New data on reference ranges for transgender men

Total testosterone and estradiol were measured using immunoassay and liquid chromatography coupled with tandem mass spectrometry. FSH, LH, SHBG, prolactin, progesterone, AMH, and DHEAS were measured using immunoassay. LH, AMH, and DHEAS were measured on the Roche Cobas immunoassay instrument, while the other analytes, including total testosterone and estradiol, were measured on the Roche and Beckman Coulter DxI immunoassay instruments. Free testosterone was calculated.

The authors measured many of the analytes on multiple instruments to follow “in a limited way what’s been done in other areas, like pediatrics, where the samples are harder to get so you analyze them on multiple instruments,” Dr. Krasowski says.

The study of transgender men was preceded by an analysis of the same endocrine markers in a cohort of 93 transgender individuals on stable feminizing hormone therapy. In that study, conducted by the same group, the distribution of results differed from those of cisgender men and women across all measurements, supporting the use of transfeminine-specific reference intervals for the sex hormones (Greene DN, et al. J Appl Lab Med. 2021;6[1]:15–26).

More than a third of participants in the feminizing cohort administered spironolactone in addition to estrogen, and a little more than 10 percent administered estrogen and progesterone. Spironolactone administration was associated with statistically significant differences in the distribution of results for AMH, FSH, LH, and progesterone. “Spironolactone is a messy drug. It affects multiple hormones and also has effects on blood pressure and other endpoints,” Dr. Krasowski says. “So it’s tricky, what it might be doing. But there’s a lot of variability in how it’s prescribed, and it does seem to be causing some differences.” The study wasn’t sufficiently large to answer with certainty how spironolactone might impact the tests, he says.

The researchers had hoped to recruit 120 participants to each of the two studies but ultimately fell short, Dr. Krasowski says. While it helped that the clinics in Seattle and Iowa City where recruitment took place had years of experience treating LGBTQ patients, it was a difficult recruitment, he says, noting that a longer recruitment period and travel reimbursements might have made it easier. One exclusion criterion—obesity—was the subject of debate, and a decision was made to stick with a BMI cutoff of less than 30. But it “cost us about 40 percent of potential subjects,” Dr. Krasowski says. Because of the limited sample size, they were unable to analyze subgroupings, such as age or mode of hormone administration. A limitation of the study of transgender men was that the timing of last testosterone dose was not reported, meaning participants may have been at different points in the cycle when measurements were taken.

Both studies are the first of their kind to prospectively derive transgender reference intervals, Dr. Krasowski says. “It’s kind of amazing that it’s taken this long.” Dr. Greene speculates it’s a lack of interdisciplinary communication that resulted in a “less robust promotion of the need for this.” It’s also hard, she adds, to get oppressed populations to trust medical institutions.

The prospective studies made it possible to get a consistent range of tests that would have been difficult to pick up retrospectively, Dr. Krasowski says, as well as complete data on the subjects who were recruited. In a retrospective analysis of 150 transgender men and 152 transgender women that he helped author, there was a wide range in the completeness of lab monitoring, he says, driven in part by infrequent care or use of telehealth because of distance or insurance or other limitations (Humble RM, et al. J Appl Lab Med. 2019;3[5]:799–814).

Dr. Krasowski describes as complicated the existing research on laboratory changes in transgender patients, but says some things are clear. Most clear-cut are the findings related to hematological parameters, shown in multiple studies, he says. In a study he and Dr. Greene coauthored with others (Greene DN, et al. Clin Chim Acta. 2019;492:84–90), individuals prescribed testosterone or estrogen had hematology parameters that were not clinically different from those of cisgender males and females, respectively, regardless of serum hormone concentration. Thus, for individuals on gender-affirming hormone therapy, hemoglobin, hematocrit, and RBC count can be interpreted using the sex-specific reference intervals for their affirmed gender. “This is the clearest cut,” Dr. Krasowski says. “It moves to the opposite range.”

Dr. Krasowski highlighted findings from the 2019 retrospective study he and colleagues did and other retrospective studies. In adults on feminizing hormone therapy, increases in prolactin and SHBG have been observed. Creatinine and FSH decrease on balance, albeit with more variability seen across studies. The data on lipids, liver enzymes, DHEAS, FSH, LH, and AMH are inconsistent or limited, or both, or seen with complex effects.

In transgender men, creatine increases are consistent across studies, though that may be related mostly to increase in muscle mass, he says. Increases in ALT and AST also have been observed but not in all studies. DHEAS, FSH, LH, and SHBG tend to decrease, while the data on AMH are inconsistent or limited, or both, or seen with complex effects.

The effects of hormone therapy on plasma lipids is unclear. “Particularly for triglycerides, LDL, and total cholesterol, the changes are complex across studies,” Dr. Krasowski says, noting it may have to do with inclusion criteria, such as BMI. The data on HDL in studies of transgender men are fairly consistent, with multiple retrospective studies reporting decreases, but the data are less consistent in studies of transgender women.

Microbiology is likely to be a focus of future research, Dr. Krasowski says. “Are there changes in genitourinary flora due to hormones or surgery?” Dr. Greene and others have published the only clinical study on this topic to date, comparing the vaginal floras of transgender men on testosterone therapy with those of cisgender women. They found that the vaginal microbiome of transgender men may differ from that of cisgender women, with the transgender male participants in the study less likely to have Lactobacillus as their primary genus. They also had a significantly increased relative abundance of more than 30 species and a significantly higher alpha diversity according to the Shannon diversity index (McPherson GW, et al. Clin Chem. 2019;65[1]:199–207).

Further research is needed, too, to resolve how route of administration may affect laboratory values. Oral estradiol, for example, has a first pass through the liver, which doesn’t occur with intramuscular, subcutaneous, or topical administration. More research is also needed on the impact of additional medications such as spironolactone and flutamide.

Awaiting publication now, Dr. Greene says, are the results of a study on estrone, an estrogen metabolite sometimes used in transgender medicine, the clinical utility of which she says is highly controversial. “This article”—a study spearheaded by a pharmacist—“gives insight into the relative ratios of estrogen to estradiol, depending on the mode of administration of estradiol.”

In progress is a high-sensitivity troponin analysis by Drs. Greene and Krasowski and others. Studying the effects of gender-affirming hormone therapy on high-sensitivity troponin may shed light on the physiological mechanism that accounts for the sex-based difference in high-sensitivity troponin reference intervals.

“It’s unclear whether the sex-based differences in high-sensitivity troponin affect clinical outcomes,” Dr. Greene says, and how gender discrimination, like long-held assumptions that women present with atypical heart attack symptoms more often than men, “bleeds into evaluating people based on their feminine or masculine characteristics when they present with signs and symptoms of acute myocardial infarction.”

“There’s a lot that can be gleaned from looking at the distribution of high-sensitivity troponin in transgender populations, and how gender-affirming hormones do or do not shift high-sensitivity troponin concentration,” she continues. Says Dr. Krasowski: “There, having a reference range is important because you’re going to trigger off it. So we’ll see what that finds.”

Charna Albert is CAP TODAY associate contributing editor.