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A pilot study to evaluate a device for the intravaginal culture of embryos

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Abstract The aim of this comparative randomized embryology trial was to determine if an intravaginal culture device (IVC) can provide acceptable embryo development compared with conventional IVF. Ten women between the ages of 27 and 37 years with an
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  ARTICLE A pilot study to evaluate a device for theintravaginal culture of embryos Frederic Mitri  a , Navid Esfandiari  a , Joan Coogan-Prewer  a , Paul Chang  a ,Yaakov Bentov  a , John McNaught  b , Anat H Klement  a , Robert F Casper  a, * a TCART Fertility Partners, Division of Reproductive Sciences, Department of Obstetrics and Gynecology, Lunenfeld-Tanenbaum Research Institute, Mount Sinai Hospital, University of Toronto, Toronto M5S 2X9, Canada;  b Fertility Ontario,London N6C 5Z2, Canada * Corresponding author. Tel.: 416-972-0777; Fax: 416-972-0036.  E-mail address:  casper@lunenfeld.ca (RF Casper).Dr Frederic Mitri completed his residency training in Obstetrics and Gynecology in Beirut, Lebanon. He is cur-rently completing a clinical fellowship in Reproductive Endocrinology and Infertility at TCART, Toronto. His re-search interests include molecular and genetic biology, fertility preservation and poor responders. Abstract  The aim of this comparative randomized embryology trial was to determine if an intravaginal culture device (IVC) can provideacceptable embryo development compared with conventional IVF. Ten women between the ages of 27 and 37 years with an indica-tion for IVF treatment were included in this study. After ovarian stimulation, oocytes were randomized to fertilization in the IVCdevice or using conventional IVF. Fertilization rates were higher in the IVF group compared with the IVC device (68.7%  ±  36 % versus40.7%  ±  27%), respectively, whereas cleavage rates were similar (93%  ±  1.5% versus 97%  ±  6%) for both groups. A significantly lowernumber of embryos of suitable quality for transfer was obtained from the IVC device compared with conventional IVF (OR, 0.47; 95%CI, 0.26 to 0.87). The clinical pregnancy rate from transfer of IVC device embryos was 30%. Satisfaction questionnaires were alsocompleted by all participants. Most women (70%) placed high importance on having had fertilization and embryo development occurwhile carrying the device. Overall, the IVC device produced reasonable pregnancy rates suggesting this technology may have a placeunder certain circumstances. Cost-benefit analysis, psychological factors and future studies must be considered. © 2015 Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved. KEYWORDS:  culture system, embryo score, INVOcell, IVC device, IVFhttp://dx.doi.org/10.1016/j.rbmo.2015.09.0051472-6483/© 2015 Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved.Reproductive BioMedicine Online (2015) ■■ , ■■ – ■■ ARTICLE IN PRESS Please cite this article in press as: F. Mitri, et al., A pilot study to evaluate a device for the intravaginal culture of embryos, Reproductive BioMedicine Online (2015), doi:10.1016/j.rbmo.2015.09.005 www.sciencedirect.comwww.rbmonline.com  Introduction During conventional IVF, oocytes and spermatozoa are incu-bated together in the laboratory, where fertilization and em-bryonic development are carefully monitored at several timepoints. Over the years, improvements in IVF methodology andculture media have been established with the aim of devel-oping the most efficient incubator and providing optimumculture conditions for fertilization and embryo develop-ment (Gardner, 2008; Summers and Biggers, 2003). Twenty-four hour availability of laboratory personnel and equipment,sophisticated sequential culture media and highly trained em-bryologists are, however, unavailable in some areas. Fur-thermore, IVF is quite costly, and this may pose a barrier forsome couples who are trying to conceive (Collins, 2001). Onthe basis of cost analysis, the embryology and laboratory ser-vices are the most expensive. The average cost of one stan-dard IVF cycle in Canada can exceed $12,000 depending onthe woman’s age and quantity of fertility medication used;this figure may be higher in other areas. The cost per live birthcan be several fold greater and may depend on other vari-ables, including per cycle pregnancy rate and the number of embryos transferred (Bhatt and Baibergenova, 2008). Intravaginal culture (IVC) technology was initially pro-posed and developed in 1988. Many improvements and modi-ficationshavebeenmadetoanearlierprototypedevice,whichevolvedintoINVOcell TM, aHealthCanadaapprovedIVCsystem,which has been ISO 10993 tested and received the