Steven T. Nakajima, M.D.1, Sam Najmabadi, M.D.2, Jose Luis Rivas, M.D3, Alexander Nadal, B.A.4, Santiago Munné, Ph.D.5,6, Sandra A. Carson, M.D.6, John E. Buster, M.D.4,7
1Department of Obstetrics and Gynecology, Stanford University School of Medicine
2Center for Reproductive Health and Gynecology
3Punta Mita Hospital, Punta Mita Fertility Center/Center for Reproductive Health and Gynecology
4Previvo Genetics, Inc.
6Department of Obstetrics, Gynecology and Reproductive Sciences, Yale University
7Department of Obstetrics and Gynecology, Warren Alpert Medical School of Brown University
We recently reported the first successful in vivo lavages (IVLs) with preimplantation genetic testing for aneuploidy (PGT-A) on human blastocysts (1). This was a series of 134 uterine lavages with the recovery of 96 in vivo conceived blastocysts available for aneuploidy screening. This research was a proof of concept study and has resulted in the delivery of five healthy infants.
In this editorial, we outline clinical applications of IVL and propose it as a new method for “elective embryo freezing” for women who would like to preserve their fertility. We feel IVL could expand fertility preservation to a wider group of women at a lower expense due to the use of their natural menstrual cycle and the need for less medication than in vitro fertilization (IVF).
The IVL Procedure
The lavage system is comprised of a disposable single use sterile catheter and a reusable controller that automates pulses of sterile lavage media into the uterine cavity manufactured by Previvo Genetics, Inc. (Figure 1). IVL can be performed in a monitored natural ovulatory cycle or after controlled ovarian stimulation (COS) followed by an ovulation trigger and intrauterine insemination (IUI) of sperm. The IVL is scheduled for five days (range 115-129 hours) after the IUI. Due to the short duration of the procedure (less than 2 minutes) and the smaller diameter of the tip of the catheter (ball tip 4.4 mm and shaft 4.1 mm in diameter) conscious sedation is not necessary. The vagina and cervix is rinsed with normal saline. A tenaculum is placed on the cervix to ensure a snug contact between the cervix and uterine lavage catheter’s acorn tip preventing backflow of infused media into the vagina. With the patient awake, the operator inserts the catheter tip to the central portion of the uterus with the assistance of a simultaneous transabdominal ultrasound similar to an ultrasound-guided embryo transfer procedure. When the catheter tip is positioned within the central portion of the uterus, the automated lavage process is initiated. During this procedure, the operator can slide and/or rotate the catheter tip from the fundus to the internal os. Cells within the cavity, embryos and unfertilized oocytes, flow passively into the fluid recovery port to an attached collection bottle. The duration of the automated lavage is one minute, followed by a gentle suction without media to vacuum up any residual fluid within the uterine cavity and/or recovery line into the collection bottle.
IVL offers simple, less expensive, office-based access to in vivo conceived human blastocysts in comparison to IVF. The uterine lavage can be performed in remote locations and the identified embryos sent to a central laboratory in a portable incubator within 24-36 hours for potential biopsy and/or cryopreservation. IVL can be easily established in fertility centers already performing assisted reproductive technology (ART) procedures.
IVL Research Trial Results
In the study by Munné and colleagues (1), 134 consecutive uterine lavages were performed after COS and IUI of sperm. Women were primarily oocyte donors and donor sperm was utilized. Both female and male donors signed written informed consent for gamete donation with the knowledge that one option for any embryos recovered would be for future embryo donation to infertile couples. IVL recovered 136 embryos in 42% (56/134) of lavage cycles performed in 81 women. In 20 lavage subjects, a single IVF cycle (control group) utilizing the same COS protocol was performed for a comparison of in vivo to in vitro blastocysts. In the women undergoing both uterine lavage and one IVF cycle, 46 and 163 blastocysts were analyzed, respectively. There was no significant difference in the rate of euploid blastocysts (57% versus 51%, respectively), however, there was a significant improvement in embryo morphology evaluated by the Gardner classification (2). More in vivo blastocysts were of good quality (3BB or higher) when compared to in vitro blastocysts (81% versus 43%, respectively, Table 1).
