was defined as total number of blastocysts formed per total number of 2PN zygotes, whereas, blastocyst utilisation rate was defined as total number of blastocysts transferred or cryopreserved per total number of blastocysts formed.

Results: In this study, 992 oocytes from 91 patients were fertilised on Day 1. The average number of fertilised oocyte per patient was 10.9 (range 4-22). Of these, 72.5% (720/992) formed blastocysts on Day 5 or Day 6. The blastocyst utilisation rate was 62.8% (452/720), with 184 blastocysts transferred and 268 blastocyst cryopreserved.

Conclusion: This study shows that with good warm chain system and optimal laboratory environment, we are pleased to note that our blastulation rate and blastocyst utilisation rate are comparable to leading IVF centres internationally.

Lee, CSS.; Lim YX; Low SY. High Blastulation Rate and Blastocyst Utilisation Rate at Alpha Fertility Centre. Presented at the 23rd Congress of the Obstetrical & Gynaecological Society of Malaysia, 5-8 June 2014, Malaysia

Lee, CSS.; Lim YX; Low SY

Introduction: Since July 2013, we started using a new embryo vitrification and thawing method (Cryotec Method, Japan) in all frozen-thawed cycles. The objective of this retrospective study was to evaluate the efficacy of the Cryotec Method in terms of post-thawed survival rate and clinical pregnancy outcome.

Material & Methods: This study was performed between July 2013 and February 2014. Twenty-eight blastocysts from 15 patients were frozen on Day 5 or 6 using Cryotech Vitrification Media and were thawed using Cryotech Warming Media. Freezing and thawing of embryos were done by Embryologists who were certified by Cryotech, Japan. The mean age was 30.9 years old (range 21-43). The mean number of embryos transferred was 1.9. In addition, 5 cleavage stage embryos from 2 patients were also frozen on Day 3 using Cryotech Vitrification Media and were thawed using Cryotech Warming Media. The age of the patients were 36 years- and 40 years old respectively. One had 2 embryos transferred and one had 3.

Results: All 28 blastocysts survived the post-thawed process (100% survival rate), enabling transfer in all cases. All blastocysts had intact inner cell mass and trophectoderm cells. The clinical pregnancy rate per embryo transfer with these blastocysts was 60.0%. For the 2 patients in the cleavage group, all 5 cleavage stage embryos survived the post-thawed process with all blastomeres intact. One of them (40 years old) had a successful pregnancy from 3 embryos transferred, resulting in a singleton pregnancy.

Conclusion: This preliminary study shows that using the Cryotec Method, we achieved a 100% post-thawed survival rate with all intact inner cell mass and trophectoderm cells, resulting in high clinical pregnancy rates. With the current trend towards fewer number of embryos transferred, a good cryopreservation method which gives reliably high rates of post-thawed survival is needed to cryopreserve the remaining surplus blastocysts of good to moderate quality. Cumulative pregnancy rate will therefore also be increased.

Lee, CSS.; Lim YX; Low SY. High 100% Post-thawed Survival Rate in Human Embryos at Alpha Fertility Centre. Presented at the 23rd Congress of the Obstetrical & Gynaecological Society of Malaysia, 5-8 June 2014, Malaysia

C.S.S. Lee, W.Y. Yap, S.Y. Low, Y.X. Lim.

Objectives: We had earlier reported the birth of a healthy baby resulting from an apronuclear (0PN) embryo following 5-probe FISH analysis and embryo transfer. The baby was confirmed chromosomally normal through amniocentesis (‘Case report: Ongoing pregnancy after transfer of a normal “apronucleated” embryo following PGD-FISH’, Khoo, G. et al., PGDIS 2007). This prompted us to perform Preimplantation Genetic Diagnosis (PGD) on 0PN and also unipronuclear (1PN) embryos in subsequent cases. We now report the results of Micro-array Comparative Genomic Hybridisation (MaCGH) on Day 3 embryos resulting from 0PN & 1PN embryos in patients who had MaCGH at Alpha International Fertility Centre (AFC) from July 2011 till January 2013.