Europeanconformity(FrydmanandRanoux,2008;Ranouxetal.,1988).With this technology, retrieved oocytes and processed sper-matozoa are placed directly into the specially prepared gaspermeableculturedevice.Thedevicecontainingoocytesandspermatozoa is placed in the vagina, where fertilization andsubsequent embryo development takes place. This proce-dure bypasses the requirement for sophisticated laboratoryequipment or highly trained embryologists and reduces thecostpertreatmentcycle.Ingeneral,lowovarianstimulationprotocolshavebeenusedinconjunctionwiththisdevice,andhaveprovidedencouragingresultsglobally(Bonaventuraetal.,2006; Lucena et al., 2012; Wiegerinck et al., 1990). Out of 125 IVC cycles conducted, 40% of women were reported tohaveachievedclinicalpregnancy,whichcomparesfavourablyto conventional IVF programmes (Lucena et al., 2012). Theobjective of the present study was to evaluate fertilizationratesandembryodevelopmentusingtheintravaginalculturesystemcomparedwithconventionalIVFinacomparativeran-domized embryology trial using sibling oocytes. Materials and methods Settings and design Patientcyclemonitoringandfollow-upwascarriedoutattheTorontoCentreforAdvancedReproductiveTechnology(TCART,Toronto,Ontario,Canada),itsaffiliatesatelliteclinicinKitch-ener,(Ontario,Canada)andFertilityOntario(London,Ontario).Interventionsandprocedures,includingoocyteretrieval,IVCdevice preparation and embryo transfer were all carried outatTCART.Thestudywasacomparativeembryologytrial,andethicsapprovalwasobtainedfromanexternalResearchEthicsBoard (Western Institutional Review Board; www.wirb.com)on January 29 2013, study number CP-004. Financial supportwas offered to women who participated in the study. Study population Women presenting to the clinic between the ages of 18 and38 years, who had failed to conceive after 1 year of unpro-tected intercourse, had a normal recent pelvic sonogram andpap smear and in which IVF had been deemed to be the nextstep of treatment were invited to take part in the study. In-clusion criteria required a diagnosis of bilateral blocked tubes,early stage endometriosis (stage I or II) or unexplained infer-tility requiring IVF and a minimum of eight mature re-trieved oocytes. Male partners were required to have a normalsemenanalysis.Willingindividualswereeligibleaslongastheydid not have any exclusion factors and understood and ac-cepted the terms of the study. Exclusion criteria included ob-taining fewer than eight mature oocytes based on morphologyof cumulus cell expansion, a history of toxic shock syn-drome, chronic illness, vaginal inflammation, infection, ana-tomic abnormalities or allergy to plastics, human proteins orgentamicin.Otherexclusionfactorsincludedahistoryofpelvicsurgery within the past 8 weeks, history of cervical infec-tion with chlamydia or gonorrhoea in the past 12 months,pelvic inflammatory disease, severe endometriosis, body massindex 36 or over, use of donor spermatozoa or eggs, lowovarian reserve, polycystic ovaries, poor responders, priorhistory of ovarian hyperstimulation, inability to wear a dia-phragm, smoking, drug or alcohol abuse and two or more pre-vious failed IVF cycles. Women were diagnosed with lowovarian reserve based on anti-Müllerian hormone less than1.1 ng/ml, FSH greater than 13 and antral follicle count lessthan five. Poor responders were identified on the basis of theBologna criteria (Ferraretti et al., 2011). Between March and September 2013, a total of 15 women agreed to participatein this study. Five of these patients were excluded as theydid not fulfill the eligibility criteria. In all of the stimulationcycles, sibling oocytes were randomized for both conven-tional IVF and for incubation with sperm in the IVC device forcomparison. When more than 20 mature oocytes were re-trieved, a maximum of 10 were placed in the IVC device andthe remaining oocytes were used for conventional IVF. Stimulation protocol A long luteal phase gonadotrophin-releasing hormone (GnRH)agonist protocol was used with the GnRH agonist buserelinacetate (Suprefact; Sanofi-Aventis, Laval, Quebec) in dailydoses of 200  μ g subcutaneously starting one week before ex-pected menses. After confirming ovarian quiescence by day3 ultrasound and a normal baseline hormone profile (FSH < 10 IU/l, oestradiol  < 200 pmol/l), two or more ampoules of 75 IU human menopausal gonadotropin (Menopur; Ferring,Toronto) were administered on day 3 of the cycle. Dose ad-justments were made according to the rate of follicle growthwith the aim of obtaining around 10 eggs. Follicle growth andhormone levels were serially monitored by ultrasound andblood tests until the dominant follicles reached an average ARTICLE IN PRESS Please cite this article in press as: F. Mitri, et al., A pilot study to evaluate a device for the intravaginal culture of