CSIRO PUBLISHING Reproduction, Fertility and Development, 2016, 28, 1580–1587 http://dx.doi.org/10.1071/RD15011 Ò MicroSort sperm sorting causes no increase in major malformation rate Donald P. MarazzoA,C,D, David KarabinusA, Lawrence A. JohnsonB and Joseph D. SchulmanA AGenetics and IVF Institute, 3015 Williams Drive, Fairfax, VA 22031, USA. BU.S. Department of Agriculture, 10300 Baltimore Ave, Bldg. 200, BARC-East Beltsville, MD 20705, USA. CInstitute for Healthy Reproductive Outcomes, 355 S Highland Avenue, Pittsburgh, PA 15206, USA. DCorresponding author. Email: [email protected] Abstract. The purpose of the present study was to evaluate the safety of MicroSort (MicroSort Division, GIVF, Fairfax, VA, USA) sperm sorting by monitoring major malformations in infants and fetuses conceived using sorted spermatozoa. Data were collected in a prospective protocol with monitoring that began from conception through birth until 1 year of life. Comprehensive ascertainment identified fetuses and stillbirths with malformations after 16 weeks gestation, pregnancies terminated for malformations and babies with major malformations. Outcomes in MicroSort pregnancies were compared with outcomes in published studies that used active and comprehensive ascertainment of malformations in the general population and in pregnancies established after assisted reproduction. Using comprehensive outcomes from all pregnancies, the rate of major malformations in MicroSort pregnancies conceived after IVF with or without intracyto- plasmic sperm injection was 7.8%; this did not differ significantly from the rates reported in the three assisted reproductive technology control studies not associated with MicroSort (8.6%, 9.2% and 8.3%). Similarly, the rate of major malformations in MicroSort pregnancies initiated with intrauterine insemination was 6.0%, not significantly different from that reported in non-assisted reproductive technology pregnancies not associated with MicroSort (6.9%, 4.6% and 5.7%). Prospective record review of pregnancy outcomes and paediatric evaluation to 1 year indicate no association between MicroSort sperm sorting and major malformations. Additional keywords: assisted reproductive technologies, birth defects, family balancing, genetic disease prevention, intracytoplasmic sperm injection, IVF, sexual selection, X-linked diseases. Received 9 January 2015, accepted 6 March 2015, published online 22 April 2015 Introduction and Schulman 1994; Schulman and Karabinus 2005; Karabinus MicroSort (MicroSort Division, GIVF, Fairfax, VA, USA) et al. 2014). allows families to increase their chances of having a baby of This method for sexing semen has been a commercial reality an indicated sex either to balance the sex ratio within their for cattle and deer in the animal production industry, along with family (family balancing) or to prevent genetic disease trans- several other species. Sexed semen may also be applied for missioninsex-linkeddisease.TheMicroSorttechnique conservation purposes in zoo animals and other threatened or (Johnson et al. 1993) uses high-speed flow cytometric sorting endangered wildlife species (Seidel 2012; Rath et al. 2013). of individual spermatozoa based on the 2.8% greater total DNA Overall, several millions of insemination doses are produced content in normal human spermatozoa bearing an X compared annually for commercial purposes (Seidel 2012). with Y chromosome (Sumner and Robinson 1976). Sperma- Morrell and Dresser (1989) found no morphological changes tozoa are briefly exposed to a dilute solution of a non- or loss of reproductive capacity with flow-sorted Hoechst intercalating fluorescent DNA dye (Hoechst 33342), which 33342-stained spermatozoa in three generations of swine and attaches to the minor groove of DNA (Latt and Stetten 1976). nine generations of rabbits. In a review of the birth status Sperm separation is based on the strength of the fluorescent of several animal species (cattle, swine, rabbits and sheep), signal after pulsing single spermatozoa with ultraviolet light. Johnson and Schulman (1994) noted that none of more than 200 Details of the MicroSort methodology and its effectiveness births from sorted spermatozoa was reported as having macro- have been reported elsewhere (Johnson et al. 1989; Johnson scopic morphological changes. Journal compilation Ó CSIRO 2016 www.publish.csiro.au/journals/rfd Major malformations after MicroSort sperm sorting Reproduction, Fertility and Development 1581 The present study focuses on the safety of this procedure in Data are comprehensive over the entire period from January humans based on the rate of major malformations in fetuses and 1995 to October 2012. During this time, more than 1300 babies babies conceived using the sorted sperm procedure. Active were born (993 females and 344 males) after MicroSort, ascertainment of major malformations with the comprehensive including 136 infants (121 females and 15 males) in whom the inclusion of fetal abnormalities indicates rates of approximately primary indication was genetic disease prevention. 