Design and Method: This is a retrospective study to assess chromosome copy number for Day 3 0PN and 1PN embryos. All oocytes underwent Intra-Cytoplasmic Sperm Injection (ICSI). Fertilisation checks were done between 12.5 and 20.5 hours post ICSI, of which 82% were carried out 16 19 hours post injection. There were 313 0PN, 71 1PN, 1300 2PN and 29 3PN embryos representing 18.3%, 4.1%, 75.9% and 1.7% of the total embryos checked. Out of these embryos, only 26 out of 313 0PN embryos (8.3%), 13 out of 71 1PN embryos (18.3%) and 1085 out of 1300 2PN embryos (83.5%) had MaCGH. 0PN and 1PN embryos were chosen for MaCGH when they were at least of average grade and when there was plot availability for hybridisation (i.e. empty sampling slots available in order to optimise the utilisation of the arrays) or when there were too few 2PN embryos available for biopsy. Embryos with 2PN were selected for biopsy based on morphological features. MaCGH was performed according t manufacturer’s specifications (24Sure V3, BlueGnome, United Kingdom) and analysed using BlueFuse Multi Version 3.1 Software (BlueGnome, United Kingdom).

Results: Euploidy rates for embryos arising from 0PN, 1PN and 2PN were 23.1%, 30.8% and 30.0% (p > 0.05) respectively. Aneuploidy rates for 0PN, 1PN and 2PN embryos were 73.1%, 69.2% and 64.0% (p > 0.05) respectively. The indetermination rates for 0PN, 1PN and 2PN embryos were 3.8%, 0.0% and 6.0% (p > 0.05) respectively. No significant differences were found using Fisher’s exact test between the three groups for euploidy and aneuploidy rates.

Conclusion: This retrospective pilot study suggests that 0PN and 1PN embryos that develop to the cleavage stage have a euploidy rate similar to embryos resulting from 2PN. Therefore, cleavage stage embryos of at least average quality that arose from 0PN and 1PN should be considered for transfer. Where available, MaCGH on these embryos should also be considered.

C.S.S. Lee, W.Y. Yap, S.Y. Low, Y.X. Lim. Euploidy rates for day 3 apronuclear (0PN) and unipronuclear (1PN) embryos. Reproductive BioMedicine Online (May 2013); Vol.26 Suppl, S36.

Lim YX, Lee CSS, Yap WY, Low SY, Lui WX, Lim MW

Introduction:
1PN oocytes are often observed during fertilisation check at 18-20 hours post-insemination. This may be due to oocyte parthenogenetic activation, possible fusion of male and female pronuclei or asynchronous pronuclear formation. The development of parthenogenetic oocytes usually become impaired after activation, thus leading to early embryo arrest. A small proportion however, exhibit further cleavage and at this stage are indistinguishable from 2PN embryos. Chromosomal studies revealed that 45-48% of diploid 1PN embryos are in fact fertilised displaying Y signals and were not due to parthenogenetic activation (Staessen and Steirteghem, 1997). In our previous study, we had demonstrated that 1PN embryos that develop to the cleavage stage have euploidy rates similar to embryos resulting from 2PN (Lee et al, 2013). This case report describes a successful birth following frozen embryo transfer (FET) of a 1PN embryo after microarray comparative genomic hybridisation (MaCGH) analysis.

Method:
The patient, aged 37 years, who presented with polycystic ovarian syndrome underwent IVF treatment with preimplantation genetic screening at Alpha Fertility Centre in November 2013. Following oocyte retrieval, her oocytes were inseminated and were cultured in G-1TM PLUS medium and subsequently in G-2TM PLUS medium (Vitrolife, Sweden). Fertilisation check was performed 18 hours 10 minutes post insemination. All embryos arising from 2PN or 1PN zygotes that were at least average grade were subjected to biopsy on Day 3. Biopsied cells were analysed using MaCGH (Illumina, UK). Euploid embryo was cultured to Day 5 and vitrified using Cryotec Method (Cryotech, Japan) for elective FET. No fresh transfer was carried out in view of the risk of ovarian hyperstimulation syndrome.

Results:
Eighteen oocytes were retrieved, of which, 17 oocytes were inseminated. Thirteen out of 17 (13/17) were normally fertilised (2PN), one was 1PN and three were 3PNs. There were 5 embryos (4 from 2PNs and 1 from 1PN) suitable for biopsy on Day 3. Euploidy was confirmed in the embryo resulting from 1PN zygote, while all four 2PN embryos were aneuploid. The euploid-1PN embryo displayed Y hybridisation signal, thus affirming that fertilisation had occurred. The euploid-1PN embryo survived post-thawed, was morphologically intact, and transferred. An uneventful delivery at 39 weeks resulted in birth of a normal healthy baby boy.

Conclusion:
This report shows that the transfer of a frozen-thawed euploid embryo derived from 1PN “embryo” resulted in the birth of a normal healthy baby. It appears that the initial fertilisation observation of 1PN at the normal time of fertilisation assessment cannot be the absolute indicator of development incompetence. MaCGH can be used to ascertain the chromosomal constitution of 1PN “embryos” to confirm whether fertilisation had occurred, particularly those that display Y signals. Such embryos can be selected for transfer when no 2PN euploid embryos are available. To the best of our knowledge, this is the first case report of birth following transfer of 1PN embryo after MaCGH analysis.