Differences in aneuploidy rates have been observed among fertility centers (3) and appear to be related to hormonal stimulation (4). Thus, it is not surprising that there was no significant difference in the euploid rate between in vivo and in vitro blastocysts since both groups were stimulated with the same COS protocol.
In the initial study using oocyte donors, we administered three 0.25 mg doses of gonadotropin releasing hormone (GnRH) antagonist daily after the lavage. In 11 lavages (8.2%, 11/134) there were detectable hCG levels (>2 mIU/mL) 13 days after the IUI, that regressed spontaneously in two and declined after curettage in two cases. Persistent hCG levels were noted to resolve after methotrexate (MTX) in three cases and four cases received both curettage and MTX. All subjects with persistent hCG levels were asymptomatic, and there was no evidence of a clinical intrauterine or extrauterine pregnancy in all 11 cycles. Thereafter, the protocol for GnRH antagonist administration was changed to a single dose of 0.75 mg immediately after the lavage procedure and the lavage catheter has been modified to the device depicted in Figure 1. These modifications have markedly reduced the incidence of detectable levels of hCG after the lavage to 3.3% (2/60 recent lavages). In a recent cohort of 20 lavage cycles, we have added the administration of a single 30 mg oral dose of ulipristal acetate immediately after the lavage with no detectable levels of hCG documented. We recognize that the risk of an extrauterine or an unscreened intrauterine pregnancy in the case of a PGT-A indication is a potential drawback in the use of IVL.
Clinical Applications of IVL
A. IVL as an Infertility Treatment
Augmentation of the Intrauterine Insemination Procedure
Intrauterine insemination (IUI) of sperm is a traditional first line office therapy for infertile couples. It is widely accepted that IUI is the most popular method of assisted reproduction worldwide (5) and often one of the few procedures available in resource-poor countries where IVF is either not available or accessible due to geography or high financial expense (6). IUI therapy is considered when the female partner with patent fallopian tubes fails to conceive after an adequate exposure to sperm usually 6-12 non-conceptive cycles dependent upon the patient’s age. IUI is often coupled with oral and/or injectable ovulation induction medication. In randomized controlled trials for unexplained infertility, IUI increased cycle fecundability after clomiphene citrate use (7) and gonadotropin administration (8). Observed drawbacks of this therapeutic approach are the low cycle fecundability with oral medication (7) and the risk of ovarian hyperstimulation syndrome (OHSS) and a high order multiple gestation with gonadotropin stimulation (8). IVL after COS and IUI has the potential to increase the live delivery rate per cycle and gain further access to the blastocyst for possible aneuploidy screening and embryo banking. Use of IVL in resource-poor countries may have to be limited to clinics that have the ability to closely monitor their patients before and after the IVL procedure.
IVL may have its greatest utility in improving the cycle fecundability of women with chronic anovulation. These patients are sometimes unresponsive to oral medication, but when advanced to gonadotropin therapy they have an excessive response leading to either cycle cancellation or increase morbidity if ovulated. If this particular group of high responders does conceive, they are at an increased risk for a high order multiple gestation and OHSS. Utilization of either a “rescue” or “planned” IVL after COS/IUI in this type of patient may be highly therapeutic. Many women confronted with the decision to cancel their COS cycle due to the risk of OHSS have extreme emotional difficulty in cancelling this type of robust cycle response. Many women will take needless risks despite extensive counseling, by completing their therapy with an IUI. Alternatively, converting the cycle to an IVF cycle often may not be financially possible due to the added expense of therapy and the ultimate cycle outcome may have been improved if the patient was stimulated with a planned IVF cycle in mind prospectively. It is acknowledged that a COS cycle completed with a GnRH agonist trigger injection for IVF with cryopreservation of cultured blastocysts would also be a reasonable alternative to avoid OHSS in the woman with chronic anovulation.