embryos, Reproductive BioMedicine Online (2015), doi:10.1016/j.rbmo.2015.09.005 2 F Mitri et al.  diameter of 18 – 20 mm. At that point, human chorionic go-nadotrophin (HCG) (10,000 IU Pregnyl; Merck, Kirkland,Quebec) was administered subcutaneously to trigger ovula-tion. Thirty-six hours later, oocyte retrieval was carried outunder transvaginal guided ultrasound and needle aspiration(Feichtinger and Kemeter, 1986). Oocyte assessment Mature oocytes were selected using magnification through thestereo microscope based on traditional morphological crite-ria (Mandelbaum, 2000). Cumulus, corona radiata and polarbody were all assessed. Oocytes that conformed to the abovecriteria were randomly divided into two groups by the em-bryologist for conventional IVF in the incubator or for culturein the IVC device. Intravaginal culture system and procedure The IVC device was provided disassembled, sterilized, pre-packaged and labelled (Invocell TM , Invaron Pharmaceuti-cals, Kelowna, BC). The apparatus consists of two maincomponents, an inner chamber and a rigid outer sheath(Figure 1). The inner chamber was designed to be filled with culture medium and gametes such that it was air free andallowed for easy identification and removal of the embryos,which descend into the bottom of the chamber(microchamber). Following the product instructions, the innerchamber was filled with 1.08 ml of pre-equilibrated culturemedium (G1, Vitrolife, Englewood, CO, USA) supplementedwith 5% human serum albumin (HSA, Vitrolife, Englewood, CO,USA). The gametes were then loaded into the IVC device aspreviously described with a few modifications (Frydman andRanoux, 2008). When loading the gametes, a special rotat-ing valve helped to prevent air contamination and a narroworifice prevented any sudden shift in pH.A total of 35,000 washed motile spermatozoa were firstplaced into each device using a volume of less than 50  μ l. Onaverage, eight oocytes were added to the inner chamber of each device before the components were finally reassembled.The outer chamber, which is made of polystyrene with a largesilicone O-ring, acted as a rigid shell for the inner chamber.The device was placed into the retention diaphragm, and bothwere then inserted into the vagina behind the cervix in theposterior vaginal fornix. A perforated flexible diaphragm wasthen inserted to help maintain the IVC in position. The devicewas carried in the vagina for three consecutive days duringwhich it remained completely sealed. Participants were giveninstructions, including abstinence from sexual activity orvaginal douching. After three days, the device was removedfrom the vagina, disassembled and the embryos were care-fully identified, washed and scored. No oocytes were lost ormissing on opening the device. Under trans-abdominal ultra-sound guidance, between one to three selected embryos fromthe IVC device were loaded and then transferred into theuterus using a standard embryo transfer catheter (Softpass,Cook Medical, Whitchurch-Stouffville, ON). The embryos weretransferred in G1 culture medium (G1, Vitrolife, Engle-wood, CO, USA) supplemented with human serum albumin(HSA, Vitrolife, Englewood, CO, USA). Embryos from the IVC Figure 1  The INVOcell TM device: (A) the components of the inner chamber of the device; (B) rigid outer sheath encasing the innerchamber.Adaptedfromhttp://www.canadianfertilityinstitute.com (anintroductiontoINVOcellTM);http://www.canadianfertility.com/ 152-2/an-introduction-to-invocell/. ARTICLE IN PRESS Please cite this article in press as: F. Mitri, et al., A pilot study to evaluate a device for the intravaginal culture of embryos, Reproductive BioMedicine Online (2015), doi:10.1016/j.rbmo.2015.09.005 3Intravaginal culture of embryos  device were preferentially transferred; however, when nonewere available or of suitable quality, embryos from the IVFgroup were transferred. Transfers were only carried out usingfresh embryos. The embryo transfer catheter was subse-quently examined under the microscope after each embryotransfer to exclude the possibility of retained embryos. Allsupernumerary embryos with acceptable quality were cul-tured to blastocyst and vitrified. Conventional insemination A second group of sibling oocytes was fertilized in the labo-ratory using conventional IVF. The same culture medium wasused with both the IVC device and conventional insemina-tion. Groups of four to five mature eggs were incubated with100,000 motile spermatozoa in a central-well dish (CentralWell Dish [Falcon], Corning Inc., Durham, NC, USA) and fer-tilization was documented 12 – 20 h later by the presence of two pronuclei. Embryo development was assessed on a dailybasis using the inverted microscope. Blastocyst vitrification Additional embryos of acceptable quality from either arm