References:

1. Staessen C, Van Steirteghem AC. The chromosomal constitution of embryos developing from abnormally fertilised oocytes after intracytoplasmic sperm injection and conventional in-vitro fertilization. Human Reproduction 1997;12:321-327
2. Khoo G, Lee CSS, Wong PS, Lee SA, Keith J. Case report: on-going pregnancy after transfer of a normal “apronucleated” embryo following PGD-FISH. Poster presentation, 7th Congress of PGDIS. Melbourne, Australia 13-15 June 2007

Lim YX, Lee CSS, Yap WY, Low SY, Lui WX, Lim MW. Live birth following frozen embryo transfer of a 1PN embryo after PGD-MaCGH analysis. Presented at the 14th International Conference on Preimplantation Genetic Diagnosis, 11-13 May 2015, Chicago, Illinois, USA

C.S.S. Lee, S.Y. Low, Y.X. Lim.

Introduction: Implantation failure following in-vitro fertilisation (IVF) is believed to be mainly due to chromosome abnormalities (aneuploidy). Screening of embryos using MaCGH has been implemented in an attempt to improve implantation rates. In this study, we compare the implantation rates (IR) and clinical pregnancy rates in IVF patients who had MaCGH and those who did not have MaCGH (non-MaCGH).

Materials and Methods: This is a retrospective analysis of outcomes from 152 MaCGH cycles (Day 3 biopsy) and 249 non-MaCGH cycles (cleavage-stage embryo transfer) performed at Alpha International Fertility Centre (AFC), Malaysia from July 2011 to January 2013. All embryos analysed had Intra- Cytoplasmic Sperm Injection (ICSI). The cases were divided into 4 groups, age <35, 35 37, 38 39 and 40 46. The number of MaCGH and non-MaCGH cycles in each age group was 76 vs 123, 32 vs 54, 14 vs 43 and 30 vs 29 respectively. Only one blastomere was biopsied on Day 3. Samples and reference DNAs were amplified, labelled and hybridized according to manufacturer’s (BlueGnome) specifications. Microarray slides were analysed using BlueFuse Software Version 3.1 (BlueGnome), revealing normal or whole and partial chromosome losses and gains.

Results: The mean age was comparable in all patients from all the age groups. In the Conclusions: For patients under 38 years old, the IR appears to be significantly higher in the group who had MaCGH. We were unable to demonstrate a higher IR in the older patients as the numbers were small. The clinical pregnancy rate is similar in both groups probably due to the higher number of embryos transferred in the non-MaCGH group.

C.S.S. Lee, S.Y. Low, Y.X. Lim. Implantation and clinical pregnancy rates in patients with and without micro-array comparative genomic hybridisation (MaCGH). Reproductive BioMedicine Online (May 2013); Vol.26 Suppl, S36.

Low SY, Lee CSS, Lim MW, Yap WY, Lui WX, Lim YX

Introduction:
We had previously reported on: i) Euploidy Rates of Day-3 apronuclear (0PN) and unipronuclear (1PN) embryos arising from Intra-Cytoplasmic Sperm Injection (ICSI) (CSS Lee et. al., PGDIS 2013) and ii) Blastomere biopsy results in IVF is not affected by sperm DNA (Yap WY et. al., 2014). With these studies in mind, we proceeded to perform IVF (whenever semen parameters are met) for cases where Micro-array Comparative Hybridisation (MaCGH) was planned for. All resulting Day-3 embryos even those arising from 0PN and 1PN had MaCGH when they are of at least average quality. We now report the euploidy rates of these 0PN and 1PN cleaved embryos (Alpha Fertility Centre (AFC): July 2011 to December 2014).

Material & Methods:
This is a retrospective study to assess the chromosome copy number of cleaved embryos arising from 0PN & 1PN zygotes following IVF. Fertilisation checks were done between 18 to 20 hours after insemination. There were 25 patients (mean age: 33.2 ranged 20-40) had cleaved embryos arising from 0PN & 1PN zygotes. Between them, they had a total of 28 0PN, 81 1PN, 196 2PN and 14 3PN oocytes/zygotes during fertilisation check. Out of these oocytes/zygotes, 7 0PN (25%), 9 1PN (11.1%) and 163 2PN (83.1%) zygotes cleaved and were biopsied on the morning of Day-3. All biopsied embryos were of at least average grade. MaCGH was performed according to the manufacturer’s specifications (24sureV3, BlueGnome, United Kingdom) and analysed using Bluefuse Multi Version 3.1 Software (BlueGnome, United Kingdom). Cases with known altered karyotype of inversion and translocation; and indeterminate biopsies were excluded from this study.