It is expected that with future improvements to the lavage catheter that naturally evolve with increased clinical use, the efficiency of the catheter to recover all the potential embryos will allow for less ovarian stimulation and eventually lead to performing IVL in natural ovulatory cycles. Since gonadotropin use and in vitro culturing has been suggested to contribute to an increase in aneuploidy (3,4), strategies to utilize a woman’s natural cycle would be preferred.
For women using donor semen, augmentation of the basic IUI procedure with the addition of IVL, would be helpful. Embryos could be recovered from repetitive IVL cycles for embryo cryopreservation with or without aneuploidy screening. This would allow the woman to conceive all her embryos at a younger age when planning to have two or more children. Due to the expense of donor semen, efforts to improve cycle fecundability are even more pressing as a woman ages and fertility declines.
Alterative Fertility Therapies for Lesbian Couples
In contrast to the traditional donor insemination of one lesbian partner, there is increasing interest among lesbian couples in an IVF treatment termed “Reception of Oocytes from Partner” (ROPA). This is the process where one female partner undergoes ovarian stimulation for an oocyte retrieval and the oocytes undergo subsequent intracytoplasmic sperm injection (ICSI) with donor sperm. Embryos resulting from this IVF cycle are then transferred into the patient’s female partner for her to gestate the blastocyst and deliver the infant (9). This concept could be easily adapted to inseminate one partner’s natural or stimulated cycle with IUI followed by IVL and immediate fresh transfer of a blastocyst to her female partner performing a simultaneous programed embryo transfer cycle. IVL is a much simpler and less expensive than an IVF procedure. If screening for aneuploidy was desired then the cryopreserved euploid blastocyst could be subsequently transferred in a thaw transfer cycle. Lesbian couples that have utilized IVF ROPA therapy cite a desire for both partners to have a part in their family building process. Depending on the wishes of the couple, minimal deviation from the natural conception process could be utilized. Performing an uterine lavage after donor insemination of one partner in a natural cycle timed with an ovulation predictor kit is in line with the desires of some couples to have the most natural process possible with the least required technical intervention.
Additional Selected Indications
In other selected individual cases where IVF is utilized, IVL may be an option for additional infertile couples. Sentinel to the success and the cost effectiveness of these potential indications for IVL will ultimately be the ability of the catheter to recover a majority of blastocysts in a cohort if they are present in the uterus. As catheter efficiency improves the number of IVL procedures will decrease to identify an unaffected blastocyst in a couple with a single gene defect or a normal blastocyst in a couple with recurrent pregnancy loss due to a chromosomal structural rearrangements.
B. IVL for Fertility Preservation
IVL is Particularly Suited to Clinical Strategies Directed Toward Life Long Fertility Planning
For women who would like to consider elective embryo banking with or without aneuploidy screening, IVL would be a less expensive, less invasive option than IVF. Depending on the age of the patient and her choice to augment the menstrual cycle, the number of cumulative lavage procedures to achieve her desired number of cryopreserved blastocysts will vary. One can propose a strategy to electively perform this preemptive fertility preservation procedure in natural cycles for the recovery of blastocyst(s) for embryo banking. Dependent upon a woman’s age and personal decision-making she may elect to perform PGT-A. As the ultimate efficiency of the uterine lavage procedure is established, the total cost of a series of IVL procedures with associated blastocyst vitrification would be set to be comparable to or less than the expense of a traditional IVF cycle for embryo cryopreservation. The IVL procedure is ideally suited for a woman without known infertility factors who has a chosen sperm source and desires to preserve her fertility with embryo banking. This option would empower women to participate in the timing and configuration of her future family. If performed prior to the maternal age of 35 years, high quality in vivo conceived euploid blastocysts are likely to be cryopreserved giving some further assurance of a live birth in the future. In animal models, in vivo conceived blastocysts have different characteristics (e.g. lower rate of chromosomal abnormalities, higher rates of blastocyst formation and reduced genetic aberrations) compared to in vitro developed embryos of similar species (10, 11). Recent studies performed using genetic stability measurements in bovine models have shown significantly higher chromosomal instability in in vitro cultured rather than in vivo derived embryos (12).