of the study, which had not been transferred, were further cul-tured to the blastocyst stage in the laboratory and then vit-rified as previously described (Kuwayama, 2007). Embryo scoring All of the embryos that developed into either the cleavageor blastocyst stage were individually graded. Day 3 embryosweremorphologicallyassessedaccordingtothenumberofcellsand level of fragmentation. The grade was determined basedon the fragmentation pattern with Grade 1 corresponding tominimal fragmentation and grade 5 corresponding to exten-sive fragmentation (Veeck, 1986). Embryos with acceptablescoreswereconsideredsuitablefortransfer(4CGI,4CGII,5CGI,5CGII, 6CGI, 6CGII, 6CGIII, 7CGI, 7CGII, 7CGIII, 8CGI, 8CGII,8CGIII). Day 5 embryos were also graded adopting the scoringsystemusedbyGardneretal.(2000)andRehmanetal.(2007). In brief, blastocyst expansion, trophectoderm and inner cellmass were each graded and given a numerical score (Rehmanet al., 2007). Luteal phase support Progesterone supplementation using intravaginal supposito-ries was started the day of embryo transfer up until serumbeta-HCG testing which was done 2 weeks later and if posi-tive, repeated after two days. A total of 600 mg of daily in-travaginal microinonized progesterone was administered(200 mg three times daily; Prometrium, Merck, Kirkland,Quebec). If pregnancy was confirmed after two weeks, pa-tients were instructed to continue taking progesterone andserial serum beta-HCG levels were determined after two daysand as needed thereafter. Five weeks after embryo trans-fer, transvaginal ultrasound was carried out in women whohad a positive serum pregnancy test result in order to confirmclinical pregnancy. This was defined by the presence of a ges-tational sac detected by ultrasound. Post-study questionnaire A post-study questionnaire consisting of 12 questions was dis-tributed to each participant. Scales quantifying the level of discomfort, significance of fertilization happening in the bodyrather than in the laboratory, and other questions relating toconvenience, adverse effects and adherence to the instruc-tions were all included in the questions (Table 1). All of the questionnaires were completed and analysed. The Pearsonproduct-momentcorrelationcoefficientwascalculatedfordis-comfort levels and number of eggs retrieved, in the ques-tionnaire portion of this study. Outcome measures and statistical analysis Retrieved sibling oocytes were randomized between the IVCdevice and conventional IVF. Analysis of fertilization, cleav-age rates and embryo quality comparisons between the twogroups of oocytes, was carried out using the Mantel – Haenszeltest. Mean values and standard deviations were computed andincluded in the analysis for the questionnaire portion of thestudy. Results General results Each of the 10 eligible participants between the ages of 27and 37 years underwent one ovarian stimulation cycle. Outof 10 stimulation cycles, 164 oocytes were obtained (Table 2). A total of 81 oocytes was placed into the IVC device and 33(40.7%  ±  27%) oocytes fertilized resulting in 32 cleavage stageembryos (97%  ±  6% cleavage rate). A total of 83 oocytes wasinseminated in culture dishes and kept in the incubator(Table 2). Normal fertilization occurred in 57 IVF oocytes (68.7%  ±  36%) resulting in 53 cleavage stage embryos (93%  ± 1.5%). One patient (four eggs) from the IVF group and anotherpatient (four eggs) from the IVC device group had total fer-tilization failure. A significantly smaller number of embryosthat were suitable for transfer was obtained using the IVCdevice compared with IVF (odds ratio [OR] 0.47; 95% confi-dence interval [CI] 0.26 to 0.87). The proportion of embryossuitable for transfer per fertilized oocyte was similar in bothgroups. Seven out of 10 women received IVC embryo trans-fers (Figure 2). A total of 12 cleavage stage embryos was trans- ferred into this group (average of 1.7 embryos per woman).Among the three patients who did not have embryo trans-fer, one had no fertilization using the IVC device. A secondpatient had one poor quality day three embryo, which ar-rested. The last patient was judged to be at high risk for de-veloping ovarian hyperstimulation syndrome and thereforefresh embryo transfer was avoided. IVF embryos were trans-ferred (fresh embryo transfer) in the first two patients ARTICLE IN PRESS Please cite this article in press as: F. Mitri, et al., A pilot study to evaluate a device for the intravaginal culture of embryos, Reproductive BioMedicine Online (2015), doi:10.1016/j.rbmo.2015.09.005 4 F Mitri et al.  described above who failed to have any embryos developingfrom the IVC device. One patient received two cleavage stageembryos whereas two blastocysts were transferred to thesecond patient. Four positive beta-HCG results were