Results:
Euploidy rates of Day-3 0PN, 1PN and 2PN cleaved embryos arising from IVF were 28.6%, 77.8% and 31.3% respectively. Aneuploidy rates of 0PN, 1PN and 2PN cleaved embryos arising from IVF were 71.4%, 22.2% and 68.7% respectively. All 3 groups showed no significant differences in euploidy and aneuploidy rates. We have previously reported a live birth following frozen embryo transfer of a 1PN embryo subjected to MaCGH analysis (submitted as another abstract for PGDIS 2015). Another patient whom we transferred both a 0PN and a 2PN embryo (double embryo transfer) is now in her third trimester with a twin pregnancy.

Conclusions:
0PN and 1PN oocytes/zygotes are much less likely to reach cleavage stage of at least average quality. However if they do reach that stage, then they have similar euploidy rate compared to 2PN cleaved embryos. This study shows that there are no significant differences in euploidy rates in embryos arising from Day-3 0PN, 1PN and 2PN cleaved embryos resulting from IVF. Therefore, 0PN and 1PN cleaved embryos of at least average grade should be screened via PGD rather than discarded. In IVF centers with no PGD facility and for those embryos without PGD analysis, 0PN and 1PN cleaved embryos should be considered for transfer but any resultant pregnancy subjected to full antenatal surveillance.

References:

1. CSS Lee, WY Yap, SY Low, YX Lim. Euploidy rates for Day 3 Apronuclear (0PN) and Unipronuclear (1PN) embryos. Reproductive BioMedicine Online (May 2013); Vol. 26 Suppl, S36
2. WY Yap, Lim YX, SY Low, Lui WX and CSS Lee. Sperm DNA does not contaminate blastomere biopsy results in IVF. Poster presentation, P48, 13th Congress of PGDIS, Canterbury, United Kingdom 29 April to 2 May 2014.

Low SY, Lee CSS, Lim MW, Yap WY, Lui WX, Lim YX. Euploidy rates of day 3 apronuclear (0PN) and unipronuclear (1PN) cleaved embryo arising from IVF. Presented at the 14th International Conference on Preimplantation Genetic Diagnosis, 11-13 May 2015, Chicago, Illinois, USA

Lee CSS, Yap WY, Lim MW, Lim YX, Low SY, Lui WX

Introduction:
We had previously reported on the birth of a healthy baby resulting from a 0PN embryo transfer (fertilised using IVF) screened via 5-probe FISH, and was confirmed chromosomally normal using amniocentesis (Khoo, G. et al, PGDIS 2007). We have subsequently demonstrated that fertilisation via IVF does not predispose to sperm DNA contamination for cleavage stage embryos subjected to Microarray Comparative Genomic Hybridisation (MaCGH) with SurePlex DNA amplification kit (Yap, WY. et al, 2014). With this understanding, we decided since November 2013 to perform Preimplantation Genetic Diagnosis (PGD) using MaCGH on 0PN and 1PN blastocysts resulting from IVF. This retrospective study reports the euploidy and aneuploidy rates of IVF-fertilised 0PN and 1PN blastocysts. To the best of our knowledge, this is the first such report on the euploidy and aneuploidy rates of IVF-fertilised 0PN and 1PN blastocysts using MaCGH.

Material & Methods:
For the period November 2013 till December 2014, all IVF cases had fertilisation checks done 18 – 20 hours post insemination. There were 811 zygotes of which 161 was 0PN (19.9%), 44 was 1PN (5.4%), 535 was 2PN (66.0%) and 71 was 3PN (8.7%). All zygotes were cultured to blastocysts. There were 273 blastocysts formed with at least a BG3BB grade (Gardner’s grading) from 9.9% of 0PN’s (16/161), 38.6% of 1PN’s (17/44) and 44.9% of 2PN’s (240/535). No blastocyst with at least a BG3BB grade were formed from 3PN zygotes. These blastocysts had trophectoderm cells biopsied and screened using MaCGH. The biopsied cells were amplified using SurePlex DNA Amplification System (Bluegnome), labelled and hybridised together with a set of reference DNA according to the manufacturer’s protocols (BlueGnome). The microarray slides were analysed using BlueFuse Multi Software Version 3.1 (BlueGnome) to determine the chromosomal status of the blastocysts. Cases with known altered karyotype of inversion and translocation; and indeterminate biopsies were excluded from this study.