The ability to have a less expensive and invasive fertility preservation procedure that can be performed with minimal monitoring may be especially helpful when a woman may want to minimize her exposure to infectious threats such as the current novel coronavirus (2019-nCoV). Since IVL is a short duration procedure with minimal discomfort, it can be performed without conscious sedation and the added close exposure to the provider administering the anesthesia.
Given the right social situation of the woman, we feel the IVL procedure could be a key element in a woman’s comprehensive life long fertility management plan rather than relying upon future expensive infertility therapies for subsequent age-related infertility.
- Munné S, Nakajima ST, Najmabadi S, Sauer MV, Angle MJ, Rivas JL, et al. First PGT-A using in vivo blastocysts recovered by uterine lavage: Comparison with matched IVF embryo controls. Hum Reprod 2020;35:70-80.
- Gardner DK, Lane M, Stevens J, Schlenker T, Schoolcraft WB. Blastocyst score affects implantation and pregnancy outcome: toward a single blastocyst transfer. Fertil Steril 2000;73:1155-8.
- Munné S, Alikani M, Ribustello L, Colls P, Martinez-Ortiz PA, Referring Physician Group, et al. Euploidy rates in donor egg cycles significantly differ between fertility centers. Hum Reprod 2017;32:743-9.
- McCulloh DH, Alikani M, Norian J, Kolb B, Arbones JM, Munné S. Controlled ovarian hyperstimulation (COH) parameters associated with euploidy rates in donor oocytes. Euro J Med Genet 2019;62:103707.
- Cohlen B, Bijkerk A, Van der Poel S, Ombelet W. IUI: review and systemic assessment of the evidence that supports global recommendations. Hum Reprod Update 2018;24:300-319.
- Ombelet W, Cooke I, Dyer S, Serour G, Devroey P. Infertility and the provision of infertility medical services in developing countries. Hum Reprod Update 2008;14:605-621.
- Deaton JL, Blackmer KM, Gibson M, Nakajima ST, Badger GJ, Brumsted JR: A randomized, controlled trial of clomiphene citrate and intrauterine insemination in couples with unexplained infertility or surgically corrected endometriosis. Fertil Steril 1990; 54:1083-8.
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- Bodri D, Nair S, Gill A, Lamanna G, Rahmati M, Arian-Schad M, et al. Shared motherhood IVF: high delivery rates in a large study of treatments for lesbian couples using partner-donated eggs. Reprod Biomed Online. 2018;36:130-6.
- Hyttel P, Viuff D, Laurincik J, Schmidt M, Thomsen PD, Avery B, et al. Risks of in-vitro production of cattle and swine embryos: aberrations in chromosome numbers, ribosomal RNA gene activation and perinatal physiology. Hum Reprod 2000;15 Suppl 5:87-97.
- Hamm J, Tessanne K, Murphy CN, Prather RS. Transcriptional regulators TRIM28, SETDB1, and TP53 are aberrantly expressed in porcine embryos produced by in vitro fertilization in comparison to in vivo- and somatic-cell nuclear transfer-derived embryos. Mol Reprod Dev 2014;81:552-566.
- Tšuiko O, Catteeuw M, Esteki MZ, Destouni A, Pascottini OB, Besenfelder U, et al. Genome stability of bovine in vivo-conceived cleavage-stage embryos is higher compared to in vitro-produced embryos. Hum Reprod 2017;32:2348-57.