8% in the general population, compared with approximately 4% Couples who participated in the trial, and any gamete donors in passive registries (Queißer-Luft et al. 2002). The present or gestational carriers, had negative laboratory tests for human study involved pregnancies established after either intrauterine immunodeficiency virus-1 antibody, hepatitis B surface antigen insemination (IUI) or IVF, usually with intracytoplasmic insem- and hepatitis C antibody within 1 year of their attempt at ination (ICSI). The present study compared MicroSort data with pregnancy. All couples signed informed consent before being large population-based reports (Hansen et al. 2002; Ludwig and enrolled in the clinical trial. There were exclusions for clinically Katalinic 2002; Davies et al. 2012) that actively ascertained significant diseases, including substance abuse. This category rates of major malformations in pregnancies from the general included diseases such as diabetes, cardiac disease, active population and in pregnancies conceived through assisted autoimmune disease, haemoglobinopathies and other maternal reproductive technologies (ARTs). disease that could negatively affect the health of the mother during pregnancy or that would increase the risk for pregnancy Materials and methods complications. Participants signed medical release forms so we could obtain newborn and paediatric records for children result- Herein we report data from an Investigational Device Exemp- ing from their participation in the study. tion (IDE) for testing the effectiveness and safety of MicroSort. Couples participating for the family balancing indication ‘IDE’ is the terminology used by the US Food and Drug must have had at least one child and wanted to use MicroSort to Administration (FDA) to indicate a clinical trial using a have a child of the under-represented gender among all their medical device that has not yet been approved for clinical use children. The woman was required to be between the ages of but meets the criteria of an IDE (Code of Federal Regulations 18–39 years at the time of egg retrieval or insemination. Couples Title 21; http://www.fda.gov/medicaldevices/deviceregulation were excluded if the woman or man (or gamete donor) had a andguidance/guidancedocuments/ucm080202.htm, accessed history of a major congenital malformation or chromosomal March 2015). Use of the device is limited to studies that meet abnormality in themselves or their children. the IDE criteria, and is not approved for use outside the defined Couples participating for the genetic disease prevention IDE. The Chesapeake Institutional Review Board (CIRBI) and indication had a family history and/or genetic testing supportive The Genetics and IVF Institute Institutional Review Board of a genetic diagnosis that made offspring susceptible to (IRB) approved the study protocol. The conduct of the clinical inherited disease that was X-linked or that otherwise had a trial was in accordance with the ethical standards of these IRB greater risk of significant clinical disease in one sex relative to entities. the other (i.e. sex limited). Subjects Sperm sorting The two indications for participation in the present study were Technical details of MicroSort sperm separation are provided genetic disease prevention of sex-linked or sex-limited genetic elsewhere (Johnson et al. 1993; Johnson and Schulman 1994; disorders or family balancing, defined as an attempt to increase Karabinus et al. 2014) and are described here briefly. Fresh or the less-represented gender of children within a family unit. frozen–thawed semen was processed, stained with H33342 and Participants were eligible for the genetic disease prevention arm sorted at high speed into populations enriched in either X of the study if they met one of the following criteria: (1) the chromosome-bearing spermatozoa (X-Sort) or Y chromosome- woman carried an X-linked disease that was only manifest in bearing spermatozoa (Y-Sort). During the study period, the male children or was more severe in male children; (2) the man method achieved average ratios of approximately 7 : 1 female : was affected by an X-linked disease that would only be passed male spermatozoa (87.8%/12.2%) with X-Sorting and approx- through daughters; (3) the man carried a premutation on the X imately 3 : 1 male : female spermatozoa (74.3%/25.7%) with chromosome (e.g. fragile X) that would be passed to his Y-Sorting, as determined by fluorescence in-situ hybridisation