Results:
Euploidy rates of blastocysts arising from 0PN, 1PN and 2PN were 68.8%, 52.9% and 51.7% (p>0.05) respectively. Conversely, aneuploidy rates of blastocysts resulting from 0PN, 1PN and 2PN were 31.3%, 47.1% and 48.3% (p>0.05) respectively. No significant differences were found between the 3 groups in euploidy and aneuploidy rates.

Conclusions:
It is known that 0PN and 1PN zygotes are less likely to become blastocysts with at least a BG3BB grade compared to 2PN zygotes. However, this study indicates that if 0PN and 1PN zygotes developed into at least a BG3BB blastocyst, they have euploidy rates similar to blastocysts derived from 2PN zygotes. Therefore, blastocysts arising from 0PN and 1PN zygotes should not be discarded but should be screened for euploidy. Euploid blastocysts arising from 0PN and 1PN zygotes should be considered for transfer especially when 2PN-derived blastocysts are unavailable since the former could result in pregnancies and live births of normal babies.

References:

1. Khoo G, Lee CSS, Wong PS, Lee SA, Keith J. Case report: on-going pregnancy after transfer of a normal “apronucleated” embryo following PGD-FISH. Poster presentation, 7th Congress of PGDIS. Melbourne, Australia 13-15 June 2007
2. Yap WY, Lim YX, Low SY, Lui WX, Lee CSS. Sperm DNA does not contaminate blastomere biopsy results in IVF. Poster presentation, 13th Congress of PGDIS. Canterbury, United Kingdom 29 April – 2 May 2014

Lee CSS, Yap WY, Lim MW, Lim YX, Low SY, Lui WX. Euploidy rates of apronuclear (0PN) and unipronuclear (1PN) blastocysts fertilised using in vitro fertilization (IVF). Presented at the 14th International Conference on Preimplantation Genetic Diagnosis, 11-13 May 2015, Chicago, Illinois, USA

Lee CSS, Lim YX, Lui WX

Introduction
Impaired clinical outcome from fresh Day 6 blastocyst transfers due to poor endometrial receptivity after ovarian hyperstimulation were extensively reported recently. To our knowledge, a few reported studies demonstrated that slow-growing Day 6 blastocysts using freeze-thaw technique contributes to better clinical outcome compared to fresh Day 6 blastocysts. The aim of this retrospective study is to investigate the efficacy of fresh and frozen Day 6 blastocyst transfers at Alpha International Fertility Centre (AFC), Malaysia in patients aged 40 and below.

Methods
All 30 patients who had Day 6 blastocysts transferred from the commencement of AFC (July 2011) till now were analysed. Twelve (12) had fresh Day 6 blastocyst transfer (Group A) and 18 had frozen Day 6 blastocyst transfer (Group B). These were all relatively slow-growing blastocysts and were not suitable for transfer or freezing on Day 5. Patients who had pre-implantation genetic screening were excluded in this study. The mean age of patients in each group was similar: Group A was 31.4 (ranged 21-40) and Group B was 32.3 (ranged 21-39). A total of 22 blastocysts were transferred in Group A. In Group B, 29 blastocysts were thawed. All 29 survived with morphologically intact inner cell mass and trophectoderm cells (100% post-thaw survival rate). All 29 blastocysts thawed were transferred. The mean number of blastocysts transferred was 1.8 in Group A and 1.6 in Group B. Clinical pregnancy and number of gestational sacs were determined by ultrasound.

Results
Clinical pregnancy rates per embryo transfer (ET) for Group A and Group B were 58.3% and 61.1% respectively. The implantation rates for Group A and Group B were 36.4% and 55.2% respectively. Number of babies born or expected to be born per ET (NBB-EtbB/ET) for Group A was 50.0% and for Group B was 72.2%. There were no significant differences found in clinical pregnancy rate, implantation rate and NBB-EtbB/ET between both groups, but shows a tendency for higher rates in the frozen group.

Conclusion
Our preliminary findings demonstrated that transferring frozen Day 6 blastocysts yielded a tendency towards higher clinical pregnancy rate, implantation rate and number of babies born or expected to be born per ET compared to transferring Day 6 blastocysts in fresh cycles.

Lee CSS, Lim YX, Lui WX. Clinical Outcome of Fresh versus Frozen Day 6 blastocyst transfer . Presented at the 24th Asian & Oceanic Congress of Obstetrics & Gynaecology, 3-6 June 2015, Kuching, Sarawak, Malaysia

Lee CSS, Lim YX

Introduction:
Perhaps the main cause of implantation failure is aneuploidy which increases with age. MaCGH is thus expected to improve clinical outcome by selecting only euploid blastocysts to be transferred. Lower pregnancy rates in older women could therefore be avoided. Good clinical outcome was also observed following embryos vitrified using Cryotec Method which achieves 100% post-thaw survival rate. This study describes our experience of combining these 2 recent IVF technologies in elective frozen blastocyst transfer for patients less than 38 years old. It also looks at the clinical outcome at different age groups following transfer of euploid blastocysts.