ob-tained from the IVC device transfers, which resulted in threeclinical pregnancies in this group. The biochemical preg-nancy rate for the IVC group was 40% (per cycle) and 57% (perfresh embryo transfer procedure). The clinical pregnancy ratefor the IVC group was 30% (per cycle) and 43% (per patientembryo transfer procedure). The beta-HCG results were posi-tive in both women who had embryos transferred from theIVF group, and clinical pregnancies were subsequently de-tected by ultrasound.Of the remaining 20 IVC cleavage embryos, 16 were furthercultured. Successful blastocyst stage development was foundin six of these embryos (37.5 %  ±  28%), which were subse-quently vitrified, whereas the remaining 10 embryos ar-rested at the cellular stage and were discarded. Forty-five IVFcleavage stage embryos were cultured for a further 2 – 3 daysand 32 embryos developed to the blastocyst stage (71%  ±  16%blastocystrate).Thirtyblastocystswerevitrifiedandtwoweredirectly transferred (fresh transfer). The remaining embryosarrested at the cellular stage and were therefore discarded. Questionnaire Questionnaires about the acceptability and overall experi-ence with the IVC system were completed and received beforeremoving the device on the day of embryo transfer (Table 1). Most of the participants were satisfied with the device, andmost women reported mild discomfort usually in the form of pressure. On a scale of 1 to 10, the mean discomfort scorereported while carrying the device was 3.2  ±  2.4. Number of eggs retrieved did not correlate with the level of discomfortreported and it is difficult to determine whether part of thesymptoms could have been related to the preceding oocyteretrieval procedure. The device remained in the vagina but Table 1  Study questionnaire for acceptability and overall experience with the intravaginal culture device.1. While wearing the device and retention system were you aware that they were in the vagina?2. While wearing the device and retention system did you feel any discomfort? If yes, could you tell if it was related to the deviceor retention system? Please explain how you could tell.Try to quantify the discomfort: circle one of the numbers on the scale 0 being no discomfort and 10 being severe discomfort3. While wearing the device and retention system did they become dislodged? Did you have to reposition them in the vagina? If yes,were you able to reposition them? Was the repositioning easy, somewhat challenging or difficult?4. While wearing the device and retention system did they come out of the vagina? What caused them to come out of the vagina?On which day did they come out? Were you instructed by your physician to replace them? Was the replacement easy, somewhatchallenging or difficult?5. While wearing the device and retention system did you have intercourse? Bathe in a tub? Douche? Have a sauna? Go swimming?6. While wearing the device and retention system did you perform any unusual or strenuous activity? If yes, explain briefly.7. While wearing the device and retention system did you have to take them out of the vagina? If yes explain the reason.8. While wearing the device and retention system did you have any unusual vaginal discharge? If yes, answer the followingquestions: Was the discharge abundant? Was the discharge odorous? What colour was the discharge? What was the consistency?9. While wearing the device and retention system did you have any spotting?10. While wearing the device and retention system did you have any vaginal itch?11. How important was it to you to know that the fertilization was occurring in your body and not in a laboratory? (0 being notimportant at all, 10 being extremely important).12. How important was it to you to know that the fertilization was occurring naturally? (0 being not important at all, and 10 beingextremely important?). Table2  DatacomparingtheresultsbetweenIVFandtheintravaginalculturedeviceamong10 women. Variables IVF IVC Significance Number of mature oocytes (MII) 83 81  – Number of 2PN 57 33  – 2PN/MII (%) 68.7 40.7  P   =  0.002Number of cleaved embryos (day 3) 53 32  – Cleaved embryos/2PN (%) 93 97 NSNumber of usable embryos a 40 18  – Usable embryos/total number of 2PN (%) 40/57  =  70% 18/33  =  55% NS IVC  =  intravaginal culture device; MII  =  second metaphase; NS  =  not statistically significant;2PN  =  two pronuclei. a Usable embryos has been defined as embryos that were either transferred or vitrified at thecleavage or blastocyst stage. ARTICLE IN PRESS Please cite this article in press as: F. Mitri, et al., A pilot study to evaluate a device for the intravaginal culture of embryos, Reproductive BioMedicine Online (2015), doi:10.1016/j.rbmo.2015.09.005 5Intravaginal culture of embryos
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