Method:
This is a retrospective analysis of outcome from 32 frozen euploid blastocyst transfers in AFC since the introduction of Cryotec Method (November 2013 to November 2014). The cases were divided into 3 groups, age ≤30 (Group A), 31-34 (Group B) and 35-37 (Group C). All blastocysts were frozen after biopsy on Day 5 and/or Day 6 for elective FET. The biopsied cells were analysed using MaCGH. Clinical pregnancy and number of gestational sacs were determined using ultrasound.

Results:
The mean age was comparable between Group A, B and C (26.0, 32.5 and 36.0 respectively). Fourty-six frozen euploid blastocysts were thawed and all survived intact (Post-thaw survival rate = 100%). All 46 thawed blastocysts were transferred. The mean number of blastocysts transferred was 1.6 in Group A and 1.3 in Group B and C. Clinical pregnancy rates (CPR), implantation rates (IR) and live birth/on-going pregnancy rates for Group A, B and C were 66.7% vs 50.0% vs 85.7%; 54.2% vs 38.5% vs 66.7%; and 60.0% vs 50.0% vs 71.4% respectively. There was no statistical significance in CPR, IR and live birth/on-going pregnancy rates between all groups. For all the 32 patients as a whole, the CPR, IR and live birth/on-going pregnancy rates were 65.6%, 52.2% and 59.4% respectively. Of the on-going or delivered pregnancies, 16 were singleton, 3 sets were twins and there is no higher order pregnancies.

Conclusion:
This study shows that elective FET of euploid blastocysts leads to good clinical pregnancy, implantation and live birth/on-going pregnancy rates. The risk of multiple pregnancies are also reduced. By transferring only euploid blastocysts, the clinical pregnancy and implantation rates were similar and did not show a decline with advancing age.

Lee CSS, Lim YX. Clinical Outcome following Transfer of Frozen Euploid Blastocysts after Micro-Array Comparative Genomic Hybridisation (MaCGH) at Alpha Fertility Centre (AFC). Presented at the 24th Asian & Oceanic Congress of Obstetrics & Gynaecology, 3-6 June 2015, Kuching, Sarawak, Malaysia

Lee CSS, Low SY

Introduction:
It is well known that blastocysts are more robust and it is also more physiological to transfer embryos on Day 5. It is also known that blastocysts have higher euploidy rates and thus higher implantation rate (IR) and clinical pregnancy rate (CPR) compared to cleavage stage embryos. However, to enable blastocyst culture and transfer as a routine, the IVF laboratory must first achieve optimum culture conditions to enable high blastulation rates. From the commencement of AFC, we took great pains to ensure such conditions are achieved. Once we have achieved sustained blastulation rates of about 70%, we decided from May 2013 to encourage most of our patients to go for blastocyst culture and transfer. We embarked on blastocyst culture and transfer as a matter of preference since then. In this study, the main outcome measured were blastulation rate, blastocyst utilization rate, IR, CPR, multiple pregnancy rate, and number of babies born or expected to be born per embryo transfer (NBB-EtbB/ET).

Methods:
This is a retrospective analysis of patients who had fresh blastocyst culture and transfer (but without PGD) since we started AFC (July 2011) till now. The percentage of patients who had fresh blastocyst culture in year 2011, 2012, 2013 and 2014 were 7.5% (8/107), 4.9% (8/162), 46.9% (107/228) and 70.5% (203/288) respectively. The mean age of patients for blastocyst culture and transfer were 32.4, 29.4, 30.7, and 31.4 (ranged 20-37). The mean number of blastocysts transferred were 2.7, 2.1, 1.9 and 1.8 respectively. Clinical pregnancy and number of gestational-sacs were determined by ultrasound.

Results:
Over this period of time, the blastulation rate was fair consistent at about 71% and the blastocyst utilisation rate was 64.6%. The IR ranged from 47.4% to 73.3% over the period of study with no significant differences between the years. The CPR were 85.7%, 100%, 65.9% and 63.6% and were not statistically significant. Singleton pregnancy rates were 50%, 42.9%, 44.8% and 63.6% respectively. Twin pregnancy rates showed a steady decline from 50% in year 2011 to 36.4% in year 2014. There were no higher-order multiple pregnancies over the years. The NBB-EtbB/ET was 91.9%.

Conclusion:
With consistent blastulation rates over the years (around 71%), we confidently increased the percentage of patients going for blastocyst culture while maintaining the CPR, IR and NBB-EtbB/ET despite progressively reducing the mean number of blastocysts transferred year-on-year. Twin pregnancy rates were also reduced progressively. There were no higher-order multiple pregnancies.

Lee CSS, Low SY. Review of Fresh Blastocyst Culture and Transfer Program at Alpha International Fertility Centre (AFC). Presented at the 24th Asian & Oceanic Congress of Obstetrics & Gynaecology, 3-6 June 2015, Kuching, Sarawak, Malaysia

Colin S. S. Lee, S.Y. Low, W.Y. Leong

Introduction: A 37 year-old patient, whose husband has a balanced inversion of chromosome 1 (p35q42.1), had a history of 2 spontaneous miscarriages. Following PGD with MaCGH, 2 embryos with normal/ balanced chromosomes were transferred fresh in the same cycle, resulting in delivery of a live child.

Material & Methods: Twenty-two embryos were obtained following ICSI of which 12 had microarray copy number analysis PGD. Samples and reference DNAs were amplified and labelled according to manufacturer’s (BlueGnome) specifications. Labelled sample and reference DNAs were applied to microarrays (24Sure+) and co-hybridized overnight. Microarray slides were washed, dried, scanned and analysed using BlueFuse Software (BlueGnome), revealing whole and partial chromosome losses and gains, including unbalanced inversions.

Results: Results were obtained for 10 of 12 samples. Of the diagnosed samples, 5 were found to be affected with unbalanced pericentric inversion on chromosome 1, with or without other chromosome abnormalities. Three embryos with normal/ balanced chromosome 1 had other chromosome abnormalities. Only 2 embryos were found to have the complete chromosome complement, though a balanced inversion cannot be excluded. These 2 embryos were transferred on day 4 after oocyte pick-up. Both embryos implanted. One split into monozygotic twins which died at 17 weeks with associated twin-to-twin transfusion. The dead monozygotic twins were delivered at 25 weeks, while the live baby was delivered at 26 weeks and 2 days weighing 775 grams, requiring ventilation. As at the time of writing, the baby had been extubated and doing well. Preliminary karyotyping from placental tissue of the live child shows a normal chromosome complement with no inversion (46, XY). Karyotyping from placental tissue of the dead twins showed a normal chromosome complement with a balanced inversion of chromosome 1 i.e., 46, XX inv (1) (p35q42).

Conclusion: Clinical application of PGD microarray copy number analysis with fresh embryo transfer has been used successfully to detect unbalanced chromosome inversion, resulting in the live birth of a normal child.

C.S.S. Lee, S.Y. Low, W.Y. Leong. (May 2012). Birth after Preimplantation Genetic Diagnosis (PGD) using MicroAarray Comparative Genomic Hybridisation (MaCGH) for chromosome inversion (1) (p35q42). Reproductive Biomedicine Online, Vol.24 Suppl, S61, ISSN 1472-6483

Introduction: A 41 year-old patient, whose husband has a balanced chromosomal translocation t(14;21) (q22;q22.3), has prolonged infertility for 14 years. Following ICSI & Preimplantation Genetic Diagnosis (PGD) with Microarray Comparative Genomic Hybridization (MaCGH), 2 embryos with normal/ balanced chromosomes were transferred in the same cycle, resulting in an on-going pregnancy.

Material & Methods: Eleven embryos were obtained following ICSI of which 6 had microarray copy number analysis PGD. Samples and reference DNAs were amplified and labelled according to manufacturer’s (BlueGnome) specifications. Labelled sample and reference DNAs were applied to microarrays (24Sure+) and co-hybridized overnight. Microarray slides were washed, dried, scanned and analysed using BlueFuse Software Version 2.5 (BlueGnome), revealing normal or whole and partial chromosome losses and gains, including unbalanced inversions & translocations.

Results: Results were obtained for 5 of 6 the samples. Of the diagnosed samples, 1 was found to be affected with unbalanced translocation on chromosome 14 and 21. Two embryos with normal/ balanced chromosomes 14 and 21 had other chromosomal abnormalities. Only 2 embryos were found to have the complete chromosome complement, though a balanced translocation cannot be excluded. These 2 embryos were transferred at day 4 stage; resulting in a singleton pregnancy. At the time of writing, the patient is in her 25th week of pregnancy.

Conclusion: Carriers of balanced rearrangements rarely show any phenotypic effect but are at risk of producing unbalanced gametes. Chromosomally unbalanced conceptuses may miscarry or result in an abnormal live born offspring. Clinical application of PGD microarray copy number analysis with fresh embryo transfer can detect unbalanced translocation in embryos. This allows the selection of embryo with normal/ balanced chromosomes to be transferred, leading to a successful pregnancy.

Leong, WY; Lee, CSS; Low SY. (2012). On-going pregnancy after pre-implantation genetic diagnosis (PGD) for paternal balanced translocation t(14;21) (q22;q22.3) using microarray comparative genomic hybridization (MaCGH). An International Journal of Obstetrics & Gynaecology, Vol.119, Issue Supplement S1, PP.137.

Leong, WY; Lee, CSS; Low, SY

Introduction: To review the outcome of oocyte donation cases at Alpha International Fertility Centre for the year 2011.
Methods: This is a retrospective analysis of all oocyte donation treatment cycles in the Alpha International Fertility Centre from July 2011 until December 2011. Oocyte donors were assessed according to ASRM guidelines. Oocyte donation was performed for sub-fertile women with low ovarian reserve. The oocyte donors were anonymous. Oocyte recipients were down-regulated to synchronize with the donor and subsequently received both oral oestrogen and progesterone pesseries for endometrial development. The oocytes collected then underwent IVF or ICSI, and the embryos were transferred using standard embryo transfer protocols.

Result: Thirty-six oocyte retrievals were performed. Of these, all progressed to embryo transfer. The mean age of oocyte donors were 24.0 and for recipients was 40.8 respectively. Eighty-eight embryos were transferred, resulting in 42 gestational sacs. The mean number of embryos transferred was 2.4 and the implantation rate was 47.7%. There were 26 clinical pregnancies, giving a clinical pregnancy rate per embryo transfer of 72.2%. Of the 20 on-going pregnancies, seven are singleton (35%), 11 sets are twins (55%) and two sets are triplets (10%).

Conclusion: Oocyte donation offers an effective treatment option for sub-fertile women with low ovarian reserve.

Leong, WY; Lee, CSS; Low SY. Oocyte donation outcome at Alpha International Fertility Centre. An International Journal of Obstetrics & Gynaecology 2012; Vol.119, Issue Supplement S1, PP.137.

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Leong WY, Lee CSS, Low SY.

Introduction: A 35 year-old women with failure to conceive for 3 years due to profound male factor, had a successful pregnancy from a Frozen Embryo Transfer (FET) cycle using embryos fertilised earlier from frozen sperm and frozen oocytes.

Material & Methods: The patient underwent Intra-Cytoplasmic Sperm Injection (ICSI) program with a long down regulation protocol in November 2010. Her spouse who did not have a prior history of erectile dysfunction failed to produce an ejaculate on the day of oocyte pick-up. Immediate Percutaneous Epididymal Sperm Aspiration (PESA) was performed but no sperm was obtained, necessitating freezing of the available oocytes. With hindsight, sperm from an ejaculate later was frozen and they underwent a fresh embryo transfer cycle using the frozen sperm and frozen oocytes. Six thawed oocytes were fertilised from thawed sperm via ICSI and from these, 3 embryos were transferred and another 3 frozen. This cycle resulted in a viable singleton pregnancy but followed with a first trimester miscarriage. The 3 frozen embryos were utilized in a Frozen Embryo Transfer (FET) cycle 3 months later, whereby, the patient was given an ascending dose of oral ethinyl estradiol starting from day 5 of her menstrual cycle after down regulation with Intra-muscular Depot GnRH analogue. Optimal endometrial thickness was achieved after 13 days of oral ethinyl estradiol, and progesterone pessaries were then added. Three frozen embryos were thawed on day 23 of the cycle using Vitrolife Thawing Kit 1. All embryos survived and were transferred with the standard Embryo Transfer protocol, and resulted in singleton pregnancy. Luteal support continued with oral oestrogen and progesterone pessaries until 12 weeks of gestation.

Results: Of the 24 frozen oocytes, 19 (79.1%) survived the thawing process. Fertilisation rate from the frozen oocytes using frozen sperm was 31.5% (6/19). Implantation rate (IR) from the 3 fresh embryos was 33.3% albeit resulting in a miscarriage. The subsequent pregnancy from the 3 frozen embryos also resulted in a singleton pregnancy (IR of 33.3%).
Conclusion: The patient is pregnant with a baby boy and in her 24th week of pregnancy at the time of writing. A successful pregnancy can therefore be achieved via FET using frozen oocytes and frozen sperm.

Leong, WY; Lee, CSS; Low SY. (2012). First on-going pregnancy via frozen embryo transfer (FET) using frozen oocytes and frozen sperm.An International Journal of Obstetrics & Gynaecology, Vol.119, Issue Supplement S1